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In mice, variability in adult bone size and density has been observed among common inbred strains. Also, in the group of genes regulating circadian rhythmicity in mice, so called clock genes, changes in body size and skeletal parameters have been noted in knockout mice. Here, we studied the size and density of prominent bones of the axial and appendicular skeleton of clock gene Period-1-deficient (Per1 -/- ) mice by means of microcomputed tomography. Our data show shorter spinal length, smaller and less dense femora and tibiae, but no significant changes in the shape of the skull and the length of the head. Together with the significantly lower total body weight of Per1 -/- mice, we conclude that Per1-deficiency in a melatonin-proficient mouse strain is associated with an altered body phenotype with smaller appendicular (hind limb) bone size, shorter spine length and lower total body weight while normal head length and brain weight. The observed changes suggest an involvement of secondary bone mineralisation with impact on long bones, but lesser impact on those of the skull. Evidence and overall physiological implications of these findings are discussed., (© 2024 The Author(s). Journal of Pineal Research published by John Wiley & Sons Ltd.)

The suprachiasmatic nucleus of the hypothalamus (SCN) houses the central circadian oscillator of mammals. The main neurotransmitters produced in the SCN are γ-amino-butyric acid, arginine-vasopressin (AVP), vasoactive intestinal peptide (VIP), pituitary-derived adenylate cyclase-activating peptide (PACAP), prokineticin 2, neuromedin S, and gastrin-releasing peptide (GRP). Apart from these, catecholamines and their receptors were detected in the SCN as well. In this study, we confirmed the presence of β-adrenergic receptors in SCN and a mouse SCN-derived immortalized cell line by immunohistochemical, immuno-cytochemical, and pharmacological techniques. We then characterized the effects of β-adrenergic agonists and antagonists on cAMP-regulated element (CRE) signaling. Moreover, we investigated the interaction of β-adrenergic signaling with substances influencing parallel signaling pathways. Our findings have potential implications on the role of stress (elevated adrenaline) on the biological clock and may explain some of the side effects of β-blockers applied as anti-hypertensive drugs., (© 2024 The Author(s). Journal of Pineal Research published by John Wiley & Sons Ltd.)

Chronobiology investigations have revealed much about cellular and physiological clockworks but we are far from having a complete mechanistic understanding of the physiological and ecological implications. Here we present some unresolved questions in circadian biology research as posed by the editorial staff and guest contributors to the Journal of Circadian Rhythms. This collection of ideas is not meant to be comprehensive but does reveal the breadth of our observations on emerging trends in chronobiology and circadian biology. It is amazing what could be achieved with various expected innovations in technologies, techniques, and mathematical tools that are being developed. We fully expect strengthening mechanistic work will be linked to health care and environmental understandings of circadian function. Now that most clock genes are known, linking these to physiological, metabolic, and developmental traits requires investigations from the single molecule to the terrestrial ecological scales. Real answers are expected for these questions over the next decade. Where are the circadian clocks at a cellular level? How are clocks coupled cellularly to generate organism level outcomes? How do communities of circadian organisms rhythmically interact with each other? In what way does the natural genetic variation in populations sculpt community behaviors? How will methods development for circadian research be used in disparate academic and commercial endeavors? These and other questions make it a very exciting time to be working as a chronobiologist., Competing Interests: The authors have no competing interests to declare., (Copyright: © 2024 The Author(s).)

Circadian Biology intersects with diverse scientific domains, intricately woven into the fabric of organismal physiology and behavior. The rhythmic orchestration of life by the circadian clock serves as a focal point for researchers across disciplines. This retrospective examination delves into several of the scientific milestones that have fundamentally shaped our contemporary understanding of circadian rhythms. From deciphering the complexities of clock genes at a cellular level to exploring the nuances of coupled oscillators in whole organism responses to stimuli. The field has undergone significant evolution lately guided by genetics approaches. Our exploration here considers key moments in the circadian-research landscape, elucidating the trajectory of this discipline with a keen eye on scientific advancements and paradigm shifts., Competing Interests: The authors have no competing interests to declare., (Copyright: © 2024 The Author(s).)

GPR55 is involved in many physiological and pathological processes. In cancer, GPR55 has been described to show accelerating and decelerating effects in tumor progression resulting from distinct intracellular signaling pathways. GPR55 becomes activated by LPI and various plant-derived, endogenous, and synthetic cannabinoids. Cannabinoids such as THC exerted antitumor effects by inhibiting tumor cell proliferation or inducing apoptosis. Besides its effects through CB 1 and CB 2 receptors, THC modulates cellular responses among others via GPR55. Previously, we reported a reduction in Ki67-immunoreactive nuclei of human glioblastoma cells after GPR55 activation in general by THC and in particular by LPI. In the present study, we investigated intracellular mechanisms leading to an altered number of Ki67 + nuclei after stimulation of GPR55 by LPI and THC. Pharmacological analyses revealed a strongly involved PLC-IP3 signaling and cell-type-specific differences in Gα-, Gβγ-, RhoA-ROCK, and calcineurin signaling. Furthermore, immunochemical visualization of the calcineurin-dependent transcription factor NFAT revealed an unchanged subcellular localization after THC or LPI treatment. The data underline the cell-type-specific diversity of GPR55-associated signaling pathways in coupling to intracellular G proteins. Furthermore, this diversity might determine the outcome and the individual responsiveness of tumor cells to GPR55 stimulation by cannabin oids.

We evaluated the signalling framework of immortalized cells from the hypothalamic suprachiasmatic nucleus (SCN) of the mouse. We selected a vasoactive intestinal peptide (VIP)-positive sub-clone of immortalized mouse SCN-cells stably expressing a cAMP-regulated-element (CRE)-luciferase construct named SCNCRE. We characterized these cells in terms of their status as neuronal cells, as well as for important components of the cAMP-dependent signal transduction pathway and compared them to SCN ex vivo. SCNCRE cells were treated with agents that modulate different intracellular signalling pathways to investigate their potency and timing for transcriptional CRE-dependent signalling. Several activating pathways modulate SCN neuronal signalling via the cAMP-regulated-element (CRE: TGACGCTA) and phosphorylation of transcription factors such as cAMP-regulated-element-binding protein (CREB). CRE-luciferase activity induced by different cAMP-signalling pathway-modulating agents displayed a variety of substance-specific dose and time-dependent profiles and interactions relevant to the regulation of SCN physiology. Moreover, the induction of the protein kinase C (PKC) pathway by phorbol ester application modulates the CRE-dependent signalling pathway as well. In conclusion, the cAMP/PKA- and the PKC-regulated pathways individually and in combination modulate the final CRE-dependent transcriptional output.

The human pineal gland regulates the day-night dynamics of multiple physiological processes, especially through the secretion of melatonin. Recently, using mass spectrometry-based proteomics and dedicated analysis tools, we have identified regulated proteins and signaling pathways that differ between day and night and/or between control and autistic pineal glands. This large-scale proteomic approach is the method of choice to study proteins in a biological system globally. This chapter proposes a protocol for large-scale analysis of the pineal gland proteome., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cyclic nucleotides are important second messengers involved in cellular events, and analogues of this type of molecules are promising drug candidates. Some cyclic nucleotide analogues have become standard tools for the investigation of biochemical and physiological signal transduction pathways, such as the Rp -diastereomers of adenosine and guanosine 3',5'-cyclic monophosphorothioate, which are competitive inhibitors of cAMP- and cGMP-dependent protein kinases. Next generation analogues exhibit a higher membrane permeability, increased resistance against degradation, and improved target specificity, or are caged or photoactivatable for fast and/or targeted cellular imaging. Novel specific nucleotide analogues activating or inhibiting cyclic nucleotide-dependent ion channels, EPAC/GEF proteins, and bacterial target molecules have been developed, opening new avenues for basic and applied research. This review provides an overview of the current state of the field, what can be expected in the future and some practical considerations for the use of cyclic nucleotide analogues in biological systems.

The human pineal gland regulates day-night dynamics of multiple physiological processes, especially through the secretion of melatonin. Using mass-spectrometry-based proteomics and dedicated analysis tools, we identify proteins in the human pineal gland and analyze systematically their variation throughout the day and compare these changes in the pineal proteome between control specimens and donors diagnosed with autism. Results reveal diverse regulated clusters of proteins with, among others, catabolic carbohydrate process and cytoplasmic membrane-bounded vesicle-related proteins differing between day and night and/or control versus autism pineal glands. These data show novel and unexpected processes happening in the human pineal gland during the day/night rhythm as well as specific differences between autism donor pineal glands and those from controls., (© 2020 The Authors. Journal of Pineal Research published by John Wiley & Sons Ltd.)

Signaling pathways, depending on the second messenger molecule cAMP, modulate hippocampal cell signaling via influencing transcription factors like cAMP-regulated element-binding protein (CREB) or early growth response 1 EGR1/Krox24/zif268/ZENK (EGR1). Here, we investigated two reporter cell lines derived from an immortalized hippocampal neuronal cell line stably expressing a CRE- or EGR1-luciferase reporter gene (HT22CREluc and HT22EGR1luc, respectively). The cells were subjected to phosphodiesterase inhibitors and other cAMP-modulating agents to investigate dose- and time-dependent phosphodiesterase (PDE)-mediated fine-tuning of cAMP-dependent transcriptional signaling. The non-isoform-specific cyclic nucleotide phosphodiesterase (PDE) inhibitor isobutyl-methyl-xanthine (IBMX), as well as selective inhibitors of PDE3 (milrinone) and PDE4 (rolipram), were tested for their ability to elevate CRE- and EGR1-luciferase activity. Pharmacological parameters like onset of activity, maximum activity, and offset of activity were determined. In summary, phosphodiesterase inhibition appeared similarly potent in comparison to adenylate cyclase stimulation or direct activation of protein kinase A (PKA) via specific cAMP agonists and was at least partly mediated by PKA as shown by the selective PKA inhibitor Rp -8-Br-cAMPS. Moreover, transcriptional activation by PDE inhibition was also influenced by organic anion-exchanger action and interacted with fibroblast growth factor (FGF) receptor-mediated pathways.

Circadian oscillations in circulating leukocyte subsets including immature hematopoietic cells have been appreciated; the origin and nature of these alterations remain elusive. Our analysis of wild-type C57BL/6 mice under constant darkness confirmed circadian fluctuations of circulating leukocytes and clonogenic cells in blood and spleen but not bone marrow. Clock gene deficient Bmal1-/ - mice lacked this regulation. Cell cycle analyses in the different hematopoietic compartments excluded circadian changes in total cell numbers, rather favoring shifting hematopoietic cell redistribution as the underlying mechanism. Transplant chimeras demonstrate that circadian rhythms within the stroma mediate the oscillations independently of hematopoietic-intrinsic cues. We provide evidence of circadian CXCL12 regulation via clock genes in vitro and were able to confirm CXCL12 oscillation in bone marrow and blood in vivo. Our studies further implicate cortisol as the conveyor of circadian input to bone marrow stroma and mediator of the circadian leukocyte oscillation. In summary, we establish hematopoietic-extrinsic cues as causal for circadian redistribution of circulating mature/immature blood cells.

Background: Ultrasonic vocalizations (USV) of mice are produced in and emitted by the larynx. However, which anatomical elements of the mouse larynx are involved and to which aspects of USV they contribute is not clear. Frequency and amplitude parameters of mice, deficient in the clock gene Period1 (mPer1 -/- mice) are distinguishably different compared to C3H wildtype (WT) controls. Because structural differences in the larynx may be a reason for the different USV observed, we analyzed laryngeal anatomy of mPer1 -/- mice and WT control animals using micro-computed-tomography and stereology., Results: In mPer1 -/- mice, we found laryngeal cartilages to be normally arranged, and the thyroid, arytenoid and epiglottal cartilages were similar in diameter and volume measurements, compared to WT mice. However, in the cricoid cartilage, a significant difference in the dorso-ventral diameter and volume was evident., Conclusion: Our findings imply that laryngeal morphology is affected by inactivation of the clock gene Period1 in mice, which may contribute to their abnormal USV., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

A plethora of physiological processes show stable and synchronized daily oscillations that are either driven or modulated by biological clocks. A circadian pacemaker located in the suprachiasmatic nucleus of the ventral hypothalamus coordinates 24-hour oscillations of central and peripheral physiology with the environment. The circadian clockwork involved in driving rhythmic physiology is composed of various clock genes that are interlocked via a complex feedback loop to generate precise yet plastic oscillations of ∼24 hours. This review focuses on the specific role of the core clockwork gene Period1 and its paralogs on intra-oscillator and extra-oscillator functions, including, but not limited to, hippocampus-dependent processes, cardiovascular function, appetite control, as well as glucose and lipid homeostasis. Alterations in Period gene function have been implicated in a wide range of physical and mental disorders. At the same time, a variety of conditions including metabolic disorders also impact clock gene expression, resulting in circadian disruptions, which in turn often exacerbates the disease state.

Cyclic adenosine 3',5'monophosphate (cAMP) regulated element binding protein (CREB) is a transcription factor involved in many different signaling processes including memory storage and retrieval. The mouse hippocampal neuronal cell line HT22 is widely used as a model system for neuronal cell death and cellular signal pathway investigations. For the present work a variant of HT22 with a stably expressed CRE-luciferase (CRE-luc) reporter (HT22CRE) is introduced, characterized and used to investigate cAMP-dependent and independent CRE-dependent signal processes. Trehalose (Mykose or 1-α-Glucopyranosyl-1-α-glucopyranosid) is a naturally occurring disaccharide consisting of two α,α',1,1-glycosidic connected glucose molecules in a wide range of organisms but usually not found in mammals. Trehalose has been shown to activate autophagy, a process which regulates the degradation and recycling of proteins and organelles. The exact processes how trehalose application works on mammalian neuronal cells is not yet understood. The present work shows that trehalose application dose-dependently elevates CRE-luc activity in HT22 cells and acts synergistically with cAMP-elevating agents. In this pathway cAMP-dependent protein kinase (PKA) appears to be the most important factor and the stress kinase p38 and protein tyrosine kinases (PTKs) act as modulators.

Learning, memory consolidation, and retrieval are processes known to be modulated by the circadian ( circa : about; dies : day) system. The circadian regulation of memory performance is evolutionarily conserved, independent of the type and complexity of the learning paradigm tested, and not specific to crepuscular, nocturnal, or diurnal organisms. In mammals, long-term memory (LTM) formation is tightly coupled to de novo gene expression of plasticity-related proteins and posttranslational modifications and relies on intact cAMP/protein kinase A (PKA)/protein kinase C (PKC)/mitogen-activated protein kinase (MAPK)/cyclic adenosine monophosphate response element-binding protein (CREB) signaling. These memory-essential signaling components cycle rhythmically in the hippocampus across the day and night and are clearly molded by an intricate interplay between the circadian system and memory. Important components of the circadian timing mechanism and its plasticity are members of the Period clock gene family ( Per1 , Per2 ). Interestingly, Per1 is rhythmically expressed in mouse hippocampus. Observations suggest important and largely unexplored roles of the clock gene protein PER1 in synaptic plasticity and in the daytime-dependent modulation of learning and memory. Here, we review the latest findings on the role of the clock gene Period 1 ( Per1 ) as a candidate molecular and mechanistic blueprint for gating the daytime dependency of memory processing.

Autism Spectrum Disorders (ASD) are heterogeneous neurodevelopmental disorders with a complex genetic architecture. They are characterized by impaired social communication, stereotyped behaviors and restricted interests and are frequently associated with comorbidities such as intellectual disability, epilepsy and severe sleep disorders. Hyperserotonemia and low melatonin levels are among the most replicated endophenotypes reported in ASD, but their genetic causes remain largely unknown. Based on the biochemical profile of 717 individuals including 213 children with ASD, 128 unaffected siblings and 376 parents and other relatives, we estimated the heritability of whole-blood serotonin, platelet N-acetylserotonin (NAS) and plasma melatonin levels, as well as the two enzymes arylalkylamine N-acetyltransferase (AANAT) and acetylserotonin O-methyltransferase (ASMT) activities measured in platelets. Overall, heritability was higher for NAS (0.72 ± 0.091) and ASMT (0.59 ± 0.097) compared with serotonin (0.31 ± 0.078), AANAT (0.34 ± 0.077) and melatonin (0.22 ± 0.071). Bivariate analyses showed high phenotypic and genetic correlations between traits of the second step of the metabolic pathway (NAS, ASMT and melatonin) indicating the contribution of shared genetic factors. A better knowledge of the heritability of the melatonin synthesis variability constitutes an important step to identify the factors that perturb this pathway in individuals with ASD.

Autism spectrum disorders (ASD) are characterized by a wide genetic and clinical heterogeneity. However, some biochemical impairments, including decreased melatonin (crucial for circadian regulation) and elevated platelet N-acetylserotonin (the precursor of melatonin) have been reported as very frequent features in individuals with ASD. To address the mechanisms of these dysfunctions, we investigated melatonin synthesis in post-mortem pineal glands - the main source of melatonin (9 patients and 22 controls) - and gut samples - the main source of serotonin (11 patients and 13 controls), and in blood platelets from 239 individuals with ASD, their first-degree relatives and 278 controls. Our results elucidate the enzymatic mechanism for melatonin deficit in ASD, involving a reduction of both enzyme activities contributing to melatonin synthesis (AANAT and ASMT), observed in the pineal gland as well as in gut and platelets of patients. Further investigations suggest new, post-translational (reduced levels of 14-3-3 proteins which regulate AANAT and ASMT activities) and post-transcriptional (increased levels of miR-451, targeting 14-3-3ζ) mechanisms to these impairments. This study thus gives insights into the pathophysiological pathways involved in ASD.

Memory performance varies over a 24-h day/night cycle. While the detailed underlying mechanisms are yet unknown, recent evidence suggests that in the mouse hippocampus, rhythmic phosphorylation of mitogen-activated protein kinase (MAPK) and cyclic adenosine monophosphate response element-binding protein (CREB) are central to the circadian (~ 24 h) regulation of learning and memory. We recently identified the clock protein PERIOD1 (PER1) as a vehicle that translates information encoding time of day to hippocampal plasticity. We here elaborate how PER1 may gate the sensitivity of memory-relevant hippocampal signaling pathways. We found that in wild-type mice (WT), spatial learning triggers CREB phosphorylation only during the daytime, and that this effect depends on the presence of PER1. The time-of-day-dependent induction of CREB phosphorylation can be reproduced pharmacologically in acute hippocampal slices prepared from WT mice, but is absent in preparations made from Per1-knockout (Per1(-/-) ) mice. We showed that the PER1-dependent CREB phosphorylation is regulated downstream of MAPK. Stimulation of WT hippocampal neurons triggered the co-translocation of PER1 and the CREB kinase pP90RSK (pMAPK-activated ribosomal S6 kinase) into the nucleus. In hippocampal neurons from Per1(-/-) mice, however, pP90RSK remained perinuclear. A co-immunoprecipitation assay confirmed a high-affinity interaction between PER1 and pP90RSK. Knocking down endogenous PER1 in hippocampal cells inhibited adenylyl cyclase-dependent CREB activation. Taken together, the PER1-dependent modulation of cytoplasmic-to-nuclear signaling in the murine hippocampus provides a molecular explanation for how the circadian system potentially shapes a temporal framework for daytime-dependent memory performance, and adds a novel facet to the versatility of the clock gene protein PER1. We provide evidence that the circadian clock gene Period1 (Per1) regulates CREB phosphorylation in the mouse hippocampus, sculpturing time-of-day-dependent memory formation. This molecular mechanism constitutes the functional link between circadian rhythms and learning efficiency. In hippocampal neurons of wild-type mice, pP90RSK translocates into the nucleus upon stimulation with forskolin (left), whereas in Period1-knockout (Per1(-/-) ) mice (right) the kinase is trapped at the nuclear periphery, unable to efficiently phosphorylate nuclear CREB. Consequently, the presence of PER1 in hippocampal neurons is a prerequisite for the time-of-day-dependent phosphorylation of CREB, as it regulates the shuttling of pP90RSK into the nucleus. Representative immunofluorescence images show a temporal difference in phosphorylated cAMP response element-binding protein (pCREB; green color) levels in all regions of the dorsal hippocampus between a wild-type C3H mouse (WT; left) and a Period1-knockout (Per1(-/-) ; right) mouse. Images were taken 2 h after lights on, thus, when fluctuating levels of pCREB peak in WT mouse hippocampus. Insets show a representative hippocampal neuron, in response to activating cAMP signaling, stained for the neuronal marker NeuN (red), the nuclear marker DAPI (blue) and the activated CREB kinase pP90RSK (green). The image was taken 2 h after light onset (at the peak of the endogenous CREB phosphorylation that fluctuates with time of day). Magnification: 100X, inset 400X. Read the Editorial Highlight for this article on page 650. Cover image for this issue: doi: 10.1111/jnc.13332., (© 2016 International Society for Neurochemistry.)

Laboratory mice are well capable of performing innate routine behaviour programmes necessary for courtship, nest-building and exploratory activities although housed for decades in animal facilities. We found that in mice inactivation of the clock gene Period1 profoundly changes innate routine behaviour programmes like those necessary for courtship, nest building, exploration and learning. These results in wild-type and Period1 mutant mice, together with earlier findings on courtship behaviour in wild-type and period-mutant Drosophila melanogaster, suggest a conserved role of Period-genes on innate routine behaviour. Additionally, both per-mutant flies and Period1-mutant mice display spatial learning and memory deficits. The profound influence of Period1 on routine behaviour programmes in mice, including female partner choice, may be independent of its function as a circadian clock gene, since Period1-deficient mice display normal circadian behaviour.

Background: Cannabinoid receptor 1 (CB1) is expressed in certain types of malignancies. An analysis of CB1 expression and function in Hodgkin lymphoma (HL), one of the most frequent lymphomas, was not performed to date., Design and Methods: We examined the distribution of CB1 protein in primary cases of HL. Using lymphoma derived cell lines, the role of CB1 signaling on cell survival was investigated., Results: A predominant expression of CB1 was found in Hodgkin-Reed-Sternberg cells in a vast majority of classical HL cases. The HL cell lines L428, L540 and KM-H2 showed strong CB1-abundance and displayed a dose-dependent decline of viability under CB1 inhibition with AM251. Further, application of AM251 led to decrease of constitutively active NFκB/p65, a crucial survival factor of HRS-cells, and was followed by elevation of apoptotic markers in HL cells., Conclusions: The present study identifies CB1 as a feature of HL, which might serve as a potential selective target in the treatment of Hodgkin lymphoma.

Surfactant proteins (SP) have been studied intensively in the respiratory system. Surfactant protein A and surfactant protein D are proteins belonging to the family of collectins each playing a major role in the innate immune system. The ability of surfactant protein A and surfactant protein D to bind various pathogens and facilitate their elimination has been described in a vast number of studies. Surfactant proteins are very important in modulating the host's inflammatory response and participate in the clearance of apoptotic cells. Surfactant protein B and surfactant protein C are proteins responsible for lowering the surface tension in the lungs. The aim of this study was an investigation of expression of surfactant proteins in the central nervous system to assess their specific distribution patterns. The second aim was to quantify surfactant proteins in cerebrospinal fluid of healthy subjects compared to patients suffering from different neuropathologies. The expression of mRNA for the surfactant proteins was analyzed with RT-PCR done with samples from different parts of the human brain. The production of the surfactant proteins in the brain was verified using immunohistochemistry and Western blot. The concentrations of the surfactant proteins in cerebrospinal fluid from healthy subjects and patients suffering from neuropathologic conditions were quantified using ELISA. Our results revealed that surfactant proteins are present in the central nervous system and that the concentrations of one or more surfactant proteins in healthy subjects differed significantly from those of patients affected by central autoimmune processes, CNS infections or cerebral infarction. Based on the localization of the surfactant proteins in the brain, their different levels in normal versus pathologic samples of cerebrospinal fluid and their well-known functions in the lungs, it appears that the surfactant proteins may play roles in host defense of the brain, facilitation of cerebrospinal fluid secretion and maintenance of the latter's rheological properties.

Pituitary function relies on strictly timed, yet plastic mechanisms, particularly with respect to the daytime-dependent coordination of hormone synthesis and release. In other systems, clock genes and their protein products are well-described candidates to anticipate the daily demands in neuroendocrine coupling and to manage cellular adaptation on changing internal or external circumstances. To elucidate possible mechanisms of time management, a total of 52 human autoptic pituitary glands were allocated to the 4 time-of-day groups, night, dawn, day, and dusk, according to reported time of death. The observed daytime-dependent dynamics in ACTH content supports a postmortem conservation of the premortem condition, and thus, principally validates the investigation of autoptic pituitary glands. Pituitary extracts were investigated for expression of clock genes Per1, Cry1, Clock, and Bmal1 and corresponding protein products. Only the clock gene Per1 showed daytime-dependent differences in quantitative real-time PCR analyses, with decreased levels observed during dusk. Although the overall amount in clock gene protein products PER1, CRY1, and CLOCK did not fluctuate with time of day in human pituitary, an indication for a temporally parallel intracellular translocation of PER1 and CRY1 was detected by immunofluorescence. Presented data suggest that the observed clock gene expression in human pituitary cells does not provide evidence for a functional intrinsic clockwork. It is suggested that clock genes and their protein products may be directly involved in the daytime-dependent regulation and adaptation of hormone synthesis and release and within homeostatic adaptive plasticity.

Transient, severe global ischemia that arises in humans as a consequence of cardiac arrest or cardiac surgery or that is induced experimentally in animals, leads to selective and delayed neuronal death, particularly in the hippocampus. Especially, in this brain structure, clock genes are rhythmically expressed, for instance the inducible and archetypical clock gene is Period1 (Per1). An eventual involvement of its trans-activating protein products in the daytime-dependent severity of ischemia-induced cell damage is not excluded. Probably, neurons may exhibit endogenously a daytimedependent variation in the expression of predictive cell death proteins. We therefore compared the cell death machinery in the hippocampus between Per1(-/-)- and wildtype (WT) mice upon cerebral ischemia. Neuronal death in the hippocampal CA1-subfield, was observed in both types of mice, but the density of damaged cells in Per1(-/-)-mice was increased by more than 23% as compared to wildtype mice. To explore the mechanisms underlying the excessive vulnerability of the hippocampus in Per1(-/-)-mice and to address if hippocampal susceptibility inherits a daytime component, the expression of both, apoptotic and autophagic predictors of cell death was monitored. In Per1(-/-)-mice, the expression of apoptotic/autophagic markers are altered and higher levels of the proapoptotic factors such as cytochrome c and Apaf-1 were observed as compared to WT mice. Moreover, the autophagy marker LC3B was dramatically reduced in Per1(-/-)- mice. Our data suggests that basal activities of apoptosis and autophagy seem to be modulated by PER1, and that the autophagic machinery is probably slowed down when this clock gene is absent. These alterations may be causal for the observed innate vulnerability of Per1(-/-)-mice to cerebral ischemia.

The cellular uptake of L-arginine and other cationic amino acids (such as L-lysine and L-ornithine) is mainly mediated by cationic amino acid transporter (CAT) proteins. Despite the important roles of cationic amino acid transporters for normal brain functioning and various brain diseases there is currently only fragmentary knowledge about their cellular and regional distribution patterns in the human brain. We mapped the immunohistochemical localization of human cationic amino acid transporters 1, 2 and 3 (hCAT1, 2, and 3) throughout five adult human brains and found a wide but uneven distribution of these transporters. All three hCAT1s were mainly localized in neurons, but were also found in numerous astrocytes, oligodendrocytes, plexus choroideus epithelial cells, and small blood vessels. The highest density of hCAT expressing neurons was observed in the hypothalamus, in some areas of the cerebral cortex, the thalamic reticular nucleus and the caudate nucleus, whereas weak to moderate expression was detected in the hippocampus, the prefrontal cortex (hCAT1 only), pons, brain stem and cerebellum. In contrast to what has been found in rodent brain, we detected hCAT2 and hCAT3 also in astrocytes. Overall, each hCAT has its characteristic, individual cerebral expression patterns, which, however, overlap with the others.

Melatonin is a synchronizer of many physiological processes. Abnormal melatonin signaling is associated with human disorders related to sleep, metabolism, and neurodevelopment. Here, we present the X-ray crystal structure of human N-acetyl serotonin methyltransferase (ASMT), the last enzyme of the melatonin biosynthesis pathway. The polypeptide chain of ASMT consists of a C-terminal domain, which is typical of other SAM-dependent O-methyltransferases, and an N-terminal domain, which intertwines several helices with another monomer to form the physiologically active dimer. Using radioenzymology, we analyzed 20 nonsynonymous variants identified through the 1000 genomes project and in patients with neuropsychiatric disorders. We found that the majority of these mutations reduced or abolished ASMT activity including one relatively frequent polymorphism in the Han Chinese population (N17K, rs17149149). Overall, we estimate that the allelic frequency of ASMT deleterious mutations ranges from 0.66% in Europe to 2.97% in Asia. Mapping of the variants on to the 3-dimensional structure clarifies why some are harmful and provides a structural basis for understanding melatonin deficiency in humans., (© 2012 John Wiley & Sons A/S.)

Autophagy has been described as a cellular response to stressful stimuli like starvation. One of its primary functions is to recycle amino acids from degraded proteins for cellular survival under nutrient deprived conditions. Autophagy is characterized by double membrane cytosolic vesicles called autophagosomes and prolonged autophagy is known to result in autophagic (Type II) cell death. Beclin-1 is involved in the regulation of autophagy in mammalian cells. This study examined the potential impact of knockdown of beclin-1 in an autophagic response in HT22 neurons challenged with amino acid starvation (AAS). AAS exposure induced light chain-3 (LC-3)-immunopositive and monodansylcadaverine (MDC) fluorescent dye-labeled autophagosome formation in cell bodies as early as 3 h post-AAS in wild type cells. Elevated levels of the autophagosome-targeting LC3-II were also observed following AAS. In addition, neuronal death induced by AAS in HT22-cells led to a moderate activation of caspase-3, a slight upregulation of AIF and did not alter the HtrA2 levels. Autophagy inhibition by a knockdown of beclin-1 significantly reduced AAS-induced LC3-II increase, reduced accumulation of autophagosomes, and potentiated AAS-mediated neuronal death. Collectively, this study shows that the both apoptotic and autophagic machineries are inducible in cultured hippocampal HT22 neurons subjected to AAS. Our data further show that attenuation of autophagy by a knockdown of beclin-1 enhanced neurons susceptibility to proapoptotic signals induced by AAS and underlines that autophagy is per se a protective than a deleterious mechanism.

The neuroendocrine substance melatonin is a hormone synthesized rhythmically by the pineal gland under the influence of the circadian system and alternating light/dark cycles. Melatonin has been shown to have broad applications, and consequently becoming a molecule of great controversy. Undoubtedly, however, melatonin plays an important role as a time cue for the endogenous circadian system. This review focuses on melatonin as a regulator in the circadian modulation of memory processing. Memory processes (acquisition, consolidation, and retrieval) are modulated by the circadian system. However, the mechanism by which the biological clock is rhythmically influencing cognitive processes remains unknown. We also discuss, how the circadian system by generating cycling melatonin levels can implant information about daytime into memory processing, depicted as day and nighttime differences in acquisition, memory consolidation and/or retrieval.

Time of day is communicated to the body through rhythmic cues, including pineal gland melatonin synthesis, which is restricted to nighttime. Whereas in most rodents transcriptional regulation of the arylalkylamine N-acetyltransferase (Aanat) gene is essential for rhythmic melatonin synthesis, investigations into nonrodent mammalian species have shown post-transcriptional regulation to be of central importance, with molecular mechanisms still elusive. Therefore, human pineal tissues, taken from routine autopsies were allocated to four time-of-death groups (night/dawn/day/dusk) and analyzed for daytime-dependent changes in phosphorylated AANAT (p31T-AANAT) and in acetyl-serotonin-methyltransferase (ASMT) expression and activity. Protein content, intracellular localization, and colocalization of p31T-AANAT and ASMT were assessed, using immunoblotting, immunofluorescence, and immunoprecipitation techniques. Fresh sheep pineal gland preparations were used for comparative purposes. The amount of p31T-AANAT and ASMT proteins as well as their intracellular localization showed no diurnal variation in autoptic human and fresh sheep pineal glands. Moreover, in human and sheep pineal extracts, AANAT could not be dephosphorylated, which was at variance to data derived from rat pineal extracts. P31T-AANAT and ASMT were often found to colocalize in cellular rod-like structures that were also partly immunoreactive for the pinealocyte process-specific marker S-antigen (arrestin) in both, human and sheep pinealocytes. Protein-protein interaction studies with p31T-AANAT, ASMT, and S-antigen demonstrated a direct association and formation of robust complexes, involving also 14-3-3. This work provides evidence for a regulation principle for AANAT activity in the human pineal gland, which may not be based on a p31T-AANAT phosphorylation/dephosphorylation switch, as described for other mammalian species., (© 2011 John Wiley & Sons A/S.)

The human pineal gland is a neuroendocrine transducer that forms an integral part of the brain. Through the nocturnally elevated synthesis and release of the neurohormone melatonin, the pineal gland encodes and disseminates information on circadian time, thus coupling the outside world to the biochemical and physiological internal demands of the body. Approaches to better understand molecular details behind the rhythmic signalling in the human pineal gland are limited but implicitly warranted, as human chronobiological dysfunctions are often associated with alterations in melatonin synthesis. Current knowledge on melatonin synthesis in the human pineal gland is based on minimally invasive analyses, and by the comparison of signalling events between different vertebrate species, with emphasis put on data acquired in sheep and other primates. Together with investigations using autoptic pineal tissue, a remnant silhouette of premortem dynamics within the hormone's biosynthesis pathway can be constructed. The detected biochemical scenario behind the generation of dynamics in melatonin synthesis positions the human pineal gland surprisingly isolated. In this neuroendocrine brain structure, protein-protein interactions and nucleo-cytoplasmic protein shuttling indicate furthermore a novel twist in the molecular dynamics in the cells of this neuroendocrine brain structure. These findings have to be seen in the light that an impaired melatonin synthesis is observed in elderly and/or demented patients, in individuals affected by Alzheimer's disease, Smith-Magenis syndrome, autism spectrum disorder and sleep phase disorders. Already, recent advances in understanding signalling dynamics in the human pineal gland have significantly helped to counteract chronobiological dysfunctions through a proper restoration of the nocturnal melatonin surge., (© 2011 John Wiley & Sons A/S.)

Endocannabinoids like 2-arachidonoylglycerol strongly modulate the complex machinery of secondary neuronal damage and are shown to improve neuronal survival after excitotoxic lesion. Palmitoylethanolamide (PEA), the naturally occurring fatty acid amide of ethanolamine and palmitic acid, is an endogenous lipid known to mimic several effects of endocannabinoids even without binding to cannabinoid receptors. Here we show that PEA (0.001-1 μM) and the synthetic peroxisome proliferator-activated receptor (PPAR)-alpha agonist 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio acetic acid (Wy-14,643; 0.1-1 μM) reduced the number of microglial cells and protected dentate gyrus granule cells in excitotoxically lesioned organotypic hippocampal slice cultures (OHSCs). Treatment with the PPAR-alpha antagonist N-((2S)-2-(((1Z)-1-Methyl-3-oxo-3-(4-(trifluoromethyl)phenyl)prop-1-enyl)amino)-3-(4-(2-(5-methyl-2-phenyl-1,3-oxazol-4-yl)ethoxy)phenyl)propyl)propanamide (GW6471; 0.05-5 μM) blocked PEA-mediated neuroprotection and reduction of microglial cell numbers whereas the PPAR-gamma antagonist 2-chloro-5-nitro-N-phenyl-benzamide (GW9662; 0.01-1 μM) showed no effects. Immunocytochemistry and Western blot analyses revealed a strong PPAR-alpha immunoreaction in BV-2 microglial cells and in HT22 hippocampal cells. Intensity and location of PPAR-alpha immunoreaction remained constant during stimulation with PEA (0.01 μM; 1-36 h). In conclusion our data provide evidence that (1) PEA counteracted excitotoxically induced secondary neuronal damage of dentate gyrus granule cells, (2) PPAR-alpha but not PPAR-gamma is the endogenous binding site for PEA-mediated neuroprotection, and (3) PEA may activate PPAR-alpha in microglial cells and hippocampal neurons to exert its neuroprotective effects. In addition to classical endocannabinoids, PEA-mediated PPAR-alpha activation represents a possible target for therapeutic interventions to mitigate symptoms of secondary neuronal damage.

Background and Purpose: The transcription factor early growth response-1 (Egr-1) and the acidic fibroblast growth factor (FGF-1) are involved in many regulatory processes, including hippocampus-associated learning and memory. However, the intracellular signalling mechanisms regulating Egr-1 in hippocampal cells are not entirely understood., Experimental Approach: We used primary mouse hippocampal neurons and the mouse hippocampal neuronal cell line HT22 to investigate how FGF-1 transiently induces Egr-1 protein. This was accomplished by a range of techniques including Western blotting, immunofluorescence, specific protein kinase inhibitors and transfectable constitutively active protein kinase constructs., Key Results: Protein kinase B (PKB) and mitogen-activated protein kinase (MAPK) were both initially phosphorylated and activated by FGF-1 treatment, but when phosphorylated MAPK reached maximal activation, phosphorylated PKB was at its lowest levels, suggesting an interaction between MAPK kinase (MEK-1/2) and phosphatidyl inositol-3-kinase (PI3K) during Egr-1 induction. Interestingly, pharmacological inhibition of MEK-1/2 resulted in a robust increase in the phosphorylation of PKB, which was repressed in the presence of increasing doses of a PI3K inhibitor. FGF-1-mediated Egr-1 induction was impaired by inhibition of MEK-1/2, but not of PI3K. However, elevated levels of PKB, induced by transfection of constitutively active PKB (myrAkt) into hippocampal neuronal HT22 cells, led to reduced levels of Egr-1 after FGF-1 application., Conclusions and Implications: Our data indicate a contribution of inactive (dephosphorylated) PKB to FGF-1-mediated induction of Egr-1, and strongly suggest a functionally and pharmacologically interesting cross-talk between MEK-1/2 and PI3K signalling in hippocampal neurons after FGF-1 stimulation that may play a role in hippocampal synaptic plasticity.

Background: Clock genes and their protein products regulate circadian rhythms in mammals but have also been implicated in various physiological processes, including bone formation. Osteoblasts build new mineralized bone whereas osteoclasts degrade it thereby balancing bone formation. To evaluate the contribution of clock components in this process, we investigated mice mutant in clock genes for a bone volume phenotype., Methodology/principal Findings: We found that Per2(Brdm1) mutant mice as well as mice lacking Cry2(-/-) displayed significantly increased bone volume at 12 weeks of age, when bone turnover is high. Per2(Brdm1) mutant mice showed alterations in parameters specific for osteoblasts whereas mice lacking Cry2(-/-) displayed changes in osteoclast specific parameters. Interestingly, inactivation of both Per2 and Cry2 genes leads to normal bone volume as observed in wild type animals. Importantly, osteoclast parameters affected due to the lack of Cry2, remained at the level seen in the Cry2(-/-) mutants despite the simultaneous inactivation of Per2., Conclusions/significance: This indicates that Cry2 and Per2 affect distinct pathways in the regulation of bone volume with Cry2 influencing mostly the osteoclastic cellular component of bone and Per2 acting on osteoblast parameters.

Objective: Critical size defects (CSDs) of bone are defined as defects that do not heal spontaneously to new bone during the lifetime of an adult individual. In contrast, immature animals are capable to heal defects of identical size. It was our hypothesis that age-related paracrine effects are relevant for this difference in regeneration., Methods: The pooled supernatant of primary rat calvarial osteoblast-like cell cultures (POBC) derived from prenatal or postnatal donors was concentrated and applied into CSDs of adult recipient organisms (n = 10). In addition, the supernatant of POBC derived from prenatal donors was pooled and purified by reverse-phase chromatography. Each pre-purified fraction was tested in a proliferation indicating bioassay. Peptide fractions containing proliferative activities were re-chromatographed and re-tested in a bioassay. Finally, a proliferative activity was purified, identified by sequence analysis and applied into CSDs of adult recipients., Results: The application of POBC derived from prenatal donors resulted in osseous regeneration of a CSD in adult recipients, while the supernatant of postnatal donors had much smaller effects. The morphologic features resembled the spontaneous osseous healing of calvarial defects of the same size in immature organisms. The polypeptide "tissue inhibitor of metalloproteinases type II"(TIMP-2) was isolated from the supernatant of cultures of POBC derived from prenatal donors by measuring the induction of their proliferation. Additionally, the application of human TIMP-2 injected into calvarial CSDs of adult organisms resulted in osseous healing., Conclusion: We conclude that one component responsible for the healing effect of CSDs of POBC supernatants derived from prenatal donors is TIMP-2.

Since its discovery glucagon-like peptide-1 (GLP-1) is investigated as a treatment for type II diabetes based on its major function as insulin secretagogue. A therapeutic use is, however, limited by its short biological half-life in the range of minutes, predominantly caused via degradation catalyzed by dipeptidyl peptidase IV (DPP-IV). Therefore, we aimed to design a GLP-1 analogue exhibiting resistance against DPP-IV-catalyzed inactivation while retaining its biological activity. By means of matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) we have studied the stability of the N-terminally blocked new analogue Ac-GLP-1-(7-34)-amide against DPP-IV and compared it with both unblocked GLP-1-(7-34)-amide and the major naturally occurring form GLP-1-(7-36)-amide. GLP-1-(7-36)-amide and the C-terminally two amino acid residues shorter GLP-1-(7-34)-amide rapidly generated peptide fragments truncated by the N-terminal dipeptide. In contrast, the N-terminal blocked Ac-GLP-1-(7-34)-amide was not degraded in the presence of DPP-IV over a period of at least two hours. Ac-GLP-1-(7-34)-amide induced a concentration-dependent increase of intracellular cAMP production and insulin release from rat insulinoma RIN-m5F cells to an extent comparable to that found for the N-terminally unblocked peptides GLP-1-(7-34)-amide and GLP-1-(7-36)-amide. Ac-GLP-1-(7-34)-amide may thus have the potential to act as a new long-acting GLP-1 analogue with significant resistance against DPP-IV and retained biological activity in vitro. Further research is required to investigate whether Ac-GLP-1-(7-34)-amide also exhibits its characteristics in animal models and humans.

The anticipation of day length and duration of darkness is necessary and advantageous for animals to survive and requires a photoperiodic memory. In the Syrian hamster this adaptation to photoperiod is mirrored by seasonal changes in the animal's reproductive state and its liver metabolism. Both events are linked to season-dependent alterations of the nocturnally elevated synthesis of the pineal hormone melatonin. To decipher molecules that are involved in this temporal gating, hamsters were exposed to long photoperiod (16 hr light:8 hr darkness; LP), or short photoperiod (8 hr light:16 hr darkness; SP). Dynamics in gene expression was investigated in the pineal gland [inducible cAMP early repressor (ICER)], and in the liver (ICER; C/EBPdelta; clock genes) using immunochemistry and reverse transcriptase PCR. While in the pineal, ICER rhythms tightly follow the prior duration of light and dark with decreasing levels at the beginning of the dark period in both LP and SP, ICER is not rhythmic in liver. In the liver, clock genes and their protein products reflect differences in photoperiodic history, with enhanced rhythm amplitudes of PER, CRY, CLOCK, and BMAL1 under SP conditions. Thus, in the Syrian hamster transcription factor expression patterns lock onto the prevailing photoperiod in two peripheral oscillators, the pineal gland and the liver, to function as mediators of suprachiasmatic nucleus-derived information on environmental light and dark. This tissue-specific gating in gene transcription represents a strategy to ameliorate consequences of altering environmental lighting conditions on endocrine and metabolic parameters that endow a strong circadian bias.

In the mammalian pineal gland, information on environmental lighting conditions that is neuronally encoded by the retina is converted into nocturnally elevated synthesis of the hormone melatonin. Evolutionary pressure has changed the morphology of vertebrate pinealocytes, eliminating direct photoreception and the endogenous clock function. Despite these changes, nocturnally elevated melatonin synthesis has remained a reliable indicator of time throughout evolution. In the photo-insensitive mammalian pineal gland this message of darkness depends on the master circadian pacemaker in the hypothalamic suprachiasmatic nuclei. The dramatic change in vertebrate pinealocytes has received little attention; here, we therefore link the known evolutionary morphodynamics and well-investigated biochemical details responsible for rhythmic synthesis of melatonin with recently characterized patterns of gene expression in the pineal gland. We also address the enigmatic function of clockwork molecules in mammalian pinealocytes.

The circulating hormonal form of human parathyroid hormone (hPTH-1-37) has been assessed in vitro as well as in vivo in the ovariectomized rat, a model for postmenopausal osteoporosis. In vitro, hPTH-1-37 induces a dose-dependent cAMP formation and increases vitality as well as alkaline phosphatase activity in UMR106 osteosarcoma cells. Differentiation and proliferation of osteoclasts in rat bone marrow-derived stem cell preparations are decreased. Daily hPTH-1-37 s.c. administration in ovariectomized rats for 60 days results in augmented formation of new bone, in amplified femural bone density, and in thickening of the calvaria.

Circadian clocks are self-sustained biochemical oscillators that autonomously generate a near-24 h cycle in the absence of external signals. The process of synchronization to the environment involves the transcriptional activation of several genes. Photic input signals from the retina are transduced via the retinohypothalamic tract to the central pacemaker located in the suprachiasmatic nuclei (SCN) of the hypothalamus. It is known that cells of peripheral organs possess similar molecular organizations, but the signal transductional pathways lack direct light entrainment. It has been assumed that the adaptation of peripheral organs to the SCN phase is achieved by the alternate usage of promoter elements. This question has been addressed by characterizing the signal transductional pathways regulating human Period-1 gene expression in human hepatoma cells (HuH-7). Plasmids coding for key modulators of circadian rhythm, hCLOCK, hBMAL1, and hCRY2 were used to analyze the activation of a human period-1 promoter luciferase (hPER1-luc) construct. Beside classical CLOCK/BMAL1 activation, hPER1-luc was also inducible by the overexpression of the catalytic subunit of PKA (Calpha). The cotransfection of dominant negative constructs to c-FOS, CREB, PKA, and C/EBP were used to characterize both regulatory pathways. It was found that hCLOCK/hBMAL1-mediated hPER1 activation was influenced by AP1, but not significantly by other regulators. Conversely, PKA-induced activation of hPER1 was reduced by the inhibition of CREB and the CCAAT-box binding protein C/EBP, but not by AP1. The present findings imply that CLOCK/BMAL1-mediated activation of hPER1 by AP1 and E-Box elements is distinct from peripheral transcriptional modulation via cAMP-induced CREB and C/EBP.

Defensins are a family of secreted antimicrobial peptides proposed to directly interfere with bacterial membranes. Here we show a functional analysis of the novel beta-defensin DEFB123. A peptide comprising the beta-defensin core region was synthesized and used for our analysis. Like other beta-defensins, DEFB123 exerted antimicrobial activity against a broad spectrum of Gram-positive and Gram-negative bacteria, which was assessed by microbroth dilution assay and radial diffusion zone assay. In addition, the peptide showed lipopolysaccharide (LPS)-binding activity in a Limulus amoebocyte lysate (LAL) assay. Moreover, DEFB123 prevented LPS-induced tumor necrosis factor (TNF)-alpha secretion in a murine monocyte cell line (RAW264.7). Accordingly, DEFB123 abolished LPS-mediated MAPK induction in these cells. Protection against LPS-mediated effects was then investigated in a murine model of acute sepsis. Our experiments show that synthetic beta-defensin DEFB123 prevents LPS-induced mortality in C57BL/6 mice in a therapeutic approach. We propose that the physiological role of beta-defensins may include interference with LPS-action on macrophages, a function formerly thought to be restricted to the family of cathelicidins, a structurally unrelated group of antimicrobial peptides.

Background: Urodilatin (ularitide), a natriuretic peptide, is produced within the kidneys. The aim of this study was to define the role of 24-hour intravenous infusions of urodilatin in the treatment of decompensated chronic heart failure (DHF)., Methods: In this randomized, double-blind, ascending-dose safety study, 24 patients with DHF (cardiac index 1.91 +/- 0.34 L/min per square meter, pulmonary capillary wedge pressure 26 +/- 6 mm Hg, right atrial pressure 11 +/- 4 mm Hg) received urodilatin (7.5, 15, or 30 ng/(kg.min)) or placebo infusions over 24 hours., Results: Compared with baseline, urodilatin decreased pulmonary capillary wedge pressure by 10 mm Hg in the 15 ng/(kg.min) group (P < .05) and by 15 mm Hg in the 30 ng/(kg.min) group (P < .05) at 6 hours. In the same dose groups, right atrial pressure decreased, and dyspnea as reported by patients tended to improve. At 24 hours, 15 and 30 ng/(kg.min) urodilatin infusions decreased N-terminal-pro-brain natriuretic peptide levels by 40% and 45%, respectively, compared with baseline. Between 1 to 12 hours, plasma cyclic guanosine monophosphate levels at 15 and 30 ng/(kg.min) urodilatin were significantly higher than both placebo and the respective baseline after infusion start (P < .05 and .01). Among the different groups, there was no obvious difference regarding total number of patients with adverse events and total number of adverse events. During infusion, 3 transient asymptomatic hypotensions occurred in the urodilatin groups., Conclusions: Our findings show that urodilatin may be a new agent for the therapy for DHF.

Reverse pharmacology is a screening technology that matches G protein-coupled receptors (GPCRs) with unknown cognate ligands in cell-based screening assays by detection of agonist-induced signaling pathways. One decade spent pursuing orphan GPCR screening by this technique assigned over 30 ligand/receptor pairs and revealed previously known or novel undescribed ligands, mostly of a peptidic nature. In this review, we describe the discovery, characterization of the structural composition, biological function, physiological role and therapeutic potential of three recently identified peptidic ligands. These are metastin, QRFP in a context of five RF-amide genes described in humans and the chemoattractant, chemerin. Metastin was initially characterized as a metastasis inhibitor. Investigations using ligand/receptor pairing revealed that metastin was involved in a variety of physiological processes, including endocrine function during pregnancy and gonad development. The novel RF-amide QRFP is implicated in food intake and aldosterone release from the adrenal cortex in the rat. Chemerin, first described as TIG2, is upregulated in tazarotene-treated psoriatic skin. By GPCR screening, bioactive chemerin was isolated from ovarial carcinoma fluid as well as hemofiltrate. Characterization as a chemoattractant for immature dendritic cells and analysis of the expression profile of metastin and its receptor suggested a physiological role of chemerin as a mediator of the immune response, inflammatory processes and bone development.

We report the isolation of a novel human circulating proopiomelanocortin-derived peptide, VA-beta-MSH, from hemofiltrate and its pharmacological characterization. Screening for lipolytic activity in differentiated 3T3-L1 adipocytes led to the isolation from a hemofiltrate peptide library by alternating reverse phase and cation exchange chromatography. In the course of this isolation, we also identified human beta-MSH-(1-22). We synthesized VA-beta-MSH by the N-(9-fluorenyl)-methoxycarbonyl (F-moc) solid phase method and used synthetic beta-MSH-(1-22) to confirm that both isolated peptides are lipolytically active in a dose-dependent manner in differentiated 3T3-L1 adipocytes in the nanomolar range. Using cAMP ELISA, we demonstrate that stimulation with both peptides caused a strong cAMP elevation in this cell system. Furthermore, we show that the selective inhibitors of cAMP-dependent protein kinase, 8-(4-Chlorophenylthio)adenosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-8-CPT-cAMPS); N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89), significantly reduce VA-beta-MSH- and beta-MSH-(1-22)-mediated lipolysis. Although isolated after its lipolytic activity on 3T3-L1 cells, this newly identified circulating human melanocortin may serve other functions in human physiology. Moreover, the fact that these peptides have been identified after a functional assay, but have been overseen in large proteomic approaches, underscores the importance of such approaches in identifying previously undescribed circulating bioactive molecules.