The nutritive value of condensed wheat distillers solubles for cattle.

The chemical composition and the energy and protein value of five batches of condensed distillers solubles (CDS) originating from wheat were determined. The net energy for lactation (NEL) was derived from digestion coefficients obtained with sheep. The true protein digested in the small intestine (DVE) and the rumen degradable protein balance (OEB) were based on the rumen degradation rate (kd D ), the rumen undegradable fraction (U) and intestinal digestibility of undegraded protein (%DVBE) predicted by regression equations derived from a data set of 28 protein feeds with kd D , U and %DVBE determined in situ. The CDS is a by-product with a high, but very variable CP content (238 to 495 g/kg DM). The CP contained on average 81% amino acids, with glutamine as main component (on average 21.8% of CP) and a relatively good lysine proportion (3.0%). Further, CDS contains quite a lot of crude fat (mean±SD: 71±14 g/kg DM), glycerol (95±52 g/kg DM) and sugars (123±24 g/kg DM) resulting in a high organic matter digestibility (88.6±3.0%) and high NEL content (8.3±0.4 MJ/kg DM). The protein value showed a large variation, with DVE ranging from 122 to 244 g/kg DM and OEB from 50 to 204 g/kg DM. Wheat CDS is a rich source of minerals and trace elements with exception of calcium.

The PLA2R1-JAK2 pathway upregulates ERRα and its mitochondrial program to exert tumor-suppressive action.

Little is known about the biological role of the phospholipase A2 receptor (PLA2R1) transmembrane protein. In recent years, PLA2R1 has been shown to have an important role in regulating tumor-suppressive responses via JAK2 activation, but the underlying mechanisms are largely undeciphered. In this study, we observed that PLA2R1 increases the mitochondrial content, judged by increased levels of numerous mitochondrial proteins, of the mitochondrial structural component cardiolipin, of the mitochondrial DNA content, and of the mitochondrial DNA replication and transcription factor TFAM. This effect of PLA2R1 relies on a transcriptional program controlled by the estrogen-related receptor alpha1 (ERRα) mitochondrial master regulator. Expression of ERRα and of its nucleus-encoded mitochondrial targets is upregulated upon PLA2R1 ectopic expression, and this effect is mediated by JAK2. Conversely, downregulation of PLA2R1 decreases the level of ERRα and of its nucleus-encoded mitochondrial targets. Finally, blocking the ERRα-controlled mitochondrial program largely inhibits the PLA2R1-induced tumor-suppressive response. Together, our data document ERRα and its mitochondrial program as downstream effectors of the PLA2R1-JAK2 pathway leading to oncosuppression.

Dental Apical Papilla as Therapy for Spinal Cord Injury.

Stem cells of the apical papilla (SCAP) represent great promise regarding treatment of neural tissue damage, such as spinal cord injury (SCI). They derive from the neural crest, express numerous neurogenic markers, and mediate neurite outgrowth and axonal targeting. The goal of the present work was to investigate for the first time their potential to promote motor recovery after SCI in a rat hemisection model when delivered in their original stem cell niche-that is, by transplantation of the human apical papilla tissue itself into the lesion. Control groups consisted of animals subjected to laminectomy only (shams) and to lesion either untreated or injected with a fibrin hydrogel with or without human SCAP. Basso-Beattie-Bresnahan locomotor scores at 1 and 3 d postsurgery confirmed early functional decline in all SCI groups. This significant impairment was reversed, as seen in CatWalk analyses, after transplantation of apical papilla into the injured spinal cord wound, whereas the other groups demonstrated persistent functional impairment. Moreover, tactile allodynia did not develop as an unwanted side effect in any of the groups, even though the SCAP hydrogel group showed higher expression of the microglial marker Iba-1, which has been frequently associated with allodynia. Notably, the apical papilla transplant group presented with reduced Iba-1 expression level. Masson trichrome and human mitochondria staining showed the preservation of the apical papilla integrity and the presence of numerous human cells, while human cells could no longer be detected in the SCAP hydrogel group at the 6-wk postsurgery time point. Altogether, our data suggest that the transplantation of a human apical papilla at the lesion site improves gait in spinally injured rats and reduces glial reactivity. It also underlines the potential interest for the application of delivering SCAP in their original niche, as compared with use of a fibrin hydrogel.

The energy and protein value of wheat, maize and blend DDGS for cattle and evaluation of prediction methods.

The chemical composition inclusive amino acids (AAs) and the energy and protein value of three wheat, three maize and seven blend (mainly wheat) dried distillers grains and solubles (DDGS) were determined. The net energy for lactation (NEL) was derived from digestion coefficients obtained with sheep. The digestible protein in the intestines (DVE) and the degraded protein balance (OEB) were determined by nylon bag incubations in the rumen and the intestines of cannulated cows. Additional chemical parameters like acid-detergent insoluble CP (ADICP), protein solubility in water, in borate-phosphate buffer and in pepsin-HCl, in vitro digestibility (cellulase, protease, rumen fluid) and colour scores (L*, a*, b*) were evaluated as potential predictors of the energy and protein value. Compared to wheat DDGS (WDDGS), maize DDGS (MDDGS) had a higher NEL-value (8.49 v. 7.38 MJ/kg DM), a higher DVE-content (216 v. 198 g/kg DM) and a lower OEB-value (14 v. 66 g/kg DM). The higher energy value of MDDGS was mainly due to the higher crude fat (CFA) content (145 v. 76 g/kg DM) and also to better digestible cell-walls, whereas the higher protein value was mainly due to the higher percentage of rumen bypass protein (RBP: 69.8 v. 55.6%). The NEL-value of blend DDGS (BDDGS) was in between that of the pure DDGS-types, whereas its DVE-value was similar to MDDGS. Although lower in CP and total AAs, MDDGS provided a similar amount of essential AAs as the other DDGS-types. Lysine content was most reduced in the production of WDDGS and cysteine in MDDGS. Fat content explained 68.6% of the variation in NEL, with hemicellulose and crude ash as extra explaining variables. The best predictor for RBP as well as for OEB was the protein solubility in pepsin-HCl (R 2=77.3% and 83.5%). Intestinal digestibility of RBP could best be predicted by ADF (R 3=73.6%) and the combination of CFA and NDF could explain 60.2% of the variation in the content of absorbable microbial protein. The availability of AAs could accurately be predicted from the rumen bypass and intestinal digestibility of CP.

Effect of supplementation on the performance of grazing Belgian Blue double-muscled heifers.

Six experiments were conducted to investigate the effect of a feed supplement on the performance of grazing Belgian Blue double-muscled (BBDM) heifers with an initial weight and age of 195 ± 43 kg and 190 ± 52 days. Treatments included were: Exp. 1: supplementation with beet pulp (BP): 2 kg/day per head v. ad libitum intake; Exp. 2: supplementation ad libitum with BP v. a mixture of BP and soybean meal (SBM; BP/SBM ratio of 80/20; FW (fresh weight) basis); Exp. 3: supplementation with 4 kg/day per head of a mixture of BP/SBM (80/20; FW basis) v. BP/formaldehyde-treated SBM (BP/FSBM); Exp. 4: supplementation with 4 kg/day per head of a mixture with a similar protein content (125 g DVE per kg dry matter (DM)), consisting of 80/20 BP/SBM v. 92/8 BP/FSBM; Exp. 5: supplementation with 3 kg/day per head of a mixture of BP/SBM (80/20; FW basis) v. BP/DDGS (dried distillers grains and solubles; 70/30, FW basis); and Exp. 6: supplementation with 3 kg/day per head of 80/20 BP/SBM v. maize silage (MS) and SBM, on the basis of a similar protein concentration in the DM as the 80/20 BP/SBM supplement, and fed at a similar amount of DM as in the BP/SBM group. Supplementing BP ad libitum did not affect daily gain (0.54 v. 0.48 kg) and partial feed conversion (3.62 kg on average) compared with 2 kg/day. Supplying SBM besides BP increased growth rate compared with BP (0.87 v. 0.62 kg/day; P < 0.001), but partial feed conversion was similar. Supplying FSBM did not affect growth rate and partial feed conversion (P > 0.10), but blood urea levels were reduced by FSBM (P < 0.05). DDGS tended to increase growth rate (0.77 v. 0.59 kg/day; P < 0.10) compared with BP/SBM, without effect on partial feed conversion. Replacing BP by MS did not affect daily gain, but partial feed conversion tended to be higher (3.21 v. 3.60 kg/kg body weight (BW) gain; P = 0.062). Increasing the supplement (80/20 BP/SBM) level from 3 to 4 kg daily, corresponding to 1.02% and 1.18% of the mean BW, respectively, resulted in a tendency (P = 0.121) for an increased growth rate. Grazing BBDM heifers of <1 year of age necessitate extra protein besides an energy supplement to improve their performance. DDGS can replace SBM and BP can be replaced by MS.

Alginate beads as a tool to handle, cryopreserve and culture isolated human primordial/primary follicles.

BACKGROUND: One major concern of grafting cryopreserved ovarian tissue to restore fertility in cancer patients is the possibility of reintroducing tumor cells. Cryopreservation of isolated primordial/primary follicles (PFs) may circumvent this problem. The aim of our work was to compare dimethyl sulfoxide (ME2SO) and ethylene glycol (EG) as cryoprotectants (CPAs) for slow-freezing of isolated human PFs in alginate. METHODS: Ovarian biopsies from four women were processed for follicle isolation. PFs were embedded in alginate (5-15 per group). Follicles were frozen-thawed using 1.4M ME2SO or 1.5M EG as CPAs. Fresh and cryopreserved isolated follicles were in vitro cultured (IVC) for 7 days. At different time periods (after isolation, cryopreservation and IVC), follicles were evaluated with live/dead assay (using fluorescent probes) and diameter measurement. Follicle viability was calculated according to the percentage of dead follicular cells and the presence of a live/dead oocyte. RESULTS: A total of 841 PFs were isolated, embedded in alginate and cryopreserved with ME2SO (n=424) or EG (n=259), or used as controls (n=158). After 7 days of IVC, a significant increase in follicle size was observed in the fresh and ME2SO groups, but not in the EG group. The percentage of totally viable PFs was not significantly different before or after seven days of culture in fresh (100% and 82%) or ME2SO (93.2% and 85.1%) tissue. The EG group showed significantly lower viability before (63.9%) and after IVC (66.2%) than the fresh and ME2SO groups. CONCLUSIONS: Our results show that 1.4M ME2SO yields better preservation of isolated PF viability after thawing and 7 days of IVC than 1.5M EG. Alginate constitutes an easy, safe hydrogel matrix to handle and cryopreserve isolated human follicles using ME2SO as a CPA.

Effect of cull potatoes in the diet for finishing Belgian Blue double-muscled cows.

The use of culled potatoes was investigated in Belgian Blue double-muscled finishing cows, confined in tie stalls. The control diet (Treatment 1) consisted of concentrate and maize silage (50/50 on a dry matter (DM) basis). Potatoes either replaced 60% maize silage (Treatment 2) or 60% concentrate (Treatment 3). Diets were formulated to be isocaloric and isonitrogenous. They were fed ad libitum. Approximately 18 kg potatoes were fed daily in Treatments 2 and 3. Daily gain was not significantly altered; it decreased from 1.09 kg (Treatment 1) to 1.04 kg (Treatment 2) or increased to 1.20 kg (Treatment 3), although potatoes stimulated DM intake by 5% to 8% (P < 0.05). Feed conversion was unaffected in comparison with the control diet, when expressed in terms of DM, but energy efficiency (MJ/kg live weight gain) was substantially lower for Treatment 2 compared with Treatment 1 (89.9 v. 79.0; P = 0.046). Carcass weight, grading and composition were not affected by treatments, but potatoes increased dressing percentage (P = 0.009). Treatment had no significant effect on meat quality parameters. However, potatoes (Treatments 2 and 3) tended to decrease moisture content (P = 0.090) and tended to increase drip loss (P = 0.059) compared with Treatment 1. Because of a better animal performance and a lower feed cost, it is most appropriate to use potatoes as a replacement for concentrate. Feeding large amounts of potatoes besides concentrate may have an adverse effect on the fibrousness of the diet, resulting in a tendency (-5%) for a reduced daily gain and a lower energy efficiency (P < 0.05).

Molecular chaperone complexes with antagonizing activities regulate stability and activity of the tumor suppressor LKB1.

LKB1 is a tumor suppressor that is constitutionally mutated in a cancer-prone condition, called Peutz-Jeghers syndrome, as well as somatically inactivated in a sizeable fraction of lung and cervical neoplasms. The LKB1 gene encodes a serine/threonine kinase that associates with the pseudokinase STRAD (STE-20-related pseudokinase) and the scaffolding protein MO25, the formation of this heterotrimeric complex promotes allosteric activation of LKB1. We have previously reported that the molecular chaperone heat shock protein 90 (Hsp90) binds to and stabilizes LKB1. Combining pharmacological studies and RNA interference approaches, we now provide evidence that the co-chaperone Cdc37 participates to the regulation of LKB1 stability. It is known that the Hsp90-Cdc37 complex recognizes a surface within the N-terminal catalytic lobe of client protein kinases. In agreement with this finding, we found that the chaperones Hsp90 and Cdc37 interact with an LKB1 isoform that differs in the C-terminal region, but not with a novel LKB1 variant that lacks a portion of the kinase N-terminal lobe domain. Reconstitution of the two complexes LKB1-STRAD and LKB1-Hsp90-Cdc37 with recombinant proteins revealed that the former is catalytically active whereas the latter is inactive. Furthermore, consistent with a documented repressor function of Hsp90, LKB1 kinase activity was transiently stimulated upon dissociation of Hsp90. Finally, disruption of the LKB1-Hsp90 complex favors the recruitment of both Hsp/Hsc70 and the U-box dependent E3 ubiquitin ligase CHIP (carboxyl terminus of Hsc70-interacting protein) that triggers LKB1 degradation. Taken together, our results establish that the Hsp90-Cdc37 complex controls both the stability and activity of the LKB1 kinase. This study further shows that two chaperone complexes with antagonizing activities, Hsp90-Cdc37 and Hsp/Hsc70-CHIP, finely control the cellular level of LKB1 protein.

Sex hormones and their receptors in bone homeostasis: insights from genetically modified mouse models.

In this review, we summarize available data regarding bone phenotypes in estrogen receptors alpha and beta, androgen receptor, and aromatase enzyme-deficient mice. We examine sex differences in the trabecular and cortical bone compartments and we discuss these findings in relation to known estrogen effects in humans. We also report how estrogen influences the responsiveness of the skeleton to exercise. Although uncertainties remain, it is clear that both estrogen and androgen are important for both male and female skeleton. Estrogen receptor alpha mainly through its classical signaling pathway is particularly important for the male mice skeleton while both estrogen receptors alpha and beta are required for female mice skeleton. These deletions also induce major hormonal alterations themselves impacting on bone metabolism. More investigations are needed to fully understand the respective role of all these receptors in periosteal expansion in both sexes and the way they affect the mechanical sensitivity of the periosteum.

Effect of energy restriction and re-alimentation in Belgian Blue double-muscled beef cows on digestibility and metabolites.

Four groups of five non-lactating and non-pregnant Belgian Blue double-muscled (BBDM) cows were used to investigate the effect of energy level (E) on digestion, and blood and urine metabolites. The energy levels of the groups, applied indoors during a 140-day restriction period, were 100%, 90%, 80% or 70% of their energy requirements (E100, E90, E80, E70) respectively. Afterwards, animals grazed on the same swards for 203 days (re-alimentation period). Balance trials were conducted at the end of the restriction period (BT1) and at the end of the re-alimentation period (BT2). Blood was sampled at the end of these trials. Diets consisted of maize silage and straw (80/20 on a dry matter basis) and a mineral-vitamin premix, fed at the appropriate E during BT1, or maize silage and a mineral-vitamin premix, fed at 125% of the maintenance requirements, during BT2. Significant increases of the digestibility coefficients were found during BT1 when E decreased, resulting in a better net energy capture of 7% for E70 compared with E100 (p < 0.05). Slightly, but non-significantly higher digestibility coefficients were observed for decreasing E during BT2. Plasma concentrations of glucose and creatinine did not differ between treatments during BT1, while differences were found for triacylglycerols and alpha-amino nitrogen. A tendency for a linear increase was observed for non-esterified fatty acids with decreasing E. Differences in blood metabolite concentrations disappeared in BT2. Urinary creatinine excretion was not affected by E, while body nitrogen loss increased linearly with energy restriction in BT1. No differences were found during BT2, suggesting that non-lactating and non-pregnant BBDM cows are able to adapt to a cyclic change of body weight and body reserves. These data show that restricted cows mobilized body fat as well as body protein. It is concluded that the qualitative aspects of metabolism during energy restriction are comparable in double-muscled cows with those in non-double-muscled animals, although the magnitude of the effects may be different.

Dimerization is required for transactivation by estrogen-receptor-related (ERR) orphan receptors: evidence from amphioxus ERR.

The estrogen-receptor-related (ERR) receptors are orphan members of the nuclear receptor superfamily that bind to their specific DNA target sites as homodimers. However, it has not been shown whether this mode of binding is required for the transcriptional activation they drive. We here show that heterodimerization can also occur between these receptors. Furthermore, we demonstrate that the unique amphioxus ortholog of ERR genes (AmphiERR) is expressed as two isoforms differing by an in-frame insertion. While the short isoform behaves like its mammalian counterparts, the long isoform (AmphiERR(L)) displays divergent transcriptional properties according to the target site to which it binds. Indeed, AmphiERR(L) binds as a monomer but does not activate transcription through the SF1 response element (SFRE). On the contrary, this isoform binds as a homodimer and activates transcription through the classical estrogen-response element. Our results strongly suggest that dimerization is required for transactivation exerted by the ERR receptors.

Expression of the orphan nuclear receptor ERRalpha is under circadian regulation in estrogen-responsive tissues.

Circadian gene expression has been demonstrated in many tissues and involves both positive and negative regulatory loops. The potential interferences of circadian rhythmicity with other well-known biologic rhythms, such as the ovarian cycle, at least in part controlled by estrogens, has not been questioned. The estrogen receptor-related receptor (ERR)alpha is an orphan nuclear receptor that is widely expressed in estrogen-responsive tissues such as liver, uterus and bone. In addition, expression of the ERRalpha gene has been proposed to be transcriptionally controlled by estrogens in the uterus. Here we show that the expression of ERRalpha displays a circadian rhythmicity in liver, bone and uterus. This is in contrast to other uterine estrogen-regulated genes. Analysis of clock/clock mutant mice shows that ERRalpha is an output gene of the circadian clock oscillator. The expression of clock-control genes, such as Bmal1 and Rev-erbalpha, also displays diurnal oscillations in the uterus, but not in bone. In this tissue, however, Per2 displayed a rhythmic expression, altogether suggesting unconventional loops in the regulation of circadian rhythm in bone.

Agri-environmental grass hay: nutritive value and intake in comparison with hay from intensively managed grassland.

Chemical composition, digestibility, nutritive value and intake of hay from an agri-environmental management (EH) were compared with those from hay (Lolium perenne) from an intensive management (IH). IH was of low to moderate quality because of unfavourable weather conditions. EH was harvested mid-June of 2000 (EH1) and 2001 (EH2) on the same sward that had not received mineral fertilizer for 10 years. The EH was characterized by a species-rich botanical composition. On average, it had lower contents of protein (32%), NDF (9%) and ash (35%), and a higher concentration of water-soluble carbohydrates (117%) than IH. Digestibility of dry and organic matter, determined with sheep, was not different between IH and EH and averaged 59 and 63%, respectively. Crude fibre and NDF digestibility were lower in EH (58 and 57%, respectively) than in IH (70 and 69%, respectively). Net energy value for lactation did not differ between IH and EH and amounted to 4.78 MJ per kg DM. True protein digested in the small intestine and rumen degraded protein balance were lower in EH (63 and -60 g per kg DM) than in IH (71 and -33 g per kg DM). Intake of hay was investigated in Holstein-Friesian heifers and Belgian Blue double-muscled heifers (mean BW 280 +/- 22 kg and 269 +/- 21 kg, respectively), and in Belgian Blue non-lactating and non-pregnant double-muscled cows (initial BW 642 +/- 82 kg), using a cross-over design. Hay was freely available. It was supplemented with 1 kg concentrate daily. Dry matter intake from hay was higher for EH than for IH in heifers (4% and 13%, respectively in Holstein-Friesian and Belgian Blue heifers) and in cows (22%). Hay from an agri-environmental management may be used for low-performing animals, as energy intake only exceeded maintenance requirements by 20 to 35%. Several characteristics of EH were different between years, such as dry matter digestibility, net energy value for lactation and fermentable organic matter content.

Estrogen receptor-related receptors: orphan receptors desperately seeking a ligand.

The nuclear receptor family comprises ligand-dependent and orphan receptors. To the latter group belong the estrogen receptor-related receptors (ERRs) for which conflicting results have been published concerning the nature (constitutive or liganded) of their transcriptional activities. ERRs interfere in various ways, positively and negatively, with estrogen signaling. Moreover recent data analyzing ERR expression in human breast tumors have proposed ERRalpha and ERRgamma as prognostic markers of these cancers. The identification of modulators (positive or negative) of ERR activities would therefore be highly useful in our understanding of estrogen-related pathologies. The purpose of this review is to summarize our knowledge of the nature of ERR activities and progresses in identifying synthetic ERR modulators.

Carcass and meat quality in double-muscled Belgian Blue bulls and cows.

Carcass and meat quality of 37 bulls and 91 cows of the Belgian Blue breed (double-muscled type) were compared. Age at slaughter averaged 648±73 and 1820±689 days, respectively. Both groups of cattle were finished on maize silage supplemented with concentrate, and were slaughtered at about 750 kg live weight. Females had a lower (P=0.004) cold carcass weight (469.7 kg) in comparison with bulls (500.8 kg), due to a reduced dressing percentage (63.8 vs. 66.6; P <0.001). SEUROP conformation was better for bulls. SEUROP fat covering (P=0.007) and fat content in the carcass (16.4 vs. 12.9%; P <0.001) and in the M. longissimus thoracis (LT) muscle (2.3 vs. 1.1%; P <0.001) were higher for females than for males. The LT of cows was darker (lower L* and higher a*-value; P <0.001), had a better waterholding capacity (P⩽0.063) and was slightly more tender (P=0.120) than the LT of bulls. Increasing parity reduced dressing percentage and increased LT lightness (L*-value) in cows. Several carcass (SEUROP-grading, composition, LT-area) and meat quality traits (protein and fat contents, drip and cooking losses, a*-value) were better correlated with carcass weight than parity. It is concluded that meat quality of the aged LT of cows is not negatively affected by age, while some carcass quality traits decreased with advancing age. Carcass quality traits adjusted for age at slaughter were better for bulls, but LT meat quality characteristics were at least as good for females as for males.

Decreasing the roughage:concentrate ratio of a diet to determine the critical roughage part for beef cattle.

The critical roughage part (CRP) of 2 diet types was determined in a cross-over design with 6 double-muscled and 6 normally conformed Belgian Blue bulls fitted with rumen cannulae. The roughage:concentrate ratio was lowered weekly until signs of a lack of physical structure were observed. For diet 1, consisting of maize silage and concentrates, the initial proportion of maize silage was 25% of DM but it decreased weekly with 5% units of DM. For the second diet, consisting of wheat straw and concentrate, 12% straw (DM basis) was provided during the first week and thereafter the proportion of straw decreased weekly with 3% units of DM. Several directly observable parameters (rumen pH, feed intake, bloat, faecal consistency) were evaluated weekly for each bull. Apart from these direct indicators of acidosis, also other parameters, whose results were only available after the end of the trial, were determined (volatile fatty acid profile, lactic acid concentration, chewing time). The roughage part between the part fed when signs of a lack of physical structure was first observed and the part that was fed the week before, was considered as the CRP. Most animals showed no acute signs of clinical acidosis (directly observable parameters) and finished the trial on a 100% concentrate diet. However, in sacco rumen DM-degradabilities of maize silage, grass silage and wheat grain was depressed considerably when low roughage diets were fed. Based on all observed parameters, the mean CRP was calculated to be 14.7% for diet 1 and 8.1% for diet 2. The beef type (double-muscled or not) had no influence on the CRP.

Characterization of oestrogen receptors in zebrafish (Danio rerio).

We cloned the cDNAs corresponding to three oestrogen receptors (ERs) in zebrafish (Danio rerio). Sequence analysis and phylogenetic studies demonstrated that two of these genes, ER beta.1 and ER beta.2, arose from duplication of the original ER beta in many species of the fish phylum, whereas ER alpha is unique. Zebrafish ERs behaved as oestrogen-dependent transcription factors in transactivation assays. However, their reactivity to various oestrogen modulators was different compared with that of mouse ERs. ER mRNA expression during zebrafish development is restricted to distinct time periods, as observed by RNase protection assays. ER beta.2 is initially expressed as maternally transmitted RNA, until 6 h after fertilization, when expression disappears. Between 6 and 48 h after fertilization, no ER expression could be observed. After 48 h after fertilization, all ERs, but predominantly ER alpha, began to be expressed. We conclude that oestrogen signal transduction can operate during zebrafish development only within discrete time windows.

Feed intake of young double-muscled bulls fed on grass and supplemented with sugar-beet pulp.

Daily dry matter intake in young growing double-muscled bulls, fed indoors on grass, was estimated based on forty-four intake data from 28 animals, ageing at least five months and weighing up to 400 kg live weight. Intake was measured during five consecutive days using one of eighteen cuts of grass. Fresh meadow grass (mainly Lolium perenne) was fed ad libitum and two kg dried sugar-beet pulp was offered per animal and per day. Animal live weight averaged 278 +/- 82 kg and mean total daily dry matter intake amounted to 5.05 +/- 1.59 kg or 73.6 +/- 13.7 g per kg metabolic weight, while pulp dry matter intake amounted to 1.49 +/- 0.50 kg per day. Regression analysis showed that animal as well as feed characteristics could explain up to approximately 90% of the variation in daily dry matter intake. The supplementation resulted in an extra daily dry matter intake of 0.68 g per g pulp dry matter. Intake of double-muscled animals was considerably lower than previously reported for non-double-muscled cattle. An extra supplementation of young grazing double-muscled animals could be advised from these findings, while extra protein should also be considered.

Structural and functional divergence of a nuclear receptor of the RXR family from the trematode parasite Schistosoma mansoni.

We describe the cloning and functional characterization of Schistosoma mansoni retinoid-X-receptor (SmRXR; NR2B4-B), a novel member of the nuclear receptor superfamily from S. mansoni, a homologue of vertebrate retinoid-X-receptor. The DNA-binding C domain of SmRXR shows 80% sequence identity to both human RXRalpha and Drosophila ultraspiracle (USP), but a much lower level of conservation of the ligand-binding E domain (22-25% identity). Phylogenetic analysis places SmRXR within the RXR group as an early offshoot of this clade. SmRXR mRNA is expressed at all life-cycle stages but at higher levels in the free-living larval stages. However, the SmRXR protein is expressed at markedly different levels, being almost absent from eggs while present at the highest concentration in schistosomula. Recombinant SmRXR fails to bind to the consensus direct repeat response elements, either alone, or as a heterodimer with mouse retinoic acid receptor alpha or the Drosophila ecdysone receptor. However, the use of chimaeric constructions shows that the C domain of SmRXR will bind to conventional response elements as a heterodimer, and that its specificity is modified by the presence of the D and E domains. In accordance with these results, native SmRXR failed to transactivate the transcription of a reporter gene after cotransfection of mammalian cell lines.

Amphicoup-TF, a nuclear orphan receptor of the lancelet Branchiostoma floridae, is implicated in retinoic acid signalling pathways.

In vertebrates, the orphan nuclear receptors of the COUP-TF group function as negative transcriptional regulators that inhibit the hormonal induction of target genes mediated by classical members of the nuclear hormone superfamily, such as the retinoic acid receptors (RARs) or the thyroid hormone receptors (TRs). To investigate the evolutionary conservation of the roles of COUP-TF receptors as negative regulators in the retinoid and thyroid hormone pathways, we have characterized AmphiCOUP-TF, the homologue of COUP-TFI and COUP-TFII, in the chordate amphioxus (Branchiostoma floridae), the closest living invertebrate relative of the vertebrates. Electrophoretic mobility shift assays (EMSA) showed that AmphiCOUP-TF binds to a wide variety of response elements, as do its vertebrate homologues. Furthermore, AmphiCOUP-TF is a transcriptional repressor that strongly inhibits retinoic acid-mediated transactivation. In situ hybridizations revealed expression of AmphiCOUP-TF in the nerve cord of late larvae, in a region corresponding to hindbrain and probably anterior spinal cord. Although the amphioxus nerve cord appears unsegmented at the gross anatomical level, this pattern reflects segmentation at the cellular level with stripes of expressing cells occurring adjacent to the ends and the centers of each myotomal segment, which may include visceral motor neurons and somatic motor neurons respectively, among other cells. A comparison of the expression pattern of AmphiCOUP-TF with those of its vertebrate homologues, suggests that the roles of COUP-TF in patterning of the nerve cord evolved prior to the split between the amphioxus and vertebrate lineages. Furthermore, in vitro data also suggest that Amphi-COUP-TF acts as a negative regulator of signalling by other nuclear receptors such as RAR, TR or ER.