Regulation of extracellular calcium sensing in rat osteoclasts by femtomolar calcitonin concentrations

Certain eukaryotic cells can sense changes in their extracellular Ca2+ concentration through molecular structures termed Ca(2+)-sensing receptors (CaRs). We have shown recently that in the bone-resorbing osteoclast, a unique cell surface-expressed ryanodine receptor (RyR), functions as the CaR. The present study demonstrates that the sensitivity of this receptor is modulated by physiological femtomolar concentrations of the bone-conserving hormone, calcitonin. Calcitonin was found to inhibit cytosolic Ca2+ responses to both Ca2+ and Ni2+. The latter inhibition was mimicked by amylin (10(-12) M), calcitonin gene-related peptide (10(-12) M), cholera toxin (5 micrograms/l) and dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP) (2.5 x 10(-4) or 5 x 10(-4) M) and was reversed by the protein kinase A phosphorylation inhibitor, IP-20. Finally, using a quench flow module, we showed that cellular cAMP levels rise to a peak within 25 ms of calcitonin application; this is consistent with the peptide's rapid effect on CaR activation. We conclude, therefore, that cAMP plays a critical role in the control of CaR function by calcitonin.

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Novel Template Plasmids pCyaA’-Kan and pCyaA’-Cam for Generation of Unmarked Chromosomal cyaA’ Translational Fusion to T3SS Effectors in Salmonella

Microorganisms ◽

10.3390/microorganisms9030475 ◽

2021 ◽

Vol 9 (3) ◽

pp. 475

Author(s):

Paulina A. Fernández ◽

Marcela Zabner ◽

Jaime Ortega ◽

Constanza Morgado ◽

Fernando Amaya ◽

...

Keyword(s):

Fusion Protein ◽

Western Blot ◽

Eukaryotic Cells ◽

Intracellular Camp ◽

Gene Fusions ◽

Secretion Systems ◽

Translational Fusion ◽

Flp Recombinase ◽

Recombination System ◽

Camp Levels

The type III secretion systems (T3SS) encoded in pathogenicity islands SPI-1 and SPI-2 are key virulence factors of Salmonella. These systems translocate proteins known as effectors into eukaryotic cells during infection. To characterize the functionality of T3SS effectors, gene fusions to the CyaA’ reporter of Bordetella pertussis are often used. CyaA’ is a calmodulin-dependent adenylate cyclase that is only active within eukaryotic cells. Thus, the translocation of an effector fused to CyaA’ can be evaluated by measuring cAMP levels in infected cells. Here, we report the construction of plasmids pCyaA’-Kan and pCyaA’-Cam, which contain the ORF encoding CyaA’ adjacent to a cassette that confers resistance to kanamycin or chloramphenicol, respectively, flanked by Flp recombinase target (FRT) sites. A PCR product from pCyaA’-Kan or pCyaA’-Cam containing these genetic elements can be introduced into the bacterial chromosome to generate gene fusions by homologous recombination using the Red recombination system from bacteriophage λ. Subsequently, the resistance cassette can be removed by recombination between the FRT sites using the Flp recombinase. As a proof of concept, the plasmids pCyaA’-Kan and pCyaA’-Cam were used to generate unmarked chromosomal fusions of 10 T3SS effectors to CyaA’ in S. Typhimurium. Each fusion protein was detected by Western blot using an anti-CyaA’ monoclonal antibody when the corresponding mutant strain was grown under conditions that induce the expression of the native gene. In addition, T3SS-1-dependent secretion of fusion protein SipA-CyaA’ during in vitro growth was verified by Western blot analysis of culture supernatants. Finally, efficient translocation of SipA-CyaA’ into HeLa cells was evidenced by increased intracellular cAMP levels at different times of infection. Therefore, the plasmids pCyaA’-Kan and pCyaA’-Cam can be used to generate unmarked chromosomal cyaA’ translational fusion to study regulated expression, secretion and translocation of Salmonella T3SS effectors into eukaryotic cells.

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Importance of Adult Dmbx1 in Long-Lasting Orexigenic Effect of Agouti-Related Peptide

Endocrinology ◽

10.1210/en.2015-1560 ◽

2016 ◽

Vol 157 (1) ◽

pp. 245-257 ◽

Cited By ~ 1

Author(s):

Seiichiro Hirono ◽

Eun Young Lee ◽

Shunsuke Kuribayashi ◽

Takahiro Fukuda ◽

Naokatsu Saeki ◽

...

Keyword(s):

Mouse Strain ◽

Energy Homeostasis ◽

Expression Patterns ◽

Critical Role ◽

Fetal Brain ◽

Cre Recombinase ◽

Calcitonin Gene Related Peptide ◽

Adult Brain ◽

Related Peptide ◽

Calcitonin Gene

Abstract Dmbx1 is a brain-specific homeodomain transcription factor expressed primarily during embryogenesis, and its systemic disruption (Dmbx1−/−) in the ICR mouse strain resulted in leanness associated with impaired long-lasting orexigenic effect of agouti-related peptide (AgRP). Because spatial and temporal expression patterns of Dmbx1 change dramatically during embryogenesis, it remains unknown when and where Dmbx1 plays a critical role in energy homeostasis. In the present study, the physiological roles of Dmbx1 were examined by its conditional disruption (Dmbx1loxP/loxP) in the C57BL/6 mouse strain. Although Dmbx1 disruption in fetal brain resulted in neonatal lethality, its disruption by synapsin promoter-driven Cre recombinase, which eliminated Dmbx1 expression postnatally, exempted the mice (Syn-Cre;Dmbx1loxP/loxP mice) from lethality. Syn-Cre;Dmbx1loxP/loxP mice show mild leanness and impaired long-lasting orexigenic action of AgRP, demonstrating the physiological relevance of Dmbx1 in the adult. Visualization of Dmbx1-expressing neurons in adult brain using the mice harboring tamoxifen-inducible Cre recombinase in the Dmbx1 locus (Dmbx1CreERT2/+ mice) revealed Dmbx1 expression in small numbers of neurons in restricted regions, including the lateral parabrachial nucleus (LPB). Notably, c-Fos expression in LPB was increased at 48 hours after AgRP administration in Dmbx1loxP/loxP mice but not in Syn-Cre;Dmbx1loxP/loxP mice. These c-Fos-positive neurons in LPB did not coincide with neurons expressing Dmbx1 or melanocortin 4 receptor but did coincide with those expressing calcitonin gene-related peptide. Accordingly, Dmbx1 in the adult LPB is required for the long-lasting orexigenic effect of AgRP via the neural circuitry involving calcitonin gene-related peptide neurons.

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patched overexpression alters wing disc size and pattern: transcriptional and post-transcriptional effects on hedgehog targets

Development ◽

10.1242/dev.121.12.4161 ◽

1995 ◽

Vol 121 (12) ◽

pp. 4161-4170 ◽

Cited By ~ 26

Author(s):

R.L. Johnson ◽

J.K. Grenier ◽

M.P. Scott

Keyword(s):

Protein Kinase ◽

Protein Kinase A ◽

Target Genes ◽

Critical Role ◽

Wing Disc ◽

Wing Vein ◽

Anterior Compartment ◽

Protein Levels ◽

Anterior Posterior ◽

Kinase A

The membrane protein, Patched, plays a critical role in patterning embryonic and imaginal tissues in Drosophila. patched constitutively inactivates the transcription of target genes such as wingless, decapentaplegic, and patched itself. The secreted protein, Hedgehog, induces transcription of target genes by opposing the Patched signaling pathway. Using the Gal4 UAS system we have overexpressed patched in wing imaginal discs and found that high Patched levels, expressed in either normal or ectopic patterns, result in loss of wing vein patterning in both compartments centering at the anterior/posterior border. In addition, patched inhibits the formation of the mechanosensory neurons, the campaniform sensilla, in the wing blade. The patched wing vein phenotype is modulated by mutations in hedgehog and cubitus interruptus (ci). Patched overexpression inhibits transcription of patched and decapentaplegic and post-transcriptionally decreases the amount of Ci protein at the anterior/posterior boundary. In hedgehogMrt wing discs, which express ectopic hedgehog, Ci levels are correspondingly elevated, suggesting that hedgehog relieves patched repression of Ci accumulation. Protein kinase A also regulates Ci; protein kinase A mutant clones in the anterior compartment have increased levels of Ci protein. Thus patched influences wing disc patterning by decreasing Ci protein levels and inactivating hedgehog target genes in the anterior compartment.

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B-Raf-dependent regulation of the MEK-1/mitogen-activated protein kinase pathway in PC12 cells and regulation by cyclic AMP

Molecular and Cellular Biology ◽

10.1128/mcb.14.10.6522-6530.1994 ◽

1994 ◽

Vol 14 (10) ◽

pp. 6522-6530

Author(s):

R R Vaillancourt ◽

A M Gardner ◽

G L Johnson

Keyword(s):

Growth Factor ◽

Protein Kinase ◽

Map Kinase ◽

Protein Kinase A ◽

Pc12 Cells ◽

Kinase Activation ◽

Mitogen Activated Protein ◽

Epidermal Growth ◽

Camp Levels ◽

Kinase A

Growth factor receptor tyrosine kinase regulation of the sequential phosphorylation reactions leading to mitogen-activated protein (MAP) kinase activation in PC12 cells has been investigated. In response to epidermal growth factor, nerve growth factor, and platelet-derived growth factor, B-Raf and Raf-1 are activated, phosphorylate recombinant kinase-inactive MEK-1, and activate wild-type MEK-1. MEK-1 is the dual-specificity protein kinase that selectively phosphorylates MAP kinase on tyrosine and threonine, resulting in MAP kinase activation. B-Raf and Raf-1 are growth factor-regulated Raf family members which regulate MEK-1 and MAP kinase activity in PC12 cells. Protein kinase A activation in response to elevated cyclic AMP (cAMP) levels inhibited B-Raf and Raf-1 stimulation in response to growth factors. Ras.GTP loading in response to epidermal growth factor, nerve growth factor, or platelet-derived growth factor was unaffected by protein kinase A activation. Even though elevated cAMP levels inhibited Raf activation, the growth factor activation of MEK-1 and MAP kinase was unaffected in PC12 cells. The results demonstrate that tyrosine kinase receptor activation of MEK-1 and MAP kinase in PC12 cells is regulated by B-Raf and Raf-1, whose activation is inhibited by protein kinase A, and MEK activators, whose activation is independent of cAMP regulation.

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Protein kinase A mediates activation of ATP-sensitive K+ currents by CGRP in gallbladder smooth muscle

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1994.267.3.g494 ◽

1994 ◽

Vol 267 (3) ◽

pp. G494-G499 ◽

Cited By ~ 10

Author(s):

L. Zhang ◽

A. D. Bonev ◽

G. M. Mawe ◽

M. T. Nelson

Keyword(s):

Smooth Muscle ◽

Protein Kinase ◽

Protein Kinase A ◽

Whole Cell Patch Clamp ◽

Camp Analogue ◽

Calcitonin Gene ◽

Transduction Mechanisms ◽

Spontaneous Action ◽

Spontaneous Action Potentials ◽

Kinase A

The signal transduction mechanisms underlying the activation of ATP-sensitive potassium (KATP) current by calcitonin gene-related peptide (CGRP) in gallbladder smooth muscle were examined with intracellular microelectrode recording and whole cell patch-clamp techniques. In the intact gallbladder preparation, the adenylyl cyclase activator forskolin hyperpolarized the membrane potential and abolished spontaneous action potentials. This response was inhibited by the KATP channel blocker glibenclamide. CGRP (10 nM), forskolin (10 microM), the membrane-permeable adenosine 3',5'-cyclic monophosphate (cAMP) analogue adenosine 3',5'-cyclic monophosphothioate (Sp-cAMP[S]; 500 microM), and the catalytic subunit of protein kinase A (100 U/ml) activated glibenclamide-sensitive currents in enzymatically dissociated gallbladder smooth muscle cells. CGRP activation of potassium currents was prevented by dialysis of the cell cytoplasm with guanosine 5'-O-(2-thiodiphosphate) (5 mM) or a specific peptide inhibitor of protein kinase A (2.3 microM). Okadaic acid (5 microM), a phosphatase inhibitor, slowed the deactivation of the KATP current, following removal of CGRP. The results of this study indicate that CGRP hyperpolarizes gallbladder smooth muscle by elevation of cAMP and subsequent stimulation of protein kinase A.

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Cyclic AMP-dependent Protein Lysine Acylation in Mycobacteria Regulates Fatty Acid and Propionate Metabolism

Journal of Biological Chemistry ◽

10.1074/jbc.m113.463992 ◽

2013 ◽

Vol 288 (20) ◽

pp. 14114-14124 ◽

Cited By ~ 69

Author(s):

Subhalaxmi Nambi ◽

Kallol Gupta ◽

Moitrayee Bhattacharyya ◽

Parvathy Ramakrishnan ◽

Vaishnavi Ravikumar ◽

...

Keyword(s):

Critical Role ◽

Fatty Acyl ◽

Intracellular Camp ◽

Dependent Manner ◽

Multiple Substrates ◽

Propionate Metabolism ◽

Lysine Residues ◽

Dependent Protein ◽

First Time ◽

Camp Levels

Acetylation of lysine residues is a posttranslational modification that is used by both eukaryotes and prokaryotes to regulate a variety of biological processes. Here we identify multiple substrates for the cAMP-dependent protein lysine acetyltransferase from Mycobacterium tuberculosis (KATmt). We demonstrate that a catalytically important lysine residue in a number of FadD (fatty acyl CoA synthetase) enzymes is acetylated by KATmt in a cAMP-dependent manner and that acetylation inhibits the activity of FadD enzymes. A sirtuin-like enzyme can deacetylate multiple FadDs, thus completing the regulatory cycle. Using a strain deleted for the KATmt ortholog in Mycobacterium bovis Bacillus Calmette-Guérin (BCG), we show for the first time that acetylation is dependent on intracellular cAMP levels. KATmt can utilize propionyl CoA as a substrate and, therefore, plays a critical role in alleviating propionyl CoA toxicity in mycobacteria by inactivating acyl CoA synthetase (ACS). The precision by which mycobacteria can regulate the metabolism of fatty acids in a cAMP-dependent manner appears to be unparalleled in other biological organisms and is ideally suited to adapt to the complex environment that pathogenic mycobacteria experience in the host.

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Recent Advances in the Management of Cluster Headache

Current Treatment Options in Neurology ◽

10.1007/s11940-020-00655-z ◽

2020 ◽

Vol 22 (12) ◽

Author(s):

María Dolores Villar-Martínez ◽

Francesca Puledda ◽

Peter J. Goadsby

Keyword(s):

Cluster Headache ◽

Serotonin Receptor ◽

Critical Role ◽

Therapeutic Approaches ◽

New Era ◽

Novel Treatments ◽

Difficult Condition ◽

Calcitonin Gene ◽

Cgrp Receptor Antagonists ◽

Autonomic Reflex

Abstract Purpose of review Among the spectrum of pain conditions, cluster headache represents one of the most severe. Targeted therapies for cluster headache are evolving thus improving the available therapeutic armamentarium. A better understanding of the currently available therapies, as well as new and emerging options, may aide physicians to manage affected sufferers better by evolving treatment guidance. Recent findings While classic first-line medications are useful in some patients with cluster headache, they are often accompanied by significant side effects that limit their use. Recently, novel treatments with better tolerability and decreased medication interactions have proven to be effective. A remarkable example of this is the blockage of the calcitonin gene-related peptide pathway with monoclonal antibodies, which may be a key element in the future treatment of cluster headache. The sphenopalatine ganglion and vagus nerve perform a critical role in the regulation of pain and the trigeminal autonomic reflex. Neuromodulation therapies targeting these structures have shown excellent tolerability and few significant adverse events, constituting a promising form of treatment. Finally, several potential therapeutic targets are examined in this review, such as small molecule CGRP receptor antagonists, known as gepants, and serotonin receptor 5-HT1F receptor agonists: ditans. Summary In summary, a deepening of the understanding of cluster headache mechanisms in recent years has driven the evolution of sophisticated therapeutic approaches that could allow a new era in the treatment of this difficult condition.

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Erenumab in prophylaxis of migraine – pilot study

Ból ◽

10.5604/01.3001.0014.0233 ◽

2020 ◽

Vol 20 (4) ◽

pp. 1-4

Author(s):

Marcin Kopka

Keyword(s):

Chronic Migraine ◽

Primary Endpoint ◽

Migraine Headache ◽

Critical Role ◽

Preventive Treatment ◽

Tolerability Profile ◽

Good Tolerability ◽

New Class ◽

Calcitonin Gene

The aim of preventive treatment of episodic and chronic migraine is to reduce the frequency and severity of attacks and thereby improve patient’s quality of life. It is suggested that calcitonin gene related peptide (CGRP) plays a critical role in migraine patophysiology. In last few decades a new class of drugs was developed – monoclonal antibodies against CGRP. One of them is erenumab, which has been available in Poland since November 2018. The aim of this study was to assess the efficacy and tolerability of erenumab in migraine prophylaxis in polish patients with episodic and chronic migraine. Adult patients diagnosed with migraine (according to ICHD 3) with at least 4 migraine headache days in month were included in this study. The primary endpoint was the proportion of patients with more than 50% reduction in number of migraine headache days after the first month of treatment. From December 2018 to April 2019 9 women (24–66 years; average 43.8 years) were included. The average migraine headache days in this study group was 9 (5–20). The participants were treated with subcutaneously delivered erenumab in daily dose of 70 mg. After the first month of treatment in 7 from 9 women (77.5 %) primary endpoint was reached. In 3 of 9 (30%) patients itch was noted. The results of this study suggest efficacy and good tolerability profile of erenumab in polish patients with episodic and chronic migraine. The future study are needed with larger groups of participants.

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Surfaces and Interfaces of Nanoscale Silicon Materials

MRS Proceedings ◽

10.1557/opl.2013.609 ◽

2013 ◽

Vol 1550 ◽

Cited By ~ 6

Author(s):

Sean R. Wagner ◽

Pengpeng Zhang

Keyword(s):

Electrical Transport ◽

Rational Design ◽

Critical Role ◽

Volume Ratio ◽

Molecular Structures ◽

Prototype System ◽

Electrical Transport Properties ◽

Surface And Interface ◽

Surfaces And Interfaces ◽

Si Nanostructures

AbstractSurfaces and interfaces play a critical role in determining properties and functions of nanomaterials, in many cases dominating bulk properties, owing to the large surface- and interface-area-to-volume ratio. Using Si nanomembranes, a well-controlled two-dimensional single-crystalline semiconductor, as a prototype system, we discuss how surfaces and interfaces influence electrical transport properties at the nanoscale. We show that electronic conduction in Si nanomembranes is not determined by bulk dopants but by the interplay of surface and interface electronic structures with the “bulk” band structure of the thin Si membrane. Additionally, we describe our recent experimental results on the control of highly ordered molecular structures on Si surfaces, which is of intense interest for the integration of ordered organic thin films in silicon-based electronics. This could also potentially lead to the rational design of Si nanostructures with controlled properties through regulation of the surface chemistry.

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