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S J Quinn

Publications and source records attributed to S J Quinn.

At least 19 recordsLinked to original sources
PubMed2005

How we do it: free conchal cartilage revisited for primary reconstruction of attic defects in combined approach tympanoplasty.

Keypoints * There are concerns over the efficacy of free cartilage autografts in attic reconstruction (scutumplasty) for combined approach tympanoplasty (CAT). * Our operative technique uses free conchal cartilage autografts and an additional piece of conchal cartilage to buttress the reconstruction. * Of the 16 cases where this technique was used, there were two cases (13%) of failure of the attic reconstruction and a keratin pearl (residual disease) in one case (6%). This compares favourably with comparable series using a variety of reconstruction techniques.

Cholesteatoma, Middle Ear↗
PubMed2002

L-amino acid sensing by the calcium-sensing receptor: a general mechanism for coupling protein and calcium metabolism?

Cellular sensing of L-amino acids is widespread and controls diverse cellular responses regulating, for example, rates of hormone secretion, amino acid uptake, protein synthesis and protein degradation (autophagy). However, the nature of the sensing mechanisms involved has been elusive. One important sensing mechanism is selective for branched chain amino acids, acts via mTOR (mammalian target of rapamycin) and regulates the rates of insulin and IGF-1 secretion as well as hepatic, and possibly muscle, autophagy. A second sensing mechanism is selective for aromatic L-amino acids and regulates the rate of gastric acid secretion and other responses in the gastro-intestinal tract. Interactions between calcium and protein metabolism, including accelerated urinary calcium excretion in subjects consuming high-protein diets and secondary hyperparathyroidism in subjects consuming low-protein diets, suggest an additional amino acid sensing mechanism linked to the control of urinary calcium excretion and parathyroid hormone (PTH) release. New data demonstrating L-amino acid-dependent activation of the calcium-sensing receptor (CaR), which regulates PTH secretion and urinary calcium excretion, suggests an unexpected explanation for these links between calcium and protein metabolism. Furthermore, expression of the CaR in gastrin-secreting G-cells and acid-secreting parietal cells, together with data indicating that the CaR exhibits selectivity for aromatic amino acids, would appear to provide a molecular explanation for amino acid sensing in the gastrointestinal tract. This review examines what is known about the CaR as a gene, a receptor, a physiological regulator and, now, as an amino acid sensor. Possible new roles for the CaR are also considered.

Amino Acids↗
PubMed2000

The extracellular calcium-sensing receptor dimerizes through multiple types of intermolecular interactions.

Recent studies have shown that the G protein-coupled, extracellular calcium ([Ca(2+)](o))-sensing receptor (CaR) forms disulfide-linked dimers through cysteine residues within its extracellular domain and that dimerization of the CaR has functional implications. In this study, we have investigated which of these disulfide linkages are essential for dimerization of the CaR and whether they are required for these functional interactions. Our results confirm the key roles of Cys(129) and Cys(131) in CaR dimerization. However, utilizing cross-linking of the CaR or immunoprecipitation of a non-FLAG-tagged CaR with a FLAG-tagged CaR using anti-FLAG antibody, we demonstrate that CaRs with or without these two cysteines form dimers on the cell surface to a similar extent. In addition, reconstitution of CaR-mediated signaling by cotransfection of two individually inactive mutant CaRs is nearly identical in the presence or absence of both Cys(129) and Cys(131), showing that covalent linkage of CaR dimers is not needed for functional interactions between CaR monomers. These findings suggest that the CaR has at least two distinct types of motifs mediating dimerization and functional interactions, i.e. covalent interactions involving intermolecular disulfide bonds and noncovalent, possibly hydrophobic, interactions.

Calcium↗
PubMed2000

L-amino acid sensing by the extracellular Ca2+-sensing receptor.

The extracellular calcium (Ca(2+)(o))-sensing receptor (CaR) recognizes and responds to (i.e., "senses") Ca(2+)(o) as its principal physiological ligand. In the present studies, we document that the CaR is activated not only by extracellular calcium ions but also by amino acids, establishing its capacity to sense nutrients of two totally different classes. l-Amino acids, especially aromatic amino acids, including l-phenylalanine and l-tryptophan, stereoselectively mobilized Ca(2+) ions in the presence of the CaR agonists, Ca(2+)(o), gadolinium (Gd(3+)(o)), and spermine in fura-2-loaded human embryonic kidney (HEK-293) cells stably transfected with the human CaR. l-amino acid-dependent effects were observed above, but not below, a threshold level of Ca(2+)(o) of approximately 1.0 mM. l-Amino acids, particularly aromatic amino acids, also stereoselectively enhanced the sensitivity of the CaR to its agonists, Ca(2+)(o) and spermine. Branched-chain amino acids were almost inactive, and charged amino acids, including arginine and lysine, were much less effective than aromatic and other amino acids. l-amino acid mixtures emulating the amino acid composition of fasting human plasma reproduced the effects of high concentrations of individual l-amino acids on Ca(2+) mobilization and enhanced the sensitivity of the CaR to Ca(2+)(o). The data presented herein identify the CaR as a molecular target for aromatic and other l-amino acids. Thus, the CaR can integrate signals arising from distinct classes of nutrients: mineral ions and amino acids. The actions of l-amino acids on the CaR may provide explanations for several long recognized but poorly understood actions of dietary protein on calcium metabolism.

Amino Acids↗
PubMed2000

Cooperative multi-modal sensing and therapeutic implications of the extracellular Ca(2+) sensing receptor.

The extracellular Ca(2+)-sensing receptor (CaR) is an unusual member of the diverse superfamily of seven-transmembrane domain G-protein-coupled receptors. Originally identified as the receptor providing the calciostat for extracellular ionized Ca(2+) ¿[Ca(2+)](o)¿, the CaR corrects small changes in [Ca(2+)](o) by regulating the secretion of the hormone that controls Ca(2+) fluxes between the blood and Ca(2+) stores in bone, and between blood and the urine. Now, research is beginning to reveal the structure and function of its unusually large N-terminal head. In addition to its role as a divalent and polyvalent cation sensor, recent studies indicate that the receptor also responds sensitively to changes in ionic strength and pH. Furthermore, new work indicates that the CaR is subject to allosteric activation by L-amino acids.

Amino Acids↗
PubMed1999

Intermolecular interactions between dimeric calcium-sensing receptor monomers are important for its normal function.

We recently demonstrated that the G protein-coupled, extracellular calcium-sensing receptor (CaR) forms disulfide-linked dimers. The functional significance of dimerization of this receptor was suggested by our earlier observations that CaRs carrying certain point mutations exert dominant negative effects on the function of the coexpressed wild-type receptor both in vivo and when cotransfected in human embryonic kidney cells. In this study, we explored the functional consequences of CaR dimerization. Coexpression in human embryonic kidney cells of specific pairs of mutant CaRs, each with reduced or absent activity because of distinct loss-of-function mutations, results in the formation of heterodimers and partially reconstitutes extracellular calcium-dependent signaling. Moreover, our results suggest that the CaR has at least two functionally separable domains. However, the presence of an abnormal domain in each mutant monomer substantially impairs the function of the CaR heterodimer, resulting in the reconstituted CaRs having characteristics distinct from those of the wild-type CaR. Our study suggests that intermolecular interactions within the dimeric CaR are important for the receptor's function.

Calcium Signaling↗
PubMed1998

Protein kinase C phosphorylation of threonine at position 888 in Ca2+o-sensing receptor (CaR) inhibits coupling to Ca2+ store release.

Previous studies in parathyroid cells, which express the G protein-coupled, extracellular calcium-sensing receptor (CaR), showed that activation of protein kinase C (PKC) blunts high extracellular calcium (Ca2+o)-evoked stimulation of phospholipase C and the associated increases in cytosolic calcium (Ca2+i), suggesting that PKC may directly modulate the coupling of the CaR to intracellular signaling systems. In this study, we examined the role of PKC in regulating the coupling of the CaR to Ca2+i dynamics in fura-2-loaded human embryonic kidney cells (HEK293 cells) transiently transfected with the human parathyroid CaR. We demonstrate that several PKC activators exert inhibitory effects on CaR-mediated increases in Ca2+i due to release of Ca2+ from intracellular stores. Consistent with the effect being mediated by activation of PKC, the inhibitory effect of PKC activators on Ca2+ release can be blocked by a PKC inhibitor. The use of site-directed mutagenesis reveals that threonine at amino acid position 888 is the major PKC site that mediates the inhibitory effect of PKC activators on Ca2+ mobilization. The effect of PKC activation can be maximally blocked by mutating three PKC sites (Thr888, Ser895, and Ser915) or all five PKC sites. In vitro phosphorylation shows that Thr888 is readily phosphorylated by PKC. Our results suggest that phosphorylation of the CaR is the molecular basis for the previously described effect of PKC activation on Ca2+o-evoked changes in Ca2+i dynamics in parathyroid cells.

Calcium↗
PubMed1998

Sodium and ionic strength sensing by the calcium receptor.

The calcium-sensing receptor (CaR) is activated by small changes in extracellular calcium [Ca2+]o) in the physiological range, allowing the parathyroid gland to regulate serum [Ca2+]o; however, the CaR is also distributed in a number of other tissues where it may sense other endogenous agonists and modulators. CaR agonists are polycationic molecules, and charged residues in the extracellular domain of the CaR appear critical for receptor activation through electrostatic interactions, suggesting that ionic strength could modulate CaR activation by polycationic agonists. Changes in the concentration of external NaCl potently altered the activation of the CaR by external Ca2+ and spermine. Ionic strength had an inverse effect on the sensitivity of CaR to its agonists, with lowering of ionic strength rendering the receptor more sensitive to activation by [Ca2+]o and raising of ionic strength producing the converse effect. Effects of osmolality could not account for the modulation seen with changes in NaCl. Other salts, which differed in the cationic or anionic species, showed shifts in the activation of the CaR by [Ca2+]o similar to that elicited by NaCl. Parathyroid cells were potently modulated by ionic strength, with addition of 40 mM NaCl shifting the EC50 for [Ca2+]o inhibition of parathyroid hormone by at least 0.5 mM. Several CaR-expressing tissues, including regions of the brain such as the subfornical organ and hypothalamus, could potentially use the CaR as a sensor for ionic strength and NaCl. The Journal guidelines state that the summary should be no longer than 200 words.

Adenosine Triphosphate↗
PubMed1997

Targeted inactivation of alphai2 or alphai3 disrupts activation of the cardiac muscarinic K+ channel, IK+Ach, in intact cells.

Cardiac muscarinic receptors activate an inwardly rectifying K+ channel, IK+Ach, via pertussis toxin (PT)-sensitive heterotrimeric G proteins (in heart Gi2, Gi3, or Go). We have used embryonic stem cell (ES cell)-derived cardiocytes with targeted inactivations of specific PT-sensitive alpha subunits to determine which G proteins are required for receptor-mediated regulation of IK+Ach in intact cells. The muscarinic agonist carbachol increased IK+Ach activity in ES cell-derived cardiocytes from wild-type cells, in cells lacking alphao, and in cells lacking the PT-insensitive G protein alphaq. In cells with targeted inactivation of alphai2 or alphai3, channel activation by both carbachol and adenosine was blocked. Carbachol-induced channel activation was restored in the alphai2- and alphai3-null cells by reexpressing the previously targeted gene and guanosine 5'-[gamma-thio] triphosphate was able to fully activate IK+Ach in excised membranes patches from these mutants. In contrast, negative chronotropic responses to both carbachol and adenosine were preserved in cells lacking alphai2 or alphai3. Our results show that expression of two specific PT-sensitive alpha subunits (alphai2 and alphai3 but not alphao) is required for normal agonist-dependent activation of IK+Ach and suggest that both alphai2- and alphai3-containing heterotrimeric G proteins may be involved in the signaling process. Also the generation of negative chronotropic responses to muscarinic or adenosine receptor agonists do not require activation of IK+Ach or the expression of alphai2 or alphai3.

Carbachol↗
PubMed1997

Markedly reduced activity of mutant calcium-sensing receptor with an inserted Alu element from a kindred with familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism.

Missense mutations have been identified in the coding region of the extracellular calcium-sensing receptor (CASR) gene and cause human autosomal dominant hypo- and hypercalcemic disorders. The functional effects of several of these mutations have been characterized in either Xenopus laevis oocytes or in human embryonic kidney (HEK293) cells. All of the mutations that have been examined to date, however, cause single putative amino acid substitutions. In this report, we studied a mutant CASR with an Alu-repetitive element inserted at codon 876, which was identified in affected members of families with the hypercalcemic disorders, familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT), to understand how this insertion affects CASR function. After cloning of the Alu-repetitive element into the wild-type CASR cDNA, we transiently expressed the mutant receptor in HEK293 cells. Expression of mutant and wild-type receptors was assessed by Western analysis, and the effects of the mutation on extracellular calcium (Ca2+(o)) and gadolinium (Gd3+(o)) elicited increases in the cytosolic calcium concentration (Ca2+(i)) were examined in fura-2-loaded cells using dual wavelength fluorimetry. The insertion resulted in truncated receptor species that had molecular masses some 30 kD less than that of the wild-type CASR and exhibited no Ca2+(i) responses to either Ca2+(o) or Gd3+(o). A similar result was observed with a mutated CASR truncated at residue 876. However, the Alu mutant receptor had no impact on the function of the coexpressed wild-type receptor. Interestingly, the Alu mutant receptor demonstrated decreased cell surface expression relative to the wild-type receptor, whereas the CASR (A877stop) mutant exhibited increased cell surface expression. Thus, like the missense mutations that have been characterized to date in families with FHH, the Alu insertion in this family is a loss-of-function mutation that produces hypercalcemia by reducing the number of normally functional CASRs on the surface of parathyroid and kidney cells. In vitro transcription of exon 7 of the CASR containing the Alu sequence yielded the full-length mutant product and an additional shorter product that was truncated due to stalling of the polymerase at the poly(T) tract. In vitro translation of the mutant transcript yielded three truncated protein products representing termination in all three reading frames at stop codons within the Alu insertion. Thus sequences within the Alu contribute to slippage or frameshift mutagenesis during transcription and/or translation.

Animals↗
PubMed1997

Reversible sensorineural hearing loss in Lyme disease.

We report a case of bilateral sensorineural hearing loss of two years duration which appears to have been due to late Borrelia burgdorferi infection. The 39-year-old woman presented with bilateral deafness and multiple other neurological complaints some six months after developing a 'target' lesion on the lower leg after walking in the New Forest. 'Serology' for Borrelia burgdorferi became positive and the patient made a complete recovery from both her deafness and her other neurological problems after a five-week course of oral antibiotic therapy.

Adult↗
PubMed1997

The Ca2+-sensing receptor: a target for polyamines.

The Ca2+-sensing receptor (CaR) is activated at physiological levels of external Ca2+ (Ca(o)) but is expressed in a number of tissues that do not have well-established roles in the control of Ca(o), including several regions of the brain and the intestine. Polyamines are endogenous polyvalent cations that can act as agonists for the CaR, as shown by our current studies of human embryonic kidney (HEK-293) cells transfected with the human CaR. Cellular parameters altered by polyamines included cytosolic free Ca2+ (Ca(i)), inositol phosphate production, and the activity of a nonselective cation channel. Spermine stimulated Ca(i) transients in CaR-transfected HEK cells, with a concentration producing a half-maximal response (EC50) of approximately 500 microM in the presence of 0.5 mM Ca2+, whereas sustained increases in Ca(i) had an EC50 of approximately 200 microM. The order of potency was spermine > spermidine >> putrescine. Elevation of Ca(o) shifted the EC50 for spermine sharply to the left, with substantial stimulation below 100 microM. Addition of subthreshold concentrations of spermine increased the sensitivity of CaR-expressing HEK cells to Ca(o). Parathyroid hormone secretion from bovine parathyroid cells was inhibited by 50% in the presence of 200 microM spermine, a response similar to that elicited by 2.0 mM Ca(o). These data suggest that polyamines could be effective agonists for the CaR, and several tissues, including the brain, may use the CaR as a target for the actions of spermine and other endogenous polycationic agonists.

Animals↗
PubMed1996

Functional characterization of calcium-sensing receptor mutations expressed in human embryonic kidney cells.

The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that plays a key role in extracellular calcium ion homeostasis. We have engineered 11 CaR mutants that have been described in the disorders familial benign hypercalcemia (FBH), neonatal severe hyperparathyroidism (NSHPT), and autosomal dominant hypocalcaemia (ADH), and studied their function by characterizing intracellular calcium [Ca2+]i transients in response to varying concentrations of extracellular calcium [Ca2+]o or gadolinium [Gd3+]o. The wild type receptor had an EC50 for calcium (EC50[Ca2+]o) (the value of [Ca2+]o producing half of the maximal increase in [Ca2+]i) of 4.0 mM (+/- 0.1 SEM). However, five missense mutations associated with FBH or NSHPT, (P55L, N178D, P221S, R227L, and V817I) had significantly higher EC50[Ca2+]os of between 5.5 and 9.3 mM (all P < 0.01). Another FBH mutation, Y218S, had an EC50[Ca2+]o of > 50 mM but had only a mildly attenuated response to gadolinium, while the FBH mutations, R680C and P747fs, were unresponsive to either calcium or gadolinium. In contrast, three mutations associated with ADH, (F128L, T151M, and E191K), showed significantly reduced EC50[Ca2+]os of between 2.2 and 2.8 mM (all P < 0.01). These findings provide insights into the functional domains of the CaR and demonstrate that mutations which enhance or reduce the responsiveness of the CaR to [Ca2+]o cause the disorders ADH, FBH, and NSHPT, respectively.

Calcium↗
PubMed1996

Agonists of the Ca(2+)-sensing receptor (CaR) activate nonselective cation channels in HEK293 cells stably transfected with the human CaR.

Calcium (Ca2+) ions serve multiple roles both intra- and extracellularly. We recently cloned a cell surface, Cao(2+)-sensing receptor (CaR) that plays a central role in Cao2+ homeostasis by enabling direct regulation by Cao2+ of parathyroid hormone (PTH) secretion and the function of other tissues involved in mineral ion homeostasis. In parathyroid cells, the CaR activates phospholipase C, thereby raising the levels of inositol trisphosphate (IP3) and releasing Ca2+ from intracellular stores. High Cao2+ also activates Ca2+ influx into parathyroid cells through poorly defined mechanisms that may involve Ca(2+)-permeable, nonselective cation channels (NCC). We now show that human embryonic kidney (HEK293) cells also have NCC and, furthermore, that these channels are regulated by the CaR. We have utilized the cell-attached configuration of the patch clamp technique to characterize the properties of these channels as well as their regulation by various CaR agonists added to the external bath solution. The polycationic CaR agonist, neomycin (100 microM), as well as an elevated concentration of Cao2+ (3 mM), both of which activate the cloned CaR, significantly increased the probability of channel opening (Po) in HEK cells stably transfected with the CaR but not in nontransfected HEK cells which do not contain the receptor. Thus, the activation of the CaR enhances the activity of Ca(2+)-permeable NCC in these cells, which could contribute to the sustained increase in Cai2+ in parathyroid cells which is observed in response to elevated Cao2+. The CaR may also regulate the membrane functions of other CaR-expressing cells (e.g., those in the brain), at least in part, by modulating similar channels.

Calcium-Binding Proteins↗
PubMed1996

Cervical necrotising fasciitis with pharyngeal perforation: treatment and reconstruction.

We present a case of life-threatening necrotising fasciitis of the neck with acute pharyngeal perforation following a parapharyngeal abscess caused by tonsillitis. The joint occurrence of cervical necrotising fasciitis and acute pharyngeal perforation has not been previously reported. A new way of reconstructing the pharyngeal defect using an islanded submental perforator flap is presented.

Acute Disease↗
PubMed1996

The differentiation of snoring mechanisms using sound analysis.

Ten subjects known to suffer from heavy snoring but not obstructive sleep apnoea were studied using the technique of sleep nasendoscopy. The mechanism of snoring was noted for each and sound recordings of the snoring noise were made. Six subjects were observed to snore using their soft palate only, three snored using only their tongue base and one snored using a combination of palate and tongue base. The sound recordings were subjected to computer analysis of waveform and frequency. Palatal flutter snoring and tongue base snoring appear to have distinct waveform and frequency patterns which allows them to be differentiated from each other.

Confounding Factors, Epidemiologic↗
PubMed1996

Calcium-sensing receptor expression and regulation by extracellular calcium in the AtT-20 pituitary cell line.

A 120 kDa, G protein-coupled calcium-sensing receptor (CaR) was recently identified and cloned from bovine parathyroid and rat kidney. We report here that a similar calcium-sensing receptor is also present in rat and mouse pituitary as well as in the mouse pituitary cell line, AtT-20. Fragments (383-bp) of the extracellular domain of the calcium-sensing receptor from the AtT-20 cells and mouse pituitary were amplified by RT-PCR, sequenced, and found to be identical. By Northern blot analysis, AtT-20 cells expressed a major CaR mRNA transcript of 7.5 kb and three minor transcripts of 9.5, 4.0, and 1.5 kb. Except for the 9.5-kb species, these CaR transcripts were also found to be present in mouse kidney, where the 7.5-kb transcript was again the predominant form. The presence of the CaR protein in AtT-20 cells was documented directly by fluorescence immunocytochemistry using an antibody directed against the extracellular domain of the CaR. Exposure of AtT-20 cells to increasing extracellular calcium concentrations from 0.3 t 3 mM for 24 h resulted in a 2- to 4-fold increase in the levels of CaR mRNA, but not of the RNAs for beta-actin or POMC. The CaR appeared to be functional in AtT-20 cells, since acute increases in extracellular calcium between 2 and 5 mM induced increases in the cellular content of total inositol phosphates, cytosolic calcium, and cAMP. This report suggests that pituitary cells respond to changes in extracellular calcium via a G protein-coupled CaR.

Amino Acid Sequence↗