Inhibition of N and PQ calcium channels by calcium entry through L channels in chromaffin cells

Transient receptor potential (TRP) polycystin 3 (TRPP3) is a member of the TRP superfamily of cation channels. Murine TRPP3 has been reported to form an acid-activated cation channel on the plasma membrane when coexpressed with the polycystin 1-like protein 3 (PKD1L3); however, the function and biophysical properties of TRPP3-dependent channels have not yet been characterized in detail. Here we show that overexpression of murine TRPP3 channel in HEK293 cells, without coexpression of PDK1-like proteins, leads to robust channel activity. These channels exhibit a high single-channel conductance of 184 pS at negative potentials, are Ca²⁺-permeable, and relatively nonselective between cations. Whole-cell experiments showed a characteristic form of voltage-dependent gating of TRPP3 channels, whereby repolarization after depolarization caused large transient inward TRPP3 tail currents. Moreover, we found that TRPP3 activity was increased upon cell swelling and by alkalization. Taken together, our results demonstrate that TRPP3, on its own, can act as a voltage-dependent, pH- and volume-sensitive plasma membrane cation channel. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

SLC26A4 variations among Graves' hyper-functioning thyroid gland

Deleterious mutations of SLC26A4 cause Pendred syndrome (PS), an autosomal recessive disorder comprising goitre and deafness with enlarged vestibular aqueducts (EVA), and nonsyndromic hearing loss (NSHL). However, the SLC26A4 hyperactivity was recently associated with the emergence of autoimmune thyroid diseases (AITD) and asthma among human and mouse model. Here, by direct sequencing, we investigate the sequences of the 20 coding exons (2 to 21) of SLC26A4 and their flanking intron-exon junctions among patients affected with Graves' disease (GD) hyperthyroidism. Ten mono-allelic variants were identified, seven of which are intronic and previously unreported. Two, c.898A>C (p.I300L) and c.1061T>C (p.F354S), of the three exonic variants are non synonymous. The p.F354S variant is already described to be involved in PS or NSHL inheritances. The exploration by PCR-RFLP of p.I300L and p.F354S variants among 132 GD patients, 105 Hashimoto thyroiditis (HT), 206 Healthy subjects and 102 families with NSHL have shown the presence of both variants. The p.F354S variation was identified both among patients (1~HT and 3 GD) and healthy subjects (n=5). Whereas, the p.I300L variant was identified only in GD patients (n=3). Our studies provide evidence of the importance of systematic analysis of SLC26A4 gene sequences on models other than deafness. This approach allows the identification of new variants and the review of the pathogenic effects of certain mono-allelic variants reported responsible for PS and NSHL development.     

Caspase-3 activation in the guinea pig cochlea exposed to salicylate

In the current study, we explored whether chronic salicylate exposure could induce apoptosis in outer hair cells (OHCs) and spiral ganglion neurons (SGNs) of the cochlea. Guinea pig received sodium salicylate (400 mg/kg/d) or saline vehicle for 10 consecutive days. Programmed cell death (PCD) executioner was evaluated with immunohistochemistry detection of activated caspase-3. Apoptosis was examined with a terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method. Repeated salicylate administration activated caspase-3 and caused apoptosis in OHCs and SGNs (p < 0.01 vs. saline control for both measures and in both cell types). Cell counting showed a significant loss in OHCs (p < 0.01 vs. saline control), but not in inner hair cells (IHCs). Transmission electron microscopy (TEM) revealed chromatin condensation and nucleus margination in salicylate-treated cochlea. Scanning electron microscopy (SEM) demonstrated stereociliary bundles breakdown and fusion at the apical of OHCs, villous matter was discovered to attach on the surface of SGNs. These findings suggest that long-term administration of high-dose salicylate can activate caspase-3 pathway to induce OHC and SGN apoptosis.     

Gastric carcinomas with microsatellite instability: clinical features and mutations to the TGF-β type II receptor,IGFII receptor,and BAX genes

The replication error phenotype (RER+) represents an important new form of genetic alteration characterized by widespread instability in repetitive nucleotide sequences. The aim of this study was to compare the features of RER+ gastric tumours with those of RER+ colonic tumours. RER status was determined by analysis of size alterations in the BAT-26 mononucleotide repeat microsatellite. Twelve of 121 (10 per cent) gastric carcinomas from a low-incidence region were found to be RER+. BAT-26 instability was associated with tumours showing an absence of nodal invasion (p = 0·009) and with a trend for improved prognosis. These tumours were more frequent in older, female patients. Frameshift mutations in mononucleotide repeat sequences within the transforming growth factor-β receptor II (RII), insulin-like growth factor II receptor (IGFIIR), and BAX genes were observed in 83, 33, and 25 per cent, respectively, of RER+ tumours. Only 1/12 (8 per cent) RER+ tumours contained a p53 gene mutation compared with 29/109 (27 per cent) RER− tumours. RER+ gastric carcinomas therefore share several important features with RER+ colonic tumours, including less frequent nodal invasion, improved prognosis, a similar frequency of mutation in growth control genes containing repetitive nucleotide sequences, and a low frequency of mutation of the p53 tumour suppressor gene. Copyright © 1999 John Wiley & Sons, Ltd.     

Expression of an anti‐RNA autoantibody in a mouse model of SLE increases neutrophil and monocyte numbers as well as IFN‐I expression

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of antinucleic acid autoantibodies, high levels of circulating type I interferon (IFN‐I), and an IFN‐I‐dependent elevated expression of activating FcγR. Increases in neutrophils and monocytes are often observed in clinical SLE, but how these contribute to autoantibody and IFN‐I production is poorly understood. Here, we analyzed SLE pathogenesis in 564Igi mice, an SLE‐model strain carrying gene‐targeted heavy and light chain antibody genes encoding an anti‐RNA autoantibody in a C57BL/6 background. Similar to human SLE patients, 564Igi mice produce anti‐RNA autoantibodies and expanded neutrophil and monocyte populations. These myeloid cells produced IFN‐I and exhibit increased FcγRIV expression induced via an IFN‐I autocrine loop. A direct effect of IFN‐I on 564Igi BM B cells and neutrophils was supported by their upregulation of “IFN‐I signature genes”. In addition, 564Igi developing B cells showed upregulated TLR7 resulting in IgG2a/2b class switch recombination and autoantibody production. Our results indicate that the production of anti‐RNA autoantibody is sufficient to induce an increase of BM, blood, and spleen IFN‐I‐producing neutrophils, and suggest a mechanism by which autoantibody and IFN‐I contribute to SLE by activating B lymphocytes, neutrophils, and monocyte effector cells in vivo.     

EGF receptor family: twisting targets for improved cancer therapies

Abstract

The epidermal growth factor receptor (EGFR) undergoes a conformational change in response to ligand binding. The ligand-induced changes in cell surface aggregation and mobility have a profound effect on the function of all the family members. Ligand also activates the EGFR intracellular kinase, stimulating proliferation and cell survival. The EGFR family are often activated, overexpressed or mutated in cancer cells and therapeutic drugs (including antibodies) can slow the progress of some cancers. This article provides a brief, annotated summary of the presentations and discussion which occurred at the Epidermal Growth Factor Receptor – Future Directions Conference held in Jerusalem in November 2013.