bluuebunny/crossref_metadata_2025 huggingface dataset:
The Nitric Oxide Theory of Aging Revisited
Authors: McCANN S M. , MASTRONARDI C , DE LAURENTIIS A , RETTORI V
Published: January 2006

Bacterial and viral products, such as bacterial lipopolysaccharide (LPS), cause inducible (i) NO synthase (NOS) synthesis, which in turn produces massive amounts of nitric oxide (NO). NO, by inactivating enzymes and leading to cell death, is toxic not only to invading viruses and bacteria, but also to host cells. Injection of LPS induces interleukin (IL)‐1β, IL‐1α, and iNOS synthesis in the anterior pituitary and pineal glands, meninges, and choroid plexus, regions outside the blood‐brain barrier. Thereafter, this induction occurs in the hypothalamic regions (such as the temperature‐regulating centers), paraventricular nucleus (releasing and inhibiting hormone neurons), and the arcuate nucleus (a region containing these neurons and axons bound for the median eminence). Aging of the anterior pituitary and pineal with resultant decreased secretion of pituitary hormones and the pineal hormone melatonin, respectively, may be caused by NO. The induction of iNOS in the temperature‐regulating centers by infections may cause the decreased febrile response in the aged by loss of thermosensitive neurons. NO may play a role in the progression of Alzheimer's disease and parkinsonism. LPS similarly activates cytokine and iNOS production in the cardiovascular system leading to coronary heart disease. Fat is a major source of NO stimulated by leptin. As fat stores increase, leptin and NO release increases in parallel in a circadian rhythm with maxima at night. NO could be responsible for increased coronary heart disease as obesity supervenes. Antioxidants, such as melatonin, vitamin C, and vitamin E, probably play important roles in reducing or eliminating the oxidant damage produced by NO.
Enhanced Response of Granulosa and Theca Cells from Sheep Carriers of the FecB Mutation in Vitro to Gonadotropins and Bone Morphogenic Protein-2, -4, and -6
Authors: Campbell B. K. , Souza C. J. H. , Skinner A. J. , Webb R. , Baird D. T.
Published: January 2006

The FecB (Booroola) mutation, which leads to increased ovulation rates and multiple births in sheep, is now known to occur in the signaling domain of the bone morphogenic protein (BMP)-1B receptor. We examined the effect of the mutation on the responsiveness of granulosa (GC) and theca cells (TC) to BMPs and other local regulators using tissue from animals with (FecB/B) and without (Fec+/+) the FecB mutation. Experiments examined the effect of BMP-2, -4, and -6 (0.005–50 ng/ml), and their interaction with IGF-I (0.1–10 ng/ml LR3 analog) and gonadotropins, on the proliferation and differentiation of GCs and TCs isolated from small (<2 mm) antral follicles and maintained in serum-free culture for up to 8 d. Dose-finding studies using ovaries from wild-type sheep obtained from the abbattoir showed no difference among the different BMPs in stimulating (P < 0.001) estradiol (E2) production by GCs cultured with FSH (10 ng/ml), but there was a clear interaction (P < 0.001) with IGF-I. BMPs had no effect on GC proliferation or the sensitivity of GCs to FSH. In contrast, higher doses of BMPs (5–50 ng/ml) inhibited LH-stimulated androstenedione production by TCs, whereas lower doses (0.005–0.05 ng/ml) stimulated TC proliferation (P < 0.01). Regardless of dose of IGF-I, at the end of culture (96–192 h) hormone production by GCs (E2, inhibin A) and TCs (androstenedione) was 4- to 5-fold greater (P < 0.001) by cells from FecB/B, compared with Fec+/+ ewes exposed to the same dose of gonadotropin. In the presence of low concentrations of IGF-I (0.1 ng/ml), the maximum increase in the production of E2 and inhibin A by GCs from FF ewes in response to BMPs was observed at doses that were 3- to 10-fold lower (3–10 ng/ml) than ++ (30 ng/ml; P < 0.001). Low doses of BMPs stimulated proliferation of TCs from ++ (P < 0.01) but not FF ewes. Immunohistochemistry confirmed BMP-6 protein expression in the oocyte, granulosa, and thecal layers of antral follicles from both genotypes. These results confirm a major role for BMPs in controlling ovarian somatic cell function in sheep and provide evidence to support the hypothesis that the FecB mutation increases the BMP response of somatic cells when stimulated to differentiate by gonadotropins.
Differential Mitogenic Signaling in Insulin Receptor-Deficient Fetal Pancreatic β-Cells
Authors: Guillen C. , Navarro P. , Robledo M. , Valverde A. M. , Benito M.
Published: January 2006

Insulin receptor (IR) may play an essential role in the development of β-cell mass in the mouse pancreas. To further define the function of this signaling system in β-cell development, we generated IR-deficient β-cell lines. Fetal pancreata were dissected from mice harboring a floxed allele of the insulin receptor (IRLoxP) and used to isolate islets. These islets were infected with a retrovirus to express simian virus 40 large T antigen, a strategy for establishing β-cell lines (β-IRLoxP). Subsequently, these cells were infected with adenovirus encoding cre recombinase to delete insulin receptor (β-IR−/−). β-Cells expressed insulin and Pdx-1 mRNA in response to glucose. In β-IRLoxP β-cells, p44/p42 MAPK and phosphatidylinositol 3 kinase pathways, mammalian target of rapamycin (mTOR), and p70S6K phosphorylation and β-cell proliferation were stimulated in response to insulin. Wortmannin or PD98059 had no effect on insulin-mediated mTOR/p70S6K signaling and the corresponding mitogenic response. However, the presence of both inhibitors totally impaired these signaling pathways and mitogenesis in response to insulin. Rapamycin completely blocked insulin-activated mTOR/p70S6K signaling and mitogenesis. Interestingly, in β-IR−/− β-cells, glucose failed to stimulate phosphatidylinositol 3 kinase activity but induced p44/p42 MAPKs and mTOR/p70S6K phosphorylation and β-cell mitogenesis. PD98059, but not wortmannin, inhibited glucose-induced mTOR/p70S6K signaling and mitogenesis in those cells. Finally, rapamycin blocked glucose-mediated mitogenesis of β-IR−/− cells. In conclusion, independently of glucose, insulin can mediate mitogenesis in fetal pancreatic β-cell lines. However, in the absence of the insulin receptor, glucose induces β-cell mitogenesis.
Cloning of the Chicken Pituitary Receptor for Growth Hormone-Releasing Hormone
Authors: Toogood Andrew A. , Harvey Stephen , Thorner Michael O. , Gaylinn Bruce D.
Published: January 2006

Details of the regulation of GH in birds are unclear. In this report, a receptor was cloned from chicken pituitary cDNA with 61% amino acid sequence identity to the human pituitary GHRH receptor. Phylogenies inferred from sequence alignments support that this is the chicken counterpart of the GHRH receptor known in mammals. Northern blotting shows that this receptor message is expressed in chicken pituitary, with lesser amounts seen in hypothalamus and brain but not in liver. The recombinant chicken receptor binds human GHRH with high affinity and specificity and signals cAMP accumulation. Surprisingly, available peptides synthesized to the published sequence for chicken GHRH-like peptide (cGHRH-LP) were inactive at this receptor. To address this we recloned the cDNA for this cGHRH-LP from chicken hypothalami. The revised sequence encodes lysine at position 21, which is consistent with all reported GHRH sequences from other species but different from the originally published chicken sequence. When this revised cGHRH-LP sequence was synthesized, it had improved but still weak potency at the cloned receptor. Consistent with the activity at the cloned receptor, human GHRH was potent when assayed in live chickens or on chicken pituitary membranes, but cGHRH-LP was not. We conclude that we have cloned a putative GHRH receptor that is homologous to mammalian GHRH receptors and functionally expressed in chicken pituitary, but that the identity of the endogenous ligand remains unclear. The chicken GHRH receptor cloned in this study can serve as a tool to identify its ligand and to clarify the evolutionary development of the regulation of GH.
Injury Elicited Increase in Spinal Cord Neurosteroid Content Analyzed by Gas Chromatography Mass Spectrometry
Authors: Labombarda F. , Pianos A. , Liere P. , Eychenne B. , Gonzalez S. , Cambourg A. , De Nicola A. F. , Schumacher M. , Guennoun R.
Published: January 2006

The effects of spinal cord injury (SCI), combined with castration and adrenalectomy, and of progesterone (PROG) treatment on neurosteroid levels and steroidogenic enzyme expression were investigated in the adult male rat spinal cord (SC). Steroid levels were quantified by gas chromatography/mass spectrometry in SC and plasma, and mRNAs of enzymes by quantitative real-time RT-PCR. The levels of pregnenolone (PREG), PROG, 5α-dihydroprogesterone, 3α,5α-tetrahydroprogesterone increased in SC 75 h after transection without significant increase in the plasma. After combined adrenalectomy and gonadectomy, significant levels of PREG and PROG remained in the SC, suggesting their local biosynthesis. In the SC of adrenalectomized and gonadectomized rats, there was an increase of PREG 24 h after SCI, followed at 75 h by a concomitant increase in its direct metabolite, PROG. These observations are consistent with a sequential increase of PREG biosynthesis and its conversion to PROG within the SC in response to injury. However, no significant change in P450-side chain cleavage and 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase mRNA levels was observed after SCI. Systemic PROG treatment after SCI, resulted in a very large increase in PROG, 5α-dihydroprogesterone, and 3α,5α-tetrahydroprogesterone in both plasma and SC. Furthermore, high levels of 3β,5α-tetrahydroprogesterone were detected in SC, whereas their plasma levels remained barely detectable. Because the ratio of reduced metabolites to PROG was 65-times higher in SC than in the plasma, it appears likely that reduced metabolites mainly originated from local biosynthesis. Our results strongly suggest an important role for locally biosynthesized neurosteroids in the response of the SC to injury.
Hypoactivity of the Hypothalamo-Pituitary-Adrenocortical Axis during Recovery from Chronic Variable Stress
Authors: Ostrander Michelle M. , Ulrich-Lai Yvonne M. , Choi Dennis C. , Richtand Neil M. , Herman James P.
Published: January 2006

Chronic stress induces both functional and structural adaptations within the hypothalamo-pituitary-adrenocortical (HPA) axis, suggestive of long-term alterations in neuroendocrine reactivity to subsequent stressors. We hypothesized that prior chronic stress would produce persistent enhancement of HPA axis reactivity to novel stressors. Adult male rats were exposed to chronic variable stress (CVS) for 1 wk and allowed to recover. Plasma ACTH and corticosterone levels were measured in control or CVS rats exposed to novel psychogenic (novel environment or restraint) or systemic (hypoxia) stressors at 16 h, 4 d, 7 d, or 30 d after CVS cessation. Plasma ACTH and corticosterone responses to psychogenic stressors were attenuated at 4 d (novel environment and restraint) and 7 d (novel environment only) recovery from CVS, whereas hormonal responses to the systemic stressor were largely unaffected by CVS. CRH mRNA expression was up-regulated in the paraventricular nucleus of the hypothalamus (PVN) at 16 h after cessation of CVS, but no other alterations in PVN CRH or arginine vasopressin mRNA expression were observed. Thus, in contrast to our hypothesis, reductions of HPA axis sensitivity to psychogenic stressors manifested at delayed recovery time points after CVS. The capacity of the HPA axis to respond to a systemic stressor appeared largely intact during recovery from CVS. These data suggest that chronic stress selectively targets brain circuits responsible for integration of psychogenic stimuli, resulting in decreased HPA axis responsiveness, possibly mediated in part by transitory alterations in PVN CRH expression.
Ciliary Neurotrophic Factor Prevents Acute Lipid-Induced Insulin Resistance by Attenuating Ceramide Accumulation and Phosphorylation of c-Jun N-Terminal Kinase in Peripheral Tissues
Authors: Watt Matthew J. , Hevener Andrea , Lancaster Graeme I. , Febbraio Mark A.
Published: January 2006

Ciliary neurotrophic factor (CNTF) is a member of the gp130 receptor cytokine family recently identified as an antiobesity agent in rodents and humans by mechanisms that remain unclear. We investigated the impact of acute CNTF treatment on insulin action in the presence of lipid oversupply. To avoid confounding effects of long-term high-fat feeding or genetic manipulation on whole-body insulin sensitivity, we performed a 2-h Intralipid infusion (20% heparinized Intralipid) with or without recombinant CNTF pretreatment (Axokine 0.3 mg/kg), followed by a 2-h hyperinsulinemic-euglycemic clamp (12 mU/kg·min) in fasted, male Wistar rats. Acute Intralipid infusion increased plasma free fatty acid levels from 1.0 ± 0.1 to 2.5 ± 0.3 mm, which subsequently caused reductions in skeletal muscle (insulin-stimulated glucose disposal rate) and liver (hepatic glucose production) insulin sensitivity by 30 and 45%, respectively. CNTF pretreatment completely prevented the lipid-mediated reduction in insulin-stimulated glucose disposal rate and the blunted suppression of hepatic glucose production by insulin. Although lipid infusion increased triacylglycerol and ceramide accumulation and phosphorylation of mixed linage kinase 3 and c-Jun N-terminal kinase 1 in skeletal muscle, CNTF pretreatment prevented these lipid-induced effects. Alterations in hepatic and muscle insulin signal transduction as well as phosphorylation of c-Jun N-terminal kinase 1/2 paralleled alterations in insulin sensitivity. These data support the use of CNTF as a potential therapeutic means to combat lipid-induced insulin resistance.
Atypical Expression of Type 2 Iodothyronine Deiodinase in Thyrotrophs Explains the Thyroxine-Mediated Pituitary Thyrotropin Feedback Mechanism
Authors: Christoffolete Marcelo A. , Ribeiro Rogério , Singru Praful , Fekete Csaba , da Silva Wagner S. , Gordon David F. , Huang Stephen A. , Crescenzi Alessandra , Harney John W. , Ridgway E. Chester , Larsen P. Reed , Lechan Ronald M. , Bianco Antonio C.
Published: January 2006

T4, the main product of thyroid secretion, is a critical signal in plasma that mediates the TSH-negative feedback mechanism. As a prohormone, T4 must be converted to T3 to acquire biological activity; thus, type 2 iodothyronine deiodinase (D2) is expected to play a critical role in this feedback mechanism. However, the mechanistic details of this pathway are still missing because, counterintuitively, D2 activity is rapidly lost in the presence of T4 by a ubiquitin-proteasomal mechanism. In the present study, we demonstrate that D2 and TSH are coexpressed in rat pituitary thyrotrophs and that hypothyroidism increases D2 expression in these cells. Studies using two murine-derived thyrotroph cells, TtT-97 and TαT1, demonstrate high expression of D2 in thyrotrophs and confirm its sensitivity to negative regulation by T4-induced proteasomal degradation of this enzyme. Despite this, expression of the Dio2 gene in TαT1 cells is higher than their T4-induced D2 ubiquitinating capacity. As a result, D2 activity and net T3 production in these cells are sustained, even at free T4 concentrations that are severalfold above the physiological range. In this system, free T4 concentrations and net D2-mediated T3 production correlated negatively with TSHβ gene expression. These results resolve the apparent paradox between the homeostatic regulation of D2 and its role in mediating the critical mechanism by which T4 triggers the TSH-negative feedback.
Inhibition of Phosphatidylinositol 3-Kinase Increases Efficacy of Cisplatin in in Vivo Ovarian Cancer Models
Authors: Ohta Tsuyoshi , Ohmichi Masahide , Hayasaka Tadashi , Mabuchi Seiji , Saitoh Maki , Kawagoe Jun , Takahashi Kazuhiro , Igarashi Hideki , Du Botao , Doshida Masakazu , Mirei Ishida Gabriela , Motoyama Teiichi , Tasaka Keiichi , Kurachi Hirohisa
Published: January 2006

The phosphatidylinositol 3-kinase (PI3K)/Akt cascade has an important role in the resistance of ovarian cancer cells to cisplatin in vitro; however, there have been no reports about whether blocking the PI3K/Akt cascade enhances the sensitivity to cisplatin in vivo. We investigated whether inhibition of PI3K increased the efficacy of cisplatin in an in vivo ovarian cancer model. Blocking the PI3K/Akt cascade with a PI3K inhibitor (wortmannin) increased the efficacy of cisplatin-induced inhibition of intraabdominal dissemination and production of ascites in athymic nude mice inoculated ip with the Caov-3 human ovarian cancer cell line. In addition, wortmannin increased the efficacy of cisplatin-induced apoptosis in tumors cells. There were no detectable side effects in mice treated with wortmannin. Moreover, the antitumor effect of cisplatin detected in mice inoculated with Caov-3 cells stably transfected with empty vector was significantly attenuated, compared with mice inoculated with Caov-3 cells stably transfected with a dominant-negative Akt, K179M-Akt. We confirmed that wortmannin blocked Akt phosphorylation and the downstream targets of the PI3K/Akt cascade, such as BAD (Bcl-2-associated death protein) and nuclear factor-κB in vivo by immunohistochemical staining and Western blotting. In accordance with the previously reported in vitro results, these in vivo results support the idea that combination therapy with cisplatin and a PI3K inhibitor would increase the therapeutic efficacy of cisplatin.
MED220/Thyroid Receptor-Associated Protein 220 Functions as a Transcriptional Coactivator with Pit-1 and GATA-2 on the Thyrotropin-β Promoter in Thyrotropes
Authors: Gordon David F. , Tucker Elizabeth A. , Tundwal Kavita , Hall Heather , Wood William M. , Ridgway E. C.
Published: January 2006

Mediator (MED) 220/thyroid receptor-associated protein (TRAP) 220 is a transcriptional mediator that interacts with liganded thyroid/steroid hormone receptors. MED220 haploinsufficient heterozygotes exhibited hypothyroidism and reduced TSHβ transcripts, suggesting a specific function for TSHβ transcription. We previously demonstrated that Pit-1 and GATA-2 can bind to a composite element within the proximal TSHβ promoter and synergistically activate transcription. We detected MED220 expression in TtT-97 thyrotropes by Northern and Western blot analysis. Cotransfections in CV-1 cells showed that Pit-1, GATA-2, or MED220 alone did not markedly stimulate the TSHβ promoter. However, Pit-1 plus GATA-2 resulted in an 10-fold activation, demonstrating synergistic cooperativity. Titration of MED220 resulted in a further dose-dependent stimulation up to 25-fold that was promoter specific. Glutathione-S-transferase interaction studies showed that MED220 or GATA-2 each bound the homeodomain of Pit-1, whereas MED220 interacted independently with each zinc finger of GATA-2 but not with either terminus. MED220 interacted with GATA-2 and Pit-1 over a broad region of its N terminus. These regions of interaction were also important for maximal function. Coimmunoprecipitation confirmed that all three factors can interact in thyrotropes and chromatin immunoprecipitation demonstrated in vivo occupancy on the proximal TSHβ promoter. Thus, the TSHβ gene is maximally activated by a combination of three thyrotrope transcription factors that act via both protein-DNA and protein-protein interactions.
TheDictyosteliumbZIP transcription factor DimB regulates prestalk-specific gene expression
Authors: Zhukovskaya Natasha V. , Fukuzawa Masashi , Yamada Yoko , Araki Tsuyoshi , Williams Jeffrey G.
Published: January 2006

The ecmA gene is specifically expressed in prestalk cells and its transcription is induced by the chlorinated hexaphenone DIF-1. We have purified a novel bZIP transcription factor, DimB, by affinity chromatography on two spatially separated ecmA promoter fragments. Mutagenesis of the cap-site proximal DimB-binding site (the -510 site) greatly decreases ecmA expression in the pstO cells, which comprise the rear half of the prestalk zone, and also in the Anterior-Like Cells, which lie scattered throughout the prespore region. However, DimB is not essential for normal expression of the ecmA gene, instead it spatially limits its expression; ecmA is relatively highly expressed in the subset of prestalk cells that coats the prestalk zone, but in slugs of a DimB-null strain, ecmA is highly expressed throughout the prestalk zone. Because the -510 site is required for correct ecmA expression, we posit a separate activator protein that competes with DimB for binding to the-510 site. DimB rapidly accumulates in the nucleus when cells are exposed to DIF-1, and ChIP analysis shows that, in the presence of extracellular cAMP,DIF-1 causes DimB to associate with the ecmA promoter in vivo. Thus,DIF-1 regulates DimB activity to generate a gradient of ecmAexpression in the prestalk zone of the slug.
A dominant-negative form of the E3 ubiquitin ligaseCullin-1disrupts the correct allocation of cell fate in the neural crest lineage
Authors: Voigt Jana , Papalopulu Nancy
Published: January 2006

Selective protein degradation is an efficient and rapid way of terminating protein activity. Defects in protein degradation are associated with a number of human diseases, including potentially DiGeorge syndrome, which is characterised by abnormal development of the neural crest lineage during embryogenesis. We describe the identification of Xenopus Cullin-1, an E3 ubiquitin ligase, and show that blocking the function of endogenous Cullin-1 leads to pleiotropic defects in development. Notably, there is an increased allocation of cells to a neural crest fate and within this lineage, an increase in melanocytes at the expense of cranial ganglia neurons. Most of the observed effects can be attributed to stabilisation ofβ-catenin, a known target of Cullin-1-mediated degradation from other systems. Indeed, we show that blocking the function of Cullin-1leads to a decrease in ubiquitinated β-catenin and an increase in totalβ-catenin. Our results show that Cullin-1-mediated protein degradation plays an essential role in the correct allocation of neural crest fates during embryogenesis.
Extrinsic cues orient the cell division axis inDrosophilaembryonic neuroblasts
Authors: Siegrist Sarah E. , Doe Chris Q.
Published: January 2006

Cell polarity must be integrated with tissue polarity for proper development. The Drosophila embryonic central nervous system (CNS) is a highly polarized tissue; neuroblasts occupy the most apical layer of cells within the CNS, and lie just basal to the neural epithelium. Neuroblasts are the CNS progenitor cells and undergo multiple rounds of asymmetric cell division, `budding off' smaller daughter cells (GMCs) from the side opposite the epithelium, thereby positioning neuronal/glial progeny towards the embryo interior. It is unknown whether this highly stereotypical orientation of neuroblast divisions is controlled by an intrinsic cue (e.g. cortical mark) or an extrinsic cue (e.g. cell-cell signal). Using live imaging and in vitro culture, we find that neuroblasts in contact with epithelial cells always `bud off' GMCs in the same direction, opposite from the epithelia-neuroblast contact site, identical to what is observed in vivo. By contrast, isolated neuroblasts `bud off' GMCs at random positions. Imaging of centrosome/spindle dynamics and cortical polarity shows that in neuroblasts contacting epithelial cells, centrosomes remained anchored and cortical polarity proteins localize at the same epithelia-neuroblast contact site over subsequent cell cycles. In isolated neuroblasts, centrosomes drifted between cell cycles and cortical polarity proteins showed a delay in polarization and random positioning. We conclude that embryonic neuroblasts require an extrinsic signal from the overlying epithelium to anchor the centrosome/centrosome pair at the site of epithelial-neuroblast contact and for proper temporal and spatial localization of cortical Par proteins. This ensures the proper coordination between neuroblast cell polarity and CNS tissue polarity.
Cholesterol modification is necessary for controlled planar long-range activity of Hedgehog inDrosophilaepithelia
Authors: Gallet Armel , Ruel Laurent , Staccini-Lavenant Laurence , Thérond Pascal P.
Published: January 2006

The Hedgehog morphogen is a major developmental regulator that acts at short and long range to direct cell fate decisions in invertebrate and vertebrate tissues. Hedgehog is the only known metazoan protein to possess a covalently linked cholesterol moiety. Although the role of the cholesterol group of Hedgehog remains unclear, it has been suggested to be dispensable for the its long-range activity in Drosophila. Here, we provide data in three different epithelia - ventral and dorsal embryonic ectoderm, and larval imaginal disc tissue - showing that cholesterol modification is in fact necessary for the controlled long-range activity of DrosophilaHedgehog. We provide an explanation for the discrepancy between our results and previous reports by showing that unmodified Hh can act at long range,albeit in an uncontrolled manner, only when expressed in squamous cells. Our data show that cholesterol modification controls long-range Hh activity at multiple levels. First, cholesterol increases the affinity of Hh for the plasma membrane, and consequently enhances its apparent intrinsic activity,both in vitro and in vivo. In addition, multimerisation of active Hh requires the presence of cholesterol. These multimers are correlated with the assembly of Hh into apically located, large punctate structures present in active Hh gradients in vivo. By comparing the activity of cholesterol-modified Hh in columnar epithelial cells and peripodial squamous cells, we show that epithelial cells provide the machinery necessary for the controlled planar movement of Hh, thereby preventing the unrestricted spreading of the protein within the three-dimensional space of the epithelium. We conclude that, as in vertebrates, cholesterol modification is essential for controlled long-range Hh signalling in Drosophila.
lessenencodes a zebrafishtrap100required for enteric nervous system development
Authors: Pietsch Jacy , Delalande Jean-Marie , Jakaitis Brett , Stensby James D. , Dohle Sarah , Talbot William S. , Raible David W. , Shepherd Iain T.
Published: January 2006

The zebrafish enteric nervous system (ENS), like those of all other vertebrate species, is principally derived from the vagal neural crest. The developmental controls that govern the specification and patterning of the ENS are not well understood. To identify genes required for the formation of the vertebrate ENS, we preformed a genetic screen in zebrafish. We isolated the lessen (lsn) mutation that has a significant reduction in the number of ENS neurons as well as defects in other cranial neural crest derived structures. We show that the lsn gene encodes a zebrafish orthologue of Trap100, one of the subunits of the TRAP/mediator transcriptional regulation complex. A point mutation in trap100causes a premature stop codon that truncates the protein, causing a loss of function. Antisense-mediated knockdown of trap100 causes an identical phenotype to lsn. During development trap100 is expressed in a dynamic tissue-specific expression pattern consistent with its function in ENS and jaw cartilage development. Analysis of neural crest markers revealed that the initial specification and migration of the neural crest is unaffected in lsn mutants. Phosphohistone H3 immunocytochemistry revealed that there is a significant reduction in proliferation of ENS precursors in lsn mutants. Using cell transplantation studies, we demonstrate that lsn/trap100 acts cell autonomously in the pharyngeal mesendoderm and influences the development of neural crest derived cartilages secondarily. Furthermore, we show that endoderm is essential for ENS development. These studies demonstrate that lsn/trap100 is not required for initial steps of cranial neural crest development and migration, but is essential for later proliferation of ENS precursors in the intestine.
TheCdx4mutation affects axial development and reveals an essential role of Cdx genes in the ontogenesis of the placental labyrinth in mice
Authors: van Nes Johan , de Graaff Wim , Lebrin Franck , Gerhard Markus , Beck Felix , Deschamps Jacqueline
Published: January 2006

Caudal related homeobox (Cdx) genes have so far been shown to be important for embryonic axial elongation and patterning in several vertebrate species. We have generated a targeted mutation of mouse Cdx4, the third member of this family of transcription factor encoding genes and the last one to be inactivated genetically. Cdx4-null embryos were born healthy and appeared morphologically normal. A subtle contribution of Cdx4 to anteroposterior (AP) vertebral patterning was revealed in Cdx1/Cdx4 and Cdx2/Cdx4 compound mutants. Neither Cdx4-null nor Cdx1/Cdx4 double mutants are impaired in their axial elongation, but a redundant contribution of Cdx4 in this function was unveiled when combined with a Cdx2 mutant allele. In addition, inactivation of Cdx4 combined with heterozygous loss of Cdx2 results in embryonic death around E10.5 and reveals a novel function of Cdx genes in placental ontogenesis. In a subset of Cdx2/Cdx4 compound mutants, the fully grown allantois failed to fuse with the chorion. The remaining majority of these mutants undergo successful chorio-allantois fusion but fail to properly extend their allantoic vascular network into the chorionic ectoderm and do not develop a functional placental labyrinth. We present evidence that Cdx4 plays a crucial role in the ontogenesis of the allantoic component of the placental labyrinth when one Cdx2 allele is inactivated. The axial patterning role of Cdx transcription factors thus extends posteriorly to the epiblast-derived extra-embryonic mesoderm and, consequent upon the evolution of placental mammals, is centrally involved in placental morphogenesis. The relative contribution of Cdx family members in the stepwise ontogenesis of a functional placenta is discussed, with Cdx2 playing an obligatory part, assisted by Cdx4. The possible participation of Cdx1 was not documented but cannot be ruled out until allelic combinations further decreasing Cdx dose have been analyzed. Cdx genes thus operate in a redundant way during placentogenesis, as they do during embryonic axial elongation and patterning, and independently from the previously reported early Cdx2-specific role in the trophectoderm at implantation.
Hedgehog lipid modifications are required for Hedgehog stabilization in the extracellular matrix
Authors: Callejo Ainhoa , Torroja Carlos , Quijada Luis , Guerrero Isabel
Published: January 2006

The Hedgehog (Hh) family of morphogenetic proteins has important instructional roles in metazoan development. Despite Hh being modified by Ct-cholesterol and Nt-palmitate adducts, Hh migrates far from its site of synthesis and programs cellular outcomes, depending on its local concentrations. We show that in the receiving cells of the Drosophilawing imaginal disc, lipid-unmodified Hh spreads across many more cell diameters than the wild type and this spreading leads to the activation of low but not high threshold responses. Unlipidated Hh forms become internalized through the apical plasma membrane, while wild-type Hh enters through the basolateral cell surface - in all cases via a dynamin-dependent mechanism. Full activation of the Hh pathway and the spread of Hh throughout the extracellular matrix depend on the ability of lipid-modified Hh to interact with heparan sulfate proteoglycans (HSPG). However, neither Hh-lipid modifications nor HSPG function are required to activate the targets that respond to low levels of Hh. All these data show that the interaction of lipid-modified Hh with HSPG is important both for precise Hh spreading through the epithelium surface and for correct Hh reception.
Drosophila Cornichon acts as cargo receptor for ER export of the TGFα-like growth factor Gurken
Authors: Bökel Christian , Dass Sajith , Wilsch-Bräuninger Michaela , Roth Siegfried
Published: January 2006

Drosophila Cornichon (Cni) is the founding member of a conserved protein family that also includes Erv14p, an integral component of the COPII-coated vesicles that mediate cargo export from the yeast endoplasmic reticulum (ER). During Drosophila oogenesis, Cni is required for transport of the TGFα growth factor Gurken (Grk) to the oocyte surface. Here, we show that Cni, but not the second Drosophila Cni homologue Cni-related (Cnir), binds to the extracellular domain of Grk, and propose that Cni acts as a cargo receptor recruiting Grk into COPII vesicles. Consequently,in the absence of Cni function, Grk fails to leave the oocyte ER. Proteolytic processing of Grk still occurs in cni mutant ovaries, demonstrating that release of the active growth factor from its transmembrane precursor occurs earlier during secretory transport than described for the other Drosophila TGFα homologues. Massive overexpression of Grk in a cni mutant background can overcome the requirement of Grk signalling for cni activity, confirming that cni is not essential for the production of the functional Grk ligand. However, the rescued egg chambers lack dorsoventral polarity. This demonstrates that the generation of temporally and spatially precisely coordinated Grk signals cannot be achieved by bulk flow secretion, but instead has to rely on fast and efficient ER export through cargo receptor-mediated recruitment of Grk into the secretory pathway.
GLI3-dependent transcriptional repression ofGli1, Gli2and kidney patterning genes disrupts renal morphogenesis
Authors: Hu Ming Chang , Mo Rong , Bhella Sita , Wilson Christopher W. , Chuang Pao-Tien , Hui Chi-chung , Rosenblum Norman D.
Published: January 2006

Truncating mutations in Gli3, an intracellular effector in the SHH-SMO-GLI signaling pathway, cause renal aplasia/dysplasia in humans and mice. Yet, the pathogenic mechanisms are undefined. Here, we report the effect of decreased SHH-SMO signaling on renal morphogenesis, the expression of SHH target genes and GLI binding to Shh target genes. Shhdeficiency or cyclopamine-mediated SMO inhibition disrupted renal organogenesis, decreased expression of GLI1 and GLI2 proteins, but increased expression of GLI3 repressor relative to GLI3 activator. Shhdeficiency decreased expression of kidney patterning genes (Pax2 and Sall1) and cell cycle regulators (cyclin D1 and MYCN). Elimination of Gli3 in Shh–/– mice rescued kidney malformation and restored expression of Pax2, Sall1, cyclin D1, MYCN, Gli1 and Gli2. To define mechanisms by which SHH-SMO signaling controls gene expression, we determined the binding of GLI proteins to 5′ flanking regions containing GLI consensus binding sequences in Shh target genes using chromatin immunoprecipitation. In normal embryonic kidney tissue, GLI1 and/or GLI2 were bound to each target gene. By contrast, treatment of embryonic kidney explants with cyclopamine decreased GLI1 and/or GLI2 binding, and induced binding of GLI3. However, cyclopamine failed to decrease Gli1 and Gli2 expression and branching morphogenesis in Gli3-deficient embryonic kidney tissue. Together,these results demonstrate that SHH-SMO signaling controls renal morphogenesis via transcriptional control of Gli, renal patterning and cell cycle regulator genes in a manner that is opposed by GLI3.
Development of the mesencephalic dopaminergic neuron system is compromised in the absence of neurogenin 2
Authors: Andersson E. , Jensen J. B. , Parmar M. , Guillemot F. , Björklund A.
Published: January 2006

Neurogenin 2 (Ngn2) is a proneural gene involved in neuronal differentiation and subtype specification in various regions of the nervous system. In the ventral midbrain, Ngn2 is expressed in a spatiotemporal pattern that correlates with the generation of mesencephalic dopaminergic (mesDA)neurons. We show here that lack of Ngn2 impairs the development of mesDA neurons, such that less than half of the normal mesDA neuron number remain in Ngn2 mutant mice at postnatal stages. Analysis of Ngn2mutant mice during mesDA neurogenesis show that medially located precursors are formed but are arrested in their differentiation at a stage when they have not yet acquired the characteristics of mesDA neuron precursors. Loss of Ngn2 function appears to specifically affect the generation of DA neurons, as the development of other types of neurons within the ventral midbrain is unaltered. Ngn2 is the first example of a gene expressed in progenitors in the ventricular zone of the mesDA neuron domain that is essential for proper mesDA neuron differentiation, and whose loss of function causes impaired mesDA neurogenesis without other major abnormalities in the ventral midbrain.
Timing of identity: spatiotemporal regulation ofhunchbackin neuroblast lineages ofDrosophilaby Seven-up and Prospero
Authors: Mettler Ulrike , Vogler Georg , Urban Joachim
Published: January 2006

Neural stem cells often generate different cell types in a fixed birth order as a result of temporal specification of the progenitors. In Drosophila, the first temporal identity of most neural stem cells(neuroblasts) in the embryonic ventral nerve cord is specified by the transient expression of the transcription factor Hunchback. When reaching the next temporal identity, this expression is switched off in the neuroblasts by seven up (svp) in a mitosis-dependent manner, but is maintained in their progeny (ganglion mother cells). We show that svpmRNA is already expressed in the neuroblasts before this division. After mitosis, Svp protein accumulates in both cells, but the downregulation of hunchback (hb) occurs only in the neuroblast. In the ganglion mother cell, svp is repressed by Prospero, a transcription factor asymmetrically localised to this cell during mitosis. Thus, the differential regulation of hb between the neuroblasts and the ganglion mother cells is achieved by a mechanism that integrates information created by the asymmetric distribution of a cell-fate determinant upon mitosis(Prospero) and a transcriptional repressor present in both cells (Seven-up). Strikingly, although the complete downregulation of hb is mitosis dependent, the lineage-specific timing of svp upregulation is not.
General Considerations
Authors: Hartshorn Edward A.
Published: January 2006

Drug interaction, modification of the effects of one drug by another, is a serious problem. This series of articles will deal with the interaction of drugs with other chemical agents, both exogenous and endogenous, as well as those found in foods and those utilized in diagnostic tests. To understand drug interactions and their significance, one must be familiar with the terminology, with the biological sciences, clinical use of drugs, and the current pharmaco-medical literature.
Contribution of The Annals Of Pharmacotherapy to the Development of Clinical Pharmacy
Authors: McLeod Donald C
Published: January 2006

2006 marks the 40th year of publication for The Annals. Over that time, The Annals has been an important contributor to the development of clinical pharmacy, with the journal's founder and first editor, Dr. Don E Francke, exerting a profound influence on the evolution of the profession of pharmacy. Throughout 2006, we will publish articles authored by practitioners, including those pioneering clinical pharmacy, as well as those who have more recently entered the profession and a well-established specialty. Donald C McLeod, the first chairman of the Editorial Board for The Annals, presents the initial article of the series in this issue. Edward Hartshorn, one of the first to recognize the significant impact of drug interactions on pharmacotherapy, also offers his memories of the early days of the journal and how clinical pharmacy has grown (see page 112).

In addition, we are also presenting editorials from the early history of The Annals that have given direction and shape to the practice of clinical pharmacy (see pages 114-22).
Coelution of Other Proteins with Albumin during Size-Exclusion HPLC: Implications for Analysis of Urinary Albumin
Authors: Sviridov Denis , Meilinger Bonnie , Drake Steven K , Hoehn Gerard T , Hortin Glen L
Published: January 2006

Background: Size-exclusion HPLC has been used as an alternative to immunoassays for quantifying urinary albumin (microalbumin). Systematically higher values for the HPLC method have been proposed to result from nonimmunoreactive albumin.

Methods: We evaluated separation of purified proteins and urinary components by size-exclusion HPLC using a Zorbax Bio Series GF-250 column eluted with phosphate-buffered saline. Urinary components eluting in the “albumin” peak were analyzed by mass spectrometry and reversed-phase HPLC.

Results: Several proteins, such as transferrin, α1-proteinase inhibitor, α1-acid glycoprotein, and α2-HS glycoprotein, analyzed as purified components, were not resolved from albumin by size-exclusion HPLC. Peaks for other proteins, such as IgG and urinary components identified as dimers of α1-microglobulin and immunoglobulin light chains, overlapped with the albumin peak. Profiles of urine specimens showed variable amounts of components overlapping with albumin. Furthermore, the albumin peak obtained by size-exclusion HPLC was found by mass spectrometry and reversed-phase HPLC to contain multiple components in addition to albumin.

Conclusions: Size-exclusion HPLC does not resolve albumin from several other proteins in urine. The albumin peak resolved by this technique, although predominantly composed of albumin, contains several coeluting globulins that would contribute to overestimation of albumin concentration by size-exclusion HPLC.
Allelic Losses of Chromosome 10 in Glioma Tissues Detected by Quantitative Single-Strand Conformation Polymorphism Analysis
Authors: Hata Nobuhiro , Yoshimoto Koji , Yokoyama Nobuhiko , Mizoguchi Masahiro , Shono Tadahisa , Guan Yanlei , Tahira Tomoko , Kukita Yoji , Higasa Koichiro , Nagata Shinji , Iwaki Toru , Sasaki Tomio , Hayashi Kenshi
Published: January 2006

Background: Detection of loss of heterozygosity (LOH) in clinical tissue samples is frequently difficult because samples are usually contaminated with noncancerous cells or because tumor cells in single tissues have genetic heterogeneity, and the precision of available techniques is insufficient for reliable analysis in such materials. We hypothesized that single-strand conformation polymorphism (SSCP) analysis can precisely quantify the gene dosage in mixed samples and is suitable for detection of LOH in clinical tissue samples.

Methods: We assessed the accuracy of a fluorescent SSCP method for the quantification of single-nucleotide polymorphism (SNP) alleles, using DNAs that were composed of cancerous DNA mixed with noncancerous DNA at various ratios. We applied this method to precisely characterize LOH in glioma tissue samples, using 96 SNPs that were evenly distributed throughout chromosome 10.

Results: LOH could be detected even in the cancerous DNA heavily contaminated (up to 80%) with noncancerous DNA. Using this method, we obtained LOH profiles of 56 gliomas with resolution at the SNP level (i.e., 1.5-Mbp interval). Anaplastic astrocytomas exhibited both 10p and 10q LOH, whereas diffuse astrocytomas frequently (63% of the cases) exhibited loss of 10p alone. We also found a possible new LOH region (around 10p13) in gliomas.

Conclusions: The present method is effective for precise mapping of LOH region in surgically obtained tumor tissues and could be applicable to the genetic diagnosis of cancers other than gliomas.
Rate Limits of On-Beat and Off-Beat Tapping With Simple Auditory Rhythms
Authors: Repp Bruno H.
Published: January 2006

THE RELATIVE DIFFICULTY of on-beat and off-beat finger tapping with simple auditory rhythms was assessed in four experiments with musically trained participants. The rhythms consisted of cyclically repeated TT0 or TTT0 patterns, where T denotes the presence and 0 denotes the absence of a tone. The tasks were to tap in synchrony with one of the T ("on-beat") positions or with the 0 ("off-beat") position. Experiments 1-3 used an adaptive procedure that determined the fastest tempo at which each task could be accomplished. Experiment 1 demonstrated that it is easier to tap on tones that carry a rhythmic grouping accent (T2 in TT0, T1 and T3 in TTT0) than on other tones or in the 0 position. Off-beat tapping was more difficult in TT0 than in TTT0 sequences. Experiment 2 showed that a dynamic ( pitch) accent on one of the tones facilitates synchronization with that tone and impedes synchronization with adjacent tones. Off-beat tapping was less affected by accent location. Experiment 3 required participants to "hear" different T positions as metrically accented (i.e., to construe them as the downbeat) while carrying out the various tapping tasks. Most participants found it difficult to maintain a cognitive downbeat at fast tempi when it did not coincide with their taps. However, when such a downbeat could be maintained, it did not seem to increase the difficulty of tapping (with one exception). This suggests a unidirectional dependence of metrical structure on action. In Experiment 4, the same tasks were presented at more moderate tempi, and the dependent measure was the variability of asynchronies. Metrical downbeat location still did not have any significant effect. Thus, synchronization difficulty seems to be affected only by a rhythm's physical structure, not by the cognitive interpretation that is given to that structure.
The Third-Act Prelude of WagnerÕs Parsifal: Genesis, Form, and Dramatic Meaning
Authors: Kinderman William
Published: January 2006

The Prelude to the third act of Parsifal is one of Wagner's most advanced essays in expanded tonality. One author has described it as "set[ting] foot in atonal territory as it re-explores the melancholy, disjointed polytonal idiom of the introduction to the third act of Tristan," and a noted analyst has suggested that it is motion around the diminished-seventh chord including Bb rather than the tonic triad of Bb minor that defines the background structure of the Prelude. This music also raises issues of form and expressive meaning that have yet to be thoroughly addressed. A valuable means of approaching the Prelude is through Wagner's surviving compositional documents, particularly the individual sketches for the Prelude that preceded the writing-out of his first continuous draft for the third act (the Kompositionsskizze [Composition Draft]). These manuscripts are held in the Wagner-Archiv at Bayreuth. When these sketches are transcribed and compared with the detailed record contained in Cosima Wagner's diary entries, insight can be gained into the way that Wagner composed the Prelude, during late October 1878. This article shows in detail how the Prelude was composed on the basis of sketch sources that are virtually complete. It is supported by several facsimiles of Wagner's sketches, transcriptions, analytical graphs, and music examples. The study indicates that the "melancholy, disjointed polytonal" idiom of the Prelude is coordinated with a framework of associated tonalities reaching across vast stretches of musical time. These include not only the Bb-minor idiom of Titurel's burial, but also the associated tonality of Parsifal's Prophecy motive. The structural background of the Prelude to act III of Parsifal is not simply a diminished-seventh prolongation, but a tensional framework of motivic combinations and rotational cycles that effectively convey the bleak wandering and promise of deliverance that lie at the core of the drama.