Intracellular signal transduction by the extracellular calcium-sensing receptor of Xenopus melanotrope cells

The extracellular calcium-sensing receptor (CaR) is expressed in various types of endocrine pituitary cell, but the intracellular mechanism this G protein-coupled receptor uses in these cells is not known. In the present study we investigated possible intracellular signal transduction pathway(s) utilized by the CaR of the endocrine melanotrope cells in the intermediate pituitary lobe of the South African-clawed toad Xenopus laevis. For this purpose, the effects of various pharmacological agents on CaR-evoked secretion of radiolabeled secretory peptides from cultured melanotrope cells were assessed. CaR-evoked secretion, induced by the potent CaR agonist l-phenylalanine (l-Phe), could not be inhibited by cholera toxin, nor by NPC-15437 and PMA, indicating that neither G_s/PKA nor G_q/PKC pathways are involved. However, pertussis toxin (G_i/o protein inhibitor), genistein (inhibitor of PTKs), wortmannin/LY-294002 (PI3-K inhibitor) and U-0126 (inhibitor of extracellular signal-regulated kinase, ERK) all substantially inhibited CaR-evoked secretion, indicating that the Xenopus melanotrope cell possesses a PI3-K/MAPK system that plays some role in CaR-signaling. Since no direct effect of l-Phe on ERK phosphorylation could be shown it is concluded that CaR must act primarily through another, still unknown, signaling pathway in Xenopus melanotropes. Our results indicate that the PI3-K/MAPK system has a facilitating effect on CaR-induced secretion, possibly by sensitizing the CaR.     

A proinflammatory chemokine, CCL3, sensitizes the heat- and capsaicin-gated ion channel TRPV1

Pain, a critical component of host defense, is one hallmark of the inflammatory response. We therefore hypothesized that pain might be exacerbated by proinflammatory chemokines. To test this hypothesis, CCR1 was cotransfected into human embryonic kidney (HEK)293 cells together with transient receptor potential vanilloid 1 (TRPV1), a cation channel required for certain types of thermal hyperalgesia. In these cells, capsaicin and anandamide induced Ca(2+) influx mediated by TRPV1. When CCR1:TRPV1/HEK293 cells were pretreated with CCL3, the sensitivity of TRPV1, as indicated by the Ca(2+) influx, was increased approximately 3-fold. RT-PCR analysis showed that a spectrum of chemokine and cytokine receptors is expressed in rat dorsal root ganglia (DRG). Immunohistochemical staining of DRG showed that CCR1 is coexpressed with TRPV1 in >85% of small-diameter neurons. CCR1 on DRG neurons was functional, as demonstrated by CCL3-induced Ca(2+) ion influx and PKC activation. Pretreatment with CCL3 enhanced the response of DRG neurons to capsaicin or anandamide. This sensitization was inhibited by pertussis toxin, U73122, or chelerythrine chloride, inhibitors of Gi-protein, phospholipase C, and protein kinase C, respectively. Intraplantar injection of mice with CCL3 decreased their hot-plate response latency. That a proinflammatory chemokine, by interacting with its receptor on small-diameter neurons, sensitizes TRPV1 reveals a previously undescribed mechanism of receptor cross-sensitization that may contribute to hyperalgesia during inflammation.     

De Novo Mutations in the Motor Domain of KIF1A Cause Cognitive Impairment,Spastic Paraparesis,Axonal Neuropathy,and Cerebellar Atrophy

KIF1A is a neuron‐specific motor protein that plays important roles in cargo transport along neurites. Recessive mutations in KIF1A were previously described in families with spastic paraparesis or sensory and autonomic neuropathy type‐2. Here, we report 11 heterozygous de novo missense mutations (p.S58L, p.T99M, p.G102D, p.V144F, p.R167C, p.A202P, p.S215R, p.R216P, p.L249Q, p.E253K, and p.R316W) in KIF1A in 14 individuals, including two monozygotic twins. Two mutations (p.T99M and p.E253K) were recurrent, each being found in unrelated cases. All these de novo mutations are located in the motor domain (MD) of KIF1A. Structural modeling revealed that they alter conserved residues that are critical for the structure and function of the MD. Transfection studies suggested that at least five of these mutations affect the transport of the MD along axons. Individuals with de novo mutations in KIF1A display a phenotype characterized by cognitive impairment and variable presence of cerebellar atrophy, spastic paraparesis, optic nerve atrophy, peripheral neuropathy, and epilepsy. Our findings thus indicate that de novo missense mutations in the MD of KIF1A cause a phenotype that overlaps with, while being more severe, than that associated with recessive mutations in the same gene.     

Crystal structures of TAFI elucidate the inactivation mechanism of activated TAFI: a novel mechanism for enzyme autoregulation

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a pro-metallocarboxypeptidase that can be proteolytically activated (TAFIa). TAFIa is unique among carboxypeptidases in that it spontaneously inactivates with a short half-life, a property that is crucial for its role in controlling blood clot lysis. We studied the intrinsic instability of TAFIa by solving crystal structures of TAFI, a TAFI inhibitor (GEMSA) complex and a quadruple TAFI mutant (70-fold more stable active enzyme). The crystal structures show that TAFIa stability is directly related to the dynamics of a 55-residue segment (residues 296-350) that includes residues of the active site wall. Dynamics of this flap are markedly reduced by the inhibitor GEMSA, a known stabilizer of TAFIa, and stabilizing mutations. Our data provide the structural basis for a model of TAFI auto-regulation: in zymogen TAFI the dynamic flap is stabilized by interactions with the activation peptide. Release of the activation peptide increases dynamic flap mobility and in time this leads to conformational changes that disrupt the catalytic site and expose a cryptic thrombin-cleavage site present at Arg302. This represents a novel mechanism of enzyme control that enables TAFI to regulate its activity in plasma in the absence of specific inhibitors.     

Variation in hospital mortality after pancreatoduodenectomy is related to failure to rescue rather than major complications: a nationwide audit

Background

In the mandatory nationwide Dutch Pancreatic Cancer Audit, rates of major complications and Failure to Rescue (FTR) after pancreatoduodenectomy between low- and high-mortality hospitals are compared, and independent predictors for FTR investigated.

Genome sequencing of mucosal melanomas reveals that they are driven by distinct mechanisms from cutaneous melanoma

Mucosal melanoma displays distinct clinical and epidemiological features compared to cutaneous melanoma. Here we used whole genome and whole exome sequencing to characterize the somatic alterations and mutation spectra in the genomes of ten mucosal melanomas. We observed somatic mutation rates that are considerably lower than occur in sun‐exposed cutaneous melanoma, but comparable to the rates seen in cancers not associated with exposure to known mutagens. In particular, the mutation signatures are not indicative of ultraviolet light‐ or tobacco smoke‐induced DNA damage. Genes previously reported as mutated in other cancers were also mutated in mucosal melanoma. Notably, there were substantially more copy number and structural variations in mucosal melanoma than have been reported in cutaneous melanoma. Thus, mucosal and cutaneous melanomas are distinct diseases with discrete genetic features. Our data suggest that different mechanisms underlie the genesis of these diseases and that structural variations play a more important role in mucosal than in cutaneous melanomagenesis. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.