:  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.

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Novel (Rp)-cAMPS analogs differed widely in ability to antagonize cAMP activation of pure cAMP-dependent protein kinase I and II and to antagonize actions of cAMP on gene expression, shape change, apoptosis, DNA replication, and protein phosphorylation in intact cells. These differences were related to different abilities of the analogs to stabilize the holoenzyme form relative to the dissociated form of cAMP kinase type I and II. (Rp)-8-Br-cAMPS and (Rp)-8-Cl-cAMPS were the most potent cAMP antagonists for isolated type I kinase and for cells expressing mostly type I kinase, like IPC-81 leukemia cells, fibroblasts transfected with type I regulatory subunit (RI), and primary hepatocytes. It is proposed that (Rp)-8-Br-cAMPS or (Rp)-8-Cl-cAMPS should replace (Rp)-cAMPS as the first line cAMP antagonist, particularly for studies in cells expressing predominantly type I kinase. The phosphorylation of endogenous hepatocyte proteins was affected oppositely by (Rp)-8-Br-cAMPS and increased cAMP, indicating that (Rp)-8-Br-cAMPS inhibited basal cAMP-kinase activity. The inhibition of basal kinase activity was accompanied by enhanced DNA replication, an effect which could be reproduced by microinjected mutant cAMP-subresponsive RI. It is concluded that the basal cAMP-kinase activity exerts a tonic inhibition of hepatocyte replication. (Rp)-8-Br-cAMPS and microinjected RI also desensitized hepatocytes toward inhibition of DNA synthesis by interleukin-1 beta. This indicates that basal cAMP-kinase activity can have a permissive role for the action of another (interleukin-1 beta) signaling pathway.

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The human genome contains numerous genes whose protein products are unknown in terms of structure, interaction partner, expression, and function. To unravel the function of these orphan genes, it is of particular value to isolate native forms of protein and peptide products derived from these genes. From human blood ultrafiltrate, we characterized a novel gene-encoded, cysteine-rich, and cationic peptide that we termed liver-expressed antimicrobial peptide 2 (LEAP-2). We identified several circulating forms of LEAP-2 differing in their amino-terminal length, all containing a core structure with two disulfide bonds formed by cysteine residues in relative 1-3 and 2-4 positions. Molecular cloning of the cDNA showed that LEAP-2 is synthesized as a 77-residue precursor, which is predominantly expressed in the liver and highly conserved among mammals. This makes it a unique peptide that does not exhibit similarity with any known human peptide regarding its primary structure, disulfide motif, and expression. Analysis of the LEAP-2 gene resulted in the identification of an alternative promoter and at least four different splicing variants, with the two dominating transcripts being tissue-specifically expressed. The largest native LEAP-2 form of 40 amino acid residues is generated from the precursor at a putative cleavage site for a furin-like endoprotease. In contrast to smaller LEAP-2 variants, this peptide exhibited dose-dependent antimicrobial activity against selected microbial model organisms. LEAP-2 shares some characteristic properties with classic peptide hormones and it is expected that the isolation of this novel peptide will help to unravel its physiological role.Keywords: Alternative splicing; antimicrobial activity; disulfide bonds; hemofiltrate; liver; peptide; secretion As a consequence of the efforts to sequence and assemble the human genome (Lander et al. 2001;Venter et al. 2001), the systematic analysis of peptides and proteins as the functional gene products produced by a given cell population or tissue under defined conditions is considered to be the next milestone in molecular biology. The estimated number of genes is unexpectedly low, and the number of biologically active peptides and proteins cannot be deduced from these data because of events such as alternative splicing of mRNA precursors, usage of alternative gene promoters, pseudogenes, and alternatively processed proteins. The number of proteins is therefore estimated to be two to three orders of magnitude higher than the number of ∼40,000 genes annotated in the human genome (Harrison et al. 2002;Rappsilber and Mann 2002). Therefore, the mass-spectrometric identification of a gene product in proteomics or, even better, the isolation of novel proteins and peptides followed by a struc-5 Reprint requests to: Knut Adermann, IPF PharmaCeuticals GmbH, Hannover, Germany; e-mail: knut.adermann@ipf-pharmaceuticals.de; fax: 49 (0) 511 5466 132.Abbreviations: CFU, colony-forming unit; ESIMS, electrospray ionization mass spectrometry; LEAP, liver...

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BackgroundClock 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 FindingsWe found that Per2Brdm1 mutant mice as well as mice lacking Cry2−/− displayed significantly increased bone volume at 12 weeks of age, when bone turnover is high. Per2Brdm1 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/SignificanceThis 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.

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.