Two G protein-coupled receptors have been identified that bind corticotropin-releasing factor (CRF) and urocortin (UCN) with high affinity. Hybridization histochemical methods were used to shed light on controversies concerning their localization in rat brain, and to provide normative distributional data in mouse, the standard model for genetic manipulation in mammals. The distribution of CRF-R1 mRNA in mouse was found to be fundamentally similar to that in rat, with expression predominating in the cerebral cortex, sensory relay nuclei, and in the cerebellum and its major afferents. Pronounced species differences in distribution were few, although more subtle variations in the relative strength of R1 expression were seen in several forebrain regions. CRF-R2 mRNA displayed comparable expression in rat and mouse brain, distinct from, and more restricted than that of CRF-R1. Major neuronal sites of CRF-R2 expression included aspects of the olfactory bulb, lateral septal nucleus, bed nucleus of the stria terminalis, ventromedial hypothalamic nucleus, medial and posterior cortical nuclei of the amygdala, ventral hippocampus, mesencephalic raphe nuclei, and novel localizations in the nucleus of the solitary tract and area postrema. Several sites of expression in the limbic forebrain were found to overlap partially with ones of androgen receptor expression. In pituitary, rat and mouse displayed CRF-R1 mRNA signal continuously over the intermediate lobe and over a subset of cells in the anterior lobe, whereas CRF-R2 transcripts were expressed mainly in the posterior lobe. The distinctive expression pattern of CRF-R2 mRNA identifies additional putative central sites of action for CRF and/or UCN. Constitutive expression of CRF-R2 mRNA in the nucleus of the solitary tract, and stress-inducible expression of CRF-R1 transcripts in the paraventricular nucleus may provide a basis for understanding documented effects of CRF-related peptides at a loci shown previously to lack a capacity for CRF-R expression or CRF binding. Other such "mismatches" remain to be reconciled.

Here we describe the cloning and initial characterization of a previously unidentified CRF-related neuropeptide, urocortin II (Ucn II). Searches of the public human genome database identified a region with significant sequence homology to the CRF neuropeptide family. By using homologous primers deduced from the human sequence, a mouse cDNA was isolated from whole brain poly(A) ؉ RNA that encodes a predicted 38-aa peptide, structurally related to the other known mammalian family members, CRF and Ucn. Ucn II binds selectively to the type 2 CRF receptor (CRF-R2), with no appreciable activity on CRF-R1. Transcripts encoding Ucn II are expressed in discrete regions of the rodent central nervous system, including stress-related cell groups in the hypothalamus (paraventricular and arcuate nuclei) and brainstem (locus coeruleus). Central administration of 1-10 g of peptide elicits activational responses (Fos induction) preferentially within a core circuitry subserving autonomic and neuroendocrine regulation, but whose overall pattern does not broadly mimic the CRF-R2 distribution. Behaviorally, central Ucn II attenuates nighttime feeding, with a time course distinct from that seen in response to CRF. In contrast to CRF, however, central Ucn II failed to increase gross motor activity. These findings identify Ucn II as a new member of the CRF family of neuropeptides, which is expressed centrally and binds selectively to CRF-R2. Initial functional studies are consistent with Ucn II involvement in central autonomic and appetitive control, but not in generalized behavioral activation.C RF is a 41-aa peptide best known for its indispensable role in initiating pituitary-adrenal responses to stress, an effect mediated by type 1 CRF receptors (1). In addition, CRF is widely distributed in brain and has been shown repeatedly to participate in the mobilization of complementary autonomic and behavioral adjustments to a variety of threatening circumstances (2, 3). This has fostered the widely held hypothesis that CRF plays an important role in the integration of adaptive responses to stress. Rigorous testing of this idea has been impeded by the fact that a number of the cell groups identified as sites of peptide action in eliciting stress-like autonomic and behavioral responses have been found to be lacking or impoverished in the expression of requisite ligand(s), receptor(s), or both (4, 5). This has kindled the search for additional CRF-related signaling molecules, which currently number two ligands, G protein-coupled receptors derived from two distinct genes (CRF-R1 and CRF-R2), and a binding protein, whose function remains incompletely understood (6, 7).A second mammalian CRF-related neuropeptide, urocortin (Ucn), was discovered recently by our group (8) and shown to be bound with high affinity by both known CRF receptor types, whereas CRF is bound in a highly preferential manner by CRF-R1. Centrally administered Ucn is more potent than CRF in suppressing appetite, but it is less so in generating acute anxiety-like effects and g...

Neurons comprising the endocrine hypothalamus are disposed in several nuclei that develop in tandem with their ultimate target the pituitary gland, and arise from a primordium in which three related class III POU domain factors, Brn-2, Brn-4, and Brn-1, are initially coexpressed. Subsequently, these factors exhibit stratified patterns of ontogenic expression, correlating with the appearance of distinct neuropeptides that define three major endocrine hypothalamic cell types. Strikingly, deletion of the Brn-2 genomic locus results in loss of endocrine hypothalamic nuclei and the posterior pituitary gland. Lack of Brn-2 does not affect initial hypothalamic developmental events, but instead results in a failure of differentiation to mature neurosecretory neurons of the paraventricular and supraoptic nuclei, characterized by an inability to activate genes encoding regulatory neuropeptides or to make correct axonal projections, with subsequent loss of these neurons. Thus, both neuronal and endocrine components of the hypothalamic-pituitary axis are critically dependent on the action of specific POU domain factors at a penultimate step in the sequential events that underlie the appearance of mature cellular phenotypes.

Histochemical and axonal transport methods were used to clarify the central organization of cells and fibers that express urocortin (UCN), a recently discovered corticotropin-releasing factor (CRF)-related neuropeptide, which has been proposed as an endogenous ligand for type 2 CRF receptors (CRF-R2). Neurons that display both UCN mRNA and peptide expression were found to be centered in the Edinger-Westphal (EW), lateral superior olivary (LSO), and supraoptic nuclei; lower levels of expression are seen in certain cranial nerve and spinal motoneurons and in small populations of neurons in the forebrain. Additional sites of UCN mRNA and peptide expression detected only in colchicine-treated rats are considered to be minor ones. UCN-immunoreactive projections in brain are predominantly descending and largely consistent with central projections attributed to the EW and LSO, targeting principally accessory optic, precerebellar, and auditory structures, as well as the spinal intermediate gray. Although neither the EW nor LSO are known to project to the forebrain, UCN-ir neurons in the EW were identified that project to the lateral septal nucleus, which houses a prominent UCN-ir terminal field. Although substantial UCN-ir projections were observed to several brainstem cell groups that express CRF-R2, including the dorsal raphe and interpeduncular nuclei and the nucleus of the solitary tract (NTS), most prominent seats of CRF-R2 expression were found to contain inputs immunopositive for piscine urotensin I, but not rat UCN. The results define a central UCN system whose organization suggests a principal involvement in motor control and sensorimotor integration; its participation in stress-related mechanisms would appear to derive principally by virtue of projections to the spinal intermediolateral column, the NTS, and the paraventricular nucleus. Several observations, including the lack of a pervasive relationship of UCN-ir projections with CRF-R2-expressing targets, support the existence of still additional CRF-related peptides in mammalian brain.

We have provided evidence that the stimulatory effects of intravenous interleukin-1 (IL-1) on neurosecretory neurons in the paraventricular nucleus (PVH) that express corticotropinreleasing factor (CRF) depend specifically on the integrity of catecholaminergic projections originating in caudal medulla. Here we report on experiments designed to test alternative means by which circulating IL-1 might access medullary aminergic neurons, including mechanisms involving sensory components of the vagus, the area postrema, or perivascular cells bearing IL-1 receptors. Neither abdominal vagotomy nor area postrema lesions reliably altered Fos expression induced in the medulla or PVH in response to a moderately suprathreshold dose of IL-1␤. Cytokine-stimulated increases in CRF mRNA in the PVH were also unaffected by either ablation. By contrast, systemic administration of the cyclooxygenase inhibitor indomethacin resulted in parallel dose-related attenuations of IL-1 effects in hypothalamus and medulla. Microinjections of prostaglandin E2 (PGE2; Ն10 ng) in rostral ventrolateral medulla, the principal seat of IL-1-sensitive neurons that project to the PVH, provoked discrete patterns of cellular activation in hypothalamus and medulla that mimicked those seen in response to intravenous IL-1. We interpret these findings as supporting the hypothesis that paracrine effects of PGE2 released from perivascular cells in the medulla as a consequence of IL-1 stimulation and, acting through prostanoid receptors on or near local aminergic neurons that project to the PVH, contribute to the stimulatory effects of increased circulating IL-1 on neurons constituting the central limb of the hypothalamo-pituitaryadrenal axis.

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Perennial rhizomatous grasses are regarded as leading energy crops due to their environmental benefits and their suitability to regions with adverse conditions. In this paper, two different experiments were carried out in order to study the salinity (S) and water stress (WS) effects on biomass production in giant reed (Arundo donax L.). In Experiment 1, eight clones of giant reed were subjected to four S and WS treatments: (i) well-watered with nonsaline solution, (ii) water stress with non-saline solution, (iii) well-watered with saline solution and iv) water stress with saline solution. In Experiment 2, five clones of giant reed were subjected to increasing S levels in two locations: University of Catania (UNICT-Italy) (i)well-watered with non-saline solution and (ii)wellwatered with mild saline solution; and University of Barcelona (UB-Spain) (iii)well-watered with non-saline solution and (iv)well-watered with severe saline solution. Photosynthetic and physiological parameters as well as biomass production were measured in these plants. According to our data, giant reed seems to be more tolerant to S than WS. Both stresses mainly affected stomatal closure to prevent dehydration of the plant, and eventually decreasing the photosynthetic rate. The differential performance of the giant reed clones was ranked according to their tolerance to S and WS by using the Stress Susceptibility Index. 'Agrigento' was the most WS resistant clone and 'Martinensis' was the most S resistant. 'Martinensis' and 'Piccoplant' were found to be the most suitable clones for growing under both stress conditions. Moreover, 'Fondachello', 'Cefalú' and 'Licata' were the most resistant clones to increasing S levels.Keywords: Arundo donax L.; biomass; water stress; salinity stress; photosynthesis; Stress Susceptibility Index. ABBREVIATIONSA sat , light saturated net CO 2 assimilation rate (µmol m -2 s -1 ); DLP, complete dry leaves percentage (%); DM, dry matter (g); FC, field capacity; g s , stomatal conductance (mol m -2 s -1 ); gLA, green leaf area (m 2 ); GLP, complete green leaves percentage (%); H, height (cm); LAR, leaf area ratio (m 2 Kg -1 ); LWR, leaf weight ratio (Kg Kg -1 ); NL, number of leaves; NS, number of stems; PPFD, photosynthetic photon flux density; PRG, perennial rhizomatous grasses; RWC, relative water content (%); S, salinity; SA, stem area (m 2 ); SLA, specific leaf area (m 2 Kg -1 ); S/R, shoot/root ratio (g g -1 ); SSI, stress susceptibility index; TDW, total dry weight (g); WS, water stress; YLP, complete yellow leaves percentage (%).3

p53 expression has been examined in 89 squamous cell carcinomas of the larynx (34 glottic, 28 supraglottic, 18 transglottic, 8 pyriform sinus, and 1 subglottic) obtained from 88 patients surgically treated in our centre. In addition, 59 laryngeal samples including normal respiratory epithelium and non-invasive squamous cell lesions were also tested. Frozen sections were immunostained with PAb 1801 and the results were correlated with pathological features, DNA ploidy and S-phase of the tumours, disease-free interval, and survival of the patients. p53 immunoreactivity was observed in 57 (64 per cent) carcinomas. None of the eight samples of normal respiratory epithelium was positive. p53-positive cells were seen in 8 of 23 (35 per cent) squamous cell metaplasias, 6 of 19 (32 per cent) low-grade dysplasias and 5 of 10 (50 per cent) high-grade dysplasias. No correlation was found between p53 expression in carcinomas and their clinical and pathological characteristics, DNA ploidy, or proliferative activity. Neither disease-free nor overall survival showed differences between p53-positive and p53-negative cases. These findings indicate that p53 may play a role in an early stage of malignant transformation of a subset of squamous cell carcinomas of the larynx, but seems not to be associated with further progression of the tumours.