Amplitude and frequency modulation of pulsatile luteinizing hormone-releasing hormone release

Summary 1. A variety of neuroendocrine approaches has been used to characterize cellular mechanisms governing luteinizing hormone-releasing hormone (LHRH) pulse generation. We review recentin vivo microdialysis,in vitro superfusion, andin situ hybridization experiments in which we tested the hypothesis that the amplitude and frequency of LHRH pulses are subject to independent regulation via distinct and identifiable cellular pathways.2. Augmentation of LHRH pulse amplitude is proposed as a central feature of preovulatory LHRH surges. Three mechanisms are described which may contribute to this increase in LHRH pulse amplitude: (a) increased LHRH gene expression, (b) augmentation of facilitatory neurotransmission, and (c) increased responsiveness of LHRH neurons to afferent synaptic signals. Neuropeptide Y (NPY) is examined as a prototypical afferent transmitter regulating the generation of LHRH surges through the latter two mechanisms.3. Retardation of LHRH pulse generator frequency is postulated to mediate negative feedback actions of gonadal hormones. Evidence supporting this hypothesis is reviewed, including results ofin vivo monitoring experiments in which LHRH pulse frequency, but not amplitude, is shown to be increased following castration. A role for noradrenergic neurons as intervening targets of gonadal hormone negative feedback actions is discussed.4. Future directions for study of the LHRH pulse generator are suggested.     

Molecular Characterization of Three Canine Models of Human Rare Bone Diseases: Caffey,van den Ende-Gupta,and Raine Syndromes

One to two percent of all children are born with a developmental disorder requiring pediatric hospital admissions. For many such syndromes, the molecular pathogenesis remains poorly characterized. Parallel developmental disorders in other species could provide complementary models for human rare diseases by uncovering new candidate genes, improving the understanding of the molecular mechanisms and opening possibilities for therapeutic trials. We performed various experiments, e.g. combined genome-wide association and next generation sequencing, to investigate the clinico-pathological features and genetic causes of three developmental syndromes in dogs, including craniomandibular osteopathy (CMO), a previously undescribed skeletal syndrome, and dental hypomineralization, for which we identified pathogenic variants in the canine SLC37A2 (truncating splicing enhancer variant), SCARF2 (truncating 2-bp deletion) and FAM20C (missense variant) genes, respectively. CMO is a clinical equivalent to an infantile cortical hyperostosis (Caffey disease), for which SLC37A2 is a new candidate gene. SLC37A2 is a poorly characterized member of a glucose-phosphate transporter family without previous disease associations. It is expressed in many tissues, including cells of the macrophage lineage, e.g. osteoclasts, and suggests a disease mechanism, in which an impaired glucose homeostasis in osteoclasts compromises their function in the developing bone, leading to hyperostosis. Mutations in SCARF2 and FAM20C have been associated with the human van den Ende-Gupta and Raine syndromes that include numerous features similar to the affected dogs. Given the growing interest in the molecular characterization and treatment of human rare diseases, our study presents three novel physiologically relevant models for further research and therapy approaches, while providing the molecular identity for the canine conditions.     

BVOC Emissions From a Subarctic Ecosystem,as Controlled by Insect Herbivore Pressure and Temperature

Ecosystems - The biogenic volatile organic compounds, BVOCs have a central role in ecosystem–atmosphere interactions. High-latitude ecosystems are facing increasing temperatures and insect...     

Glycosylation, transport, and complex formation of palmitoyl protein thioesterase 1 (PPT1) – distinct characteristics in neurons

Background

About 20 % of nemaline myopathies are thus far related to skeletal muscle alpha-actin. Seven actin mutants located in different parts of the actin molecule and linked to different forms of the disease were selected and expressed as EGFP-tagged constructs in differentiated C2C12 mytoubes. Results were compared with phenotypes in patient skeletal muscle fibres and with previous expression studies in fibroblasts and C2C12 myoblasts/myotubes.

Results

Whereas EGFP wt-actin nicely incorporated into endogenous stress fibres and sarcomeric structures, the mutants showed a range of phenotypes, which generally changed upon differentiation. Many mutants appeared delocalized in myoblasts but integrated into endogenous actin structures after 4–6 days of differentiation, demonstrating a poor correlation between the appearance in myotubes and the severity of the disease. However, for some mutants, integration into stress fibres induced aberrant structures in differentiated cells, like thickening or fragmentation of stress fibres. Other mutants almost failed to integrate but formed huge aggregates in the cytoplasm of myotubes. Those did not co-stain with alpha-actinin, a main component of nemaline bodies found in patient muscle. Interestingly, nuclear aggregates as formed by two of the mutants in myoblasts were found less frequently or not at all in differentiated cells.

Conclusion

Myotubes are a suitable system to study the capacity of a mutant to incorporate into actin structures or to form or induce pathological changes. Some of the phenotypes observed in undifferentiated myoblasts may only be in vitro effects. Other phenotypes, like aberrant stress fibres or rod formation may be more directly correlated with disease phenotypes. Some mutants did not induce any changes in the cellular actin system, indicating the importance of additional studies like functional assays to fully characterize the pathological impact of a mutant.     

Neuronostatin, a novel peptide encoded by somatostatin gene, regulates cardiac contractile function and cardiomyocyte survival

Neuronostatin, a recently discovered peptide encoded by somatostatin gene, is involved in regulation of neuronal function, blood pressure, food intake, and drinking behavior. However, the biological effects of neuronostatin on cardiac myocytes are not known, and the intracellular signaling mechanisms induced by neuronostatin remain unidentified. We analyzed the effect of neuronostatin in isolated perfused rat hearts and in cultured primary cardiomyocytes. Neuronostatin infusion alone had no effect on left ventricular (LV) contractile function or on isoprenaline- or preload-induced increase in cardiac contractility. However, infusion of neuronostatin significantly decreased the positive inotropic response to endothelin-1 (ET-1). This was associated with an increase in phosphorylation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase (JNK). Treatment of both neonatal and adult cardiomyocytes with neuronostatin resulted in reduced cardiomyocyte viability. Inhibition of JNK further increased the neuronostatin-induced cell death. We conclude that neuronostatin regulates cardiac contractile function and cardiomyocyte survival. Receptors for neuronostatin need to be identified to further characterize the biological functions of the peptide.     

Distinct subcellular localization for constitutive and agonist-modulated palmitoylation of the human delta opioid receptor

Protein palmitoylation is a reversible lipid modification that plays important roles for many proteins involved in signal transduction, but relatively little is known about the regulation of this modification and the cellular location where it occurs. We demonstrate that the human delta opioid receptor is palmitoylated at two distinct cellular locations in human embryonic kidney 293 cells and undergoes dynamic regulation at one of these sites. Although palmitoylation could be readily observed for the mature receptor (Mr 55,000), [3H]palmitate incorporation into the receptor precursor (Mr 45,000) could be detected only following transport blockade with brefeldin A, nocodazole, and monensin, indicating that the modification occurs initially during or shortly after export from the endoplasmic reticulum. Blocking of palmitoylation with 2-bromopalmitate inhibited receptor cell surface expression, indicating that it is needed for efficient intracellular transport. However, cell surface biotinylation experiments showed that receptors can also be palmitoylated once they have reached the plasma membrane. At this location, palmitoylation is regulated in a receptor activation-dependent manner, as was indicated by the opioid agonist-promoted increase in the turnover of receptor-bound palmitate. This agonist-mediated effect did not require receptor-G protein coupling and occurred at the cell surface without the need for internalization or recycling. The activation-dependent modulation of receptor palmitoylation may thus contribute to the regulation of receptor function at the plasma membrane.     

Interactive effect of elevated temperature and O<Subscript>3</Subscript> on antioxidant capacity and gas exchange in <Emphasis Type="Italic">Betula pendula</Emphasis> saplings

We studied the effects of slightly elevated temperature (T), O₃ concentration (O₃) and their combination (T + O₃) on the antioxidant defense, gas exchange and total leaf area of Betula pendula saplings in field conditions. During the second year of the experiment, T enhanced the total leaf area, net photosynthesis (P _n) and maximum capacity of carboxylation, redox state of ascorbate and total antioxidant capacity in the apoplast. O₃ did not affect the total leaf area, but P _n was slightly and g _s significantly reduced. The saplings responded to elevated O₃ level by closing the stomata and by developing leaves with a lower leaf area per mass, rather than by accumulating ascorbate in the apoplast. The effects of T and O₃ on total leaf area and P _n were counteractive. Elevated O₃ reduced the saplings’ ability to utilize the warmer growth environment by increasing the stomatal limitation for photosynthesis and by reducing the redox state of ascorbate in the apoplast in the combination treatment as compared to T alone.     

N-Glycosylation Regulates Fibroblast Growth Factor Receptor/EGL-15 Activity in Caenorhabditis elegans in Vivo

The regulation of cell function by fibroblast growth factors (FGFs) classically occurs through a dual receptor system of a tyrosine kinase receptor (FGFR) and a heparan sulfate proteoglycan co-receptor. Mutations in some consensus N-glycosylation sites in human FGFR result in skeletal disorders and craniosynostosis syndromes, and biophysical studies in vitro suggest that N-glycosylation of FGFR alters ligand and heparan sulfate binding properties. The evolutionarily conserved FGFR signaling system of Caenorhabditis elegans has been used to assess the role of N-glycosylation in the regulation of FGFR signaling in vivo. The C. elegans FGF receptor, EGL-15, is N-glycosylated in vivo, and genetic substitution of specific consensus N-glycosylation sites leads to defects in the maintenance of fluid homeostasis and differentiation of sex muscles, both of which are phenotypes previously associated with hyperactive EGL-15 signaling. These phenotypes are suppressed by hypoactive mutations in EGL-15 downstream signaling components or activating mutations in the phosphatidylinositol 3-kinase pathway, respectively. The results show that N-glycans negatively regulate FGFR activity in vivo supporting the notion that mutation of N-glycosylation sites in human FGFR may lead to inappropriate activation of the receptor.