ATP modulates interaction of syntaxin-1A with sulfonylurea receptor 1 to regulate pancreatic beta-cell KATP channels.

ATP-sensitive potassium (K(ATP)) channels are regulated by a variety of cytosolic factors (adenine nucleotides, Mg(2+), phospholipids, and pH). We previously reported that K(ATP) channels are also regulated by endogenous membrane-bound SNARE protein syntaxin-1A (Syn-1A), which binds both nucleotide-binding folds of sulfonylurea receptor (SUR)1 and 2A, causing inhibition of K(ATP) channel activity in pancreatic islet ß-cells and cardiac myocytes, respectively. In this study, we show that ATP dose-dependently inhibits Syn-1A binding to SUR1 at physiological concentrations, with the addition of Mg(2+) causing a decrease in the ATP-induced inhibitory effect. This ATP disruption of Syn-1A binding to SUR1 was confirmed by FRET analysis in living HEK293 cells. Electrophysiological studies in pancreatic ß-cells demonstrated that reduced ATP concentrations increased K(ATP) channel sensitivity to Syn-1A inhibition. Depletion of endogenous Syn-1A in insulinoma cells by botulinum neurotoxin C1 proteolysis followed by rescue with exogenous Syn-1A showed that Syn-1A modulates K(ATP) channel sensitivity to ATP. Thus, our data indicate that although both ATP and Syn-1A independently inhibit ß-cell K(ATP) channel gating, they could also influence the sensitivity of K(ATP) channels to each other. These findings provide new insight into an alternate mechanism by which ATP regulates pancreatic ß-cell K(ATP) channel activity, not only by its direct actions on Kir6.2 pore subunit, but also via ATP modulation of Syn-1A binding to SUR1.

SNAREing voltage-gated K+ and ATP-sensitive K+ channels: tuning beta-cell excitability with syntaxin-1A and other exocytotic proteins.

The three SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, syntaxin, SNAP25 (synaptosome-associated protein of 25 kDa), and synaptobrevin, constitute the minimal machinery for exocytosis in secretory cells such as neurons and neuroendocrine cells by forming a series of complexes prior to and during vesicle fusion. It was subsequently found that these SNARE proteins not only participate in vesicle fusion, but also tether with voltage-dependent Ca(2+) channels to form an excitosome that precisely regulates calcium entry at the site of exocytosis. In pancreatic islet beta-cells, ATP-sensitive K(+) (K(ATP)) channel closure by high ATP concentration leads to membrane depolarization, voltage-dependent Ca(2+) channel opening, and insulin secretion, whereas subsequent opening of voltage-gated K(+) (Kv) channels repolarizes the cell to terminate exocytosis. We have obtained evidence that syntaxin-1A physically interacts with Kv2.1 (the predominant Kv in beta-cells) and the sulfonylurea receptor subunit of beta-cell K(ATP) channel to modify their gating behaviors. A model has proposed that the conformational changes of syntaxin-1A during exocytosis induce distinct functional modulations of K(ATP) and Kv2.1 channels in a manner that optimally regulates cell excitability and insulin secretion. Other proteins involved in exocytosis, such as Munc-13, tomosyn, rab3a-interacting molecule, and guanyl nucleotide exchange factor II, have also been implicated in direct or indirect regulation of beta-cell ion channel activities and excitability. This review discusses this interesting aspect that exocytotic proteins not only promote secretion per se, but also fine-tune beta-cell excitability via modulation of ion channel gating.

Corynebacterium macginleyi conjunctivitis in Canada.

This report describes for the first time Corynebacterium macginleyi as a cause of conjunctivitis in Canada, where menaquinone analysis was done as part of the strain characterization. This species is typically isolated from ocular surfaces of patients from Europe and Japan. The isolate was resistant to erythromycin and clindamycin.

Anti-N-methyl-D-aspartate receptor encephalitis in a young woman with an ovarian tumour.

Anti-N-methyl-D-aspartate receptor encephalitis is characterised by psychiatric and neurological abnormalities and occurs in frequent association with ovarian teratoma. We report the first confirmed case of teratoma-associated anti-N-methyl-D-aspartate receptor encephalitis in Hong Kong in a young woman presenting with confusion and prominent dyskinesia, followed by a review of the current literature.

Gardnerella vaginalis bacteremia in a previously healthy man: case report and characterization of the isolate.

Gardnerella vaginalis in women causes vaginitis or infections in other sites, such as the urinary tract, but is an infrequent cause of bacteremia. Bacteremia in men is very rare and is typically associated with immunocompromised states. Here we describe G. vaginalis bacteremia in a previously healthy man with renal calculi and urosepsis.

First report of isolation and characterization of Aurantimonas altamirensis from clinical samples.

The genus Aurantimonas, proposed in 2003, encompasses four species from environmental sources, including Aurantimonas altamirensis, isolated from a cave wall in Spain. Here, we report what we believe are the first cases of the recovery of A. altamirensis from human clinical materials.

Molecular typing of a Legionella pneumophila outbreak in Ontario, Canada.

An outbreak of Legionnaires' disease at a long-term care facility in Ontario, Canada from September to October 2005 resulted in the death of 23 residents and the illness of 112 other people. In response, molecular methods were developed to detect Legionella pneumophila in clinical lung samples and to subtype isolates from clinical and environmental samples. The targeted genetic loci included Legionella-specific virulence determinants (mip, icmO, sidA and lidA) and core bacterial determinants (ftsZ, trpS and dnaX). An established amplified fragment length polymorphism typing method provided the first indication of genetic relatedness between strains recovered from clinical samples and strains cultured from environmental samples taken from the outbreak site. These associations were verified using the European Working Group for Legionella Infections sequence-based typing protocol targeting the flaA, pilE, asd, mip, mompS and proA loci. These molecular typing methods confirmed the outbreak source as a contaminated air conditioning cooling tower.

Modeling human exposures to air pollution control (APC) residues released from landfills in England and Wales.

Human exposures to air pollution control (APC) residues released from 6 landfills were modeled and assessed. Following a qualitative risk characterisation, direct and indirect exposures were quantified. Site-specific air dispersion modeling was conducted for PM(10), PCDDs/PCDFs, Pb, Cd, As and Cr(VI) concentrations at the closest residential points of exposure for 4 landfill sites accepting, in total, 75% w/w of the APC residues disposed of in 2000-2001 (UK). Inhalation risks, assessed by reference to air quality standards at residential exposure points, were assessed as insignificant. Preliminary modeling suggested that indirect exposures from PCDDs/PCDFs at the 95th percentile level for the site where APC deposition rates were highest could potentially exceed the tolerable daily soil intake (TDSI) but this warrants further study given the model limitations. These results offer an initial screen of the significance of potential risks from APC disposal, which is of value in addressing concerns about the uncertainty of potential risks to human health from bulk APC disposal at strategic locations.

Radiographic recognition and distribution of approximal carious lesions in New Zealand undergraduate dental students.

Bitewing radiographs are widely used to detect caries on the approximal surfaces of teeth. The aim of this study was to determine the prevalence of approximal caries in an educated young adult (New Zealand dental student) population, and to compare the diagnostic decisions of students and university teachers. A total of 123 students aged from 18 to 25 years (mean age 21 years) participated, having given their informed consent. Their bitewings were digitised and assessed for approximal carious lesions from the distal surface of the first premolars to the mesial surface of the second molars on the same computer screen. The following criteria were used: (0) no detectable radiolucency, (R1) lesion confined to the outer half of the enamel, (R2) lesion into the inner half of the enamel, (R3) lesion into the outer half of dentine along the amelodentinal junction, (R4) lesion beyond the outer part of the dentine. The views were assessed by two final-year dental students and two experienced university teachers, and results were compared. A total of 2710 surfaces were examined; restored and missing surfaces (tooth absent, off film or surface unreadable) were excluded. Carious lesions were found in 173 (6.38 percent) of the surfaces. Only 57 subjects presented with carious lesions radiographically, but 16 (13 percent) of subjects had 87 percent of all the lesions. Seventy-nine percent of the lesions were enamel lesions (R1 and R2), with 21 percent of the lesions having dentine involvement (R3 and R4). Inter-examiner agreement was high, Pearson's correlation coefficient r = 0.8237, and Cohen's kappa kappa = 0.76.

Difficulty in mental, neuromusculoskeletal, and movement-related school functions associated with low birthweight or preterm birth: a meta-analysis.

OBJECTIVE. Our objective was to perform a meta-analysis to investigate whether low birthweight (LBW) or preterm birth was associated with difficulty in mental, neuromusculoskeletal, and movement-related school function tasks. METHOD. Two search strategies produced 40 studies that met the inclusion criteria for the meta-analysis and yielded 549 effect sizes (d). Heterogeneity was evaluated by obtaining Q and I-squared values. Egger's regression intercept test and a funnel plot were used to check for publication bias. RESULTS. Children born LBW exhibited considerable difficulties in mental (d = -0.655, p < .0001) and neuromusculoskeletal and movement-related tasks (d = -0.391, p < .0001) compared with children of normal birthweight. Children born preterm also exhibited significant difficulties compared with full-term children in mental, neuromusculoskeletal, and movement-related tasks (d = -0.237, p < .0001). CONCLUSION. Deficits in mental and motor functions in children born LBW or preterm appear to have significant effects on school readiness and academic achievement.

Isolation and characterization of Pigmentiphaga-like isolates from human clinical material.

Species in the genus Pigmentiphaga are Gram-negative, catalase- and oxidase-positive rods derived exclusively to date from environmental sources. Features of strains most like Pigmentiphaga daeguensis or Pigmentiphaga kullae from a case of suppurative otitis media in a 6-year-old female post-transplant recipient and in a human stool sample are described.

Assignment of Brevibacterium stationis (ZoBell and Upham 1944) Breed 1953 to the genus Corynebacterium, as Corynebacterium stationis comb. nov., and emended description of the genus Corynebacterium to include isolates that can alkalinize citrate.

Brevibacterium stationis ATCC 14403(T), Corynebacterium ammoniagenes ATCC 6872 and two clinical isolates were found to form a single taxon group consistent with the genus Corynebacterium, designated here as Corynebacterium stationis comb. nov. The type strain of Corynebacterium stationis is ATCC 14403(T) =CCUG 43497( T) =CIP 104228(T) =DSM 20302(T) =NBRC 12144(T) =JCM 11611(T) =VKM B-1228(T). These strains can utilize citrate; therefore, inclusion of C. stationis requires that the description of the genus Corynebacterium be amended to include citrate-positive strains.

First report of Actinobaculum schaalii urinary tract infection in North America.

A recurrent urinary tract infection with Actinobaculum schaalii, a fastidious, facultative anaerobic, and emerging pathogen, is described. Diagnosis was delayed when routine urine cultures were initially performed yielding recurrently negative results. Resolution of symptoms occurred after anaerobic cultures were done to allow organism isolation, identification, and appropriate antimicrobial treatment.

Syntaxin-1A inhibition of P-1075, cromakalim, and diazoxide actions on mouse cardiac ATP-sensitive potassium channel.

AIMS: Syntaxin (Syn)-1A binds sulfonylurea receptor (SUR) nucleotide binding folds of cardiac myocyte (SUR2A) and islet beta-cells (SUR1) to inhibit ATP-sensitive potassium (K(ATP)) channels. We further reported that Syn-1A reduced the potency and efficacy of beta-cell-specific K(ATP) channel openers (KCOs). Here, we examined whether Syn-1A would influence non-specific (diazoxide) and SUR2-specific KCOs [N-cyano-N'-(1,1-dimethylpropyl)-N''-3-pyridylguanidine (P-1075) and cromakalim] on cardiac myocyte K(ATP) channels activation. METHODS AND RESULTS: Confocal microscopy and Western blotting verified the presence of both Syn-1A and -1B expressions on rodent cardiac ventricular myocytes. Inside-out patch-clamp electrophysiology was utilized to examine the effects of these syntaxins on K(ATP) macroscopic currents activated by various KCOs from a stable cell line expressing the potassium inward rectifier 6.2 (Kir6.2)/SUR2A and from C57BL/6 male mouse ventricular myocytes. Syn-1A inhibited the current amplitude activated by P-1075, cromakalim and diazoxide via its H3 but not Habc domain. Syn-1B exhibited similar inhibitory effects on P-1075 activation of K(ATP) currents. In examining for direct effects of Syn-1A on the KCO binding to cardiac SUR2 receptors, we found that Syn-1A did not directly affect [(3)H]-P-1075 binding to rat cardiac membrane SUR2A at maximum binding capacity, but was able to mildly reduce the affinity of cold P-1075 and cromakalim to displace [(3)H]-P-1075 binding. CONCLUSION: In conclusion, Syn-1A (and Syn-1B) could inhibit K(ATP) currents activated by SUR2A-acting KCOs. Potential fluctuations in the levels of these syntaxins in the myocardium may affect the therapeutic effectiveness of cardiac KCOs.

The actions of a novel potent islet beta-cell specific ATP-sensitive K+ channel opener can be modulated by syntaxin-1A acting on sulfonylurea receptor 1.

Islet beta-cell-specific ATP-sensitive K(+) (K(ATP)) channel openers thiadiazine dioxides induce islet rest to improve insulin secretion, but their molecular basis of action remains unclear. We reported that syntaxin-1A binds nucleotide binding folds of sulfonylurea receptor 1 (SUR1) in beta-cells to inhibit K(ATP) channels. As a strategy to elucidate the molecular mechanism of action of these K(ATP) channel openers, we explored the possibility that 6-chloro-3-(1-methylcyclobutyl)amino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (NNC55-0462) might influence syntaxin-1A-SUR1 interactions or vice versa. Whole-cell and inside-out patch-clamp electrophysiology was used to examine the effects of glutathione S-transferase (GST)-syntaxin-1A dialysis or green fluorescence protein/syntaxin-1A cotransfection on NNC55-0462 actions. In vitro pull-down binding studies were used to examine NNC55-0462 influence on syntaxin-1A-SUR1 interactions. Dialysis of GST-syntaxin-1A into the cell cytoplasm reduced both potency and efficacy of extracellularly perfused NNC55-0462 in a HEK cell line stably expressing Kir6.2/SUR1 (BA8 cells) and in rat islet beta-cells. Moreover, inside-out membrane patches excised from BA8 cells showed that both GST-syntaxin-1A and its H3 domain inhibited K(ATP) channels previously activated by NNC55-0462. This action on K(ATP) channels is isoform-specific to syntaxin-1A because syntaxin-2 was without effect. Furthermore, the parent compound diazoxide showed similar sensitivity to GST-syntaxin-1A inhibition. NNC55-0462, however, did not influence syntaxin-1A-SUR1 binding interaction. Our results demonstrated that syntaxin-1A interactions with SUR1 at its cytoplasmic domains can modulate the actions of the K(ATP) channel openers NNC55-0462 and diazoxide on K(ATP) channels. The reduced levels of islet syntaxin-1A in diabetes would thus be expected to exert a positive influence on the therapeutic effects of this class of K(ATP) channel openers.

Syntaxin-1A actions on sulfonylurea receptor 2A can block acidic pH-induced cardiac K(ATP) channel activation.

During cardiac ischemia, ATP stores are depleted, and cardiomyocyte intracellular pH lowers to <7.0. The acidic pH acts on the Kir6.2 subunit of K(ATP) channels to reduce its sensitivity to ATP, causing channel opening. We recently reported that syntaxin-1A (Syn-1A) binds nucleotide binding folds (NBF)-1 and NBF2 of sulfonylurea receptor 2A (SUR2A) to inhibit channel activity (Kang, Y., Leung, Y. M., Manning-Fox, J. E., Xia, F., Xie, H., Sheu, L., Tsushima, R. G., Light, P. E., and Gaisano, H. Y. (2004) J. Biol. Chem. 279, 47125-47131). Here, we examined Syn-1A actions on SUR2A to influence the pH regulation of cardiac K(ATP) channels. K(ATP) channel currents from inside-out patches excised from Kir6.2/SUR2A expressing HEK293 cells and freshly isolated cardiac myocytes were increased by reducing intracellular pH from 7.4 to 6.8, which could be blocked by increasing concentrations of Syn-1A added to the cytoplasmic surface. Syn-1A had no effect on C-terminal truncated Kir6.2 (Kir6.2-deltaC26) channels expressed in TSA cells without the SUR subunit. In vitro binding and co-immunoprecipitation studies show that Syn-1A binding to SUR2A or its NBF-1 and NBF-2 domain proteins increased progressively as pH was reduced from 7.4 to 6.0. The enhancement of Syn-1A binding to SUR2A by acidic pH was further regulated by Mg2+ and ATP. Therefore, pH regulates Kir.6.2/SUR2A channels not only by its direct actions on the Kir6.2 subunit but also by modulation of Syn-1A binding to SUR2A. The increased Syn-1A binding to the SUR2A at acidic pH would assert some inhibition of the K(ATP) channels, which may serve as a "brake" to temper the fluctuation of low pH-induced K(ATP) channel opening that could induce fatal reentrant arrhythmias.

Effects of NSAIDs on bladder function in normal and cystitis rats: a comparison study of aspirin, indomethacin, and ketoprofen.

To clarify the potential usefulness of non-steroidal anti-inflammatory drugs, NSAIDs, for patients with overactive bladder, we examined the effect of NSAIDs on urodynamic parameters in normal and cystitis rats and compared their ulcerogenic activity in the gastrointestinal mucosa. Cystometry was performed after administration of the conventional NSAIDs, aspirin, indomethacin, or ketoprofen. Prostaglandin levels were measured in the bladder of cystitis rats pretreated with NSAIDs. Furthermore, the ulcerogenic responses were examined. NSAIDs increased bladder capacity without any effect on micturition pressure in normal rats in the following rank order of potency: ketoprofen > or = indomethacin > or = aspirin. In cystitis rats, bladder capacity was increased and micturition frequency was decreased. The levels of prostaglandin were significantly increased in cystitis rats. All NSAIDs inhibited the increment of prostaglandin levels at doses equal to that effective in the improvement of bladder functions. When administered intraduodenally, both ketoprofen and indomethacin induced lesions in the gastrointestinal mucosa. However, aspirin had no significant effect. We demonstrate that NSAIDs are effective in animal models of disease, most likely by suppressing by prostaglandin synthesis. Since aspirin, in contrast to ketoprofen or indomethacin, did not cause any gastrointestinal lesions, aspirin might be the NSAIDs treatment of choice for overactive bladder.

Mechanism of action by which aspirin alleviates detrusor hyperactivity in rats.

We examined the effect of aspirin on urodynamic parameters in normal and cyclophosphamide-induced cystitic rats and compared them in rats with or without sensory denervation. Cystometry was performed under urethane anesthesia; and volume threshold for micturition (VT), micturition frequency (MF), micturition pressure (MP), and micturition volume (MV) were determined. Cystitis was induced by pretreatment with cyclophosphamide and sensory denervation was performed by pretreating animals with a large dose of capsaicin. PGE(2) and 6-keto-PGF(1alpha) contents in the bladder were determined by ELISA. Sensory intact, cystitic rats showed decrement of VT and increment of MF. Aspirin increased VT and decreased MF in the cystitic condition. Both PGE(2) and 6-keto-PGF(1alpha) contents in the bladder were significantly increased in cystitic rats, but such increases were completely inhibited by aspirin. In sensory denervated rats, aspirin showed a marginal tendency of increment of VT. Cystitic rats showed overflow micturition in the sensory denervated condition, but VT was the same as that of normal rats. Furthermore, following capsaicin pretreatment, aspirin had no effect on the cystometrogram in cystitic rats. From these findings, it is concluded that suppression of sensory C-fiber via inhibition of PGs synthesis in the bladder is involved in the pharmacological action of aspirin in the detrusor hyperactivity.