Variation in allergen content over time of acrylates/methacrylates in patch test preparations.

BACKGROUND: Acrylates/methacrylates are volatile substances. There might be a gradual decrease in acrylate/methacrylate allergen content over time in patch test preparations but this has not yet been documented. OBJECTIVES: To determine the allergen content of acrylates/methacrylates in patch test preparations over time under different storage conditions. METHODS: Five acrylate/methacrylate allergens [2-hydroxyethyl methacrylate (2-HEMA), methyl methacrylate (MMA), ethylene glycol dimethacrylate (EGDMA), triethylene glycol diacrylate (TREGDA) and 2-hydroxypropyl acrylate (2-HPA)] in syringes and IQ™ chambers (Chemotechnique Diagnostics, Vellinge, Sweden) were analysed using gel permeation chromatography and high-performance liquid chromatography to measure the allergen content over time in samples stored in the freezer, refrigerator and under room temperature. RESULTS: The concentration of allergens in syringes decreased with time. Those stored at room temperature had the fastest rate of decrease, followed by those in the refrigerator and freezer. In most cases, in syringes or IQ™ chambers under all storage conditions, the MMA decreased most rapidly, followed by 2-HPA, 2-HEMA, EGDMA and TREGDA. The allergens in the IQ™ chambers rapidly disappeared, with almost all samples reaching nondetectable levels by day 8. MMA was the first to reach a nondetectable level--at day 2. CONCLUSIONS: Acrylate/methacrylate allergens are lost rapidly from IQ™ chambers especially if stored at room temperature. Allergens in syringes remain above 80% of their initial concentrations for longer periods compared with IQ™ chambers. In syringes and IQ™ chambers there is a slower rate of decrease in concentration when the storage temperature is lower. Allergens should be stored refrigerated, replaced regularly, and freshly applied on to test patches on the day of use.

Scaling of musical preferences by the mentally retarded.

The ability of institutionalized retardates in scaling musical preferences was compared with that of normals. The retardates' scale values and scale forms obtained by two kinds of scaling procedures are very similar to those of normals. Deficits, however, are observed in their lower internal consistency and relative uncertainty and in higher response polarization and perseveration.

Early-onset epilepsy and postnatal lethality associated with an editing-deficient GluR-B allele in mice.

The arginine residue at position 586 of the GluR-B subunit renders heteromeric alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-sensitive glutamate receptor channels impermeable to calcium. The codon for this arginine is introduced at the precursor messenger RNA (pre-mRNA) stage by site-selective adenosine editing of a glutamine codon. Heterozygous mice engineered by gene targeting to harbor an editing-incompetent GluR-B allele synthesized unedited GluR-B subunits and, in principal neurons and interneurons, expressed AMPA receptors with increased calcium permeability. These mice developed seizures and died by 3 weeks of age, showing that GluR-B pre-mRNA editing is essential for brain function.

Relative abundance of subunit mRNAs determines gating and Ca2+ permeability of AMPA receptors in principal neurons and interneurons in rat CNS.

Recording of glutamate-activated currents in membrane patches was combined with RT-PCR-mediated AMPA receptor (AMPAR) subunit mRNA analysis in single identified cells of rat brain slices. Analysis of AMPARs in principal neurons and interneurons of hippocampus and neocortex and in auditory relay neurons and Bergmann glial cells indicates that the GluR-B subunit in its flip version determines formation of receptors with relatively slow gating, whereas the GluR-D subunit promotes assembly of more rapidly gated receptors. The relation between Ca2+ permeability of AMPAR channels and the relative GluR-B mRNA abundance is consistent with the dominance of this subunit in determining the Ca2+ permeability of native receptors. The results suggest that differential expression of GluR-B and GluR-D subunit genes, as well as splicing and editing of their mRNAs, account for the differences in gating and Ca2+ permeability of native AMPAR channels.

Trypsin activates pancreatic duct epithelial cell ion channels through proteinase-activated receptor-2.

Proteinase-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is cleaved by trypsin within the NH2-terminus, exposing a tethered ligand that binds and activates the receptor. We examined the secretory effects of trypsin, mediated through PAR-2, on well-differentiated nontransformed dog pancreatic duct epithelial cells (PDEC). Trypsin and activating peptide (AP or SLIGRL-NH2, corresponding to the PAR-2 tethered ligand) stimulated both an 125I- efflux inhibited by Ca2+-activated Cl- channel inhibitors and a 86Rb+ efflux inhibited by a Ca2+-activated K+ channel inhibitor. The reverse peptide (LRGILS-NH2) and inhibited trypsin were inactive. Thrombin had no effect, suggesting absence of PAR-1, PAR-3, or PAR-4. In Ussing chambers, trypsin and AP stimulated a short-circuit current from the basolateral, but not apical, surface of PDEC monolayers. In monolayers permeabilized basolaterally or apically with nystatin, AP activated apical Cl- and basolateral K+ conductances. PAR-2 agonists increased [Ca2+]i in PDEC, and the calcium chelator BAPTA inhibited the secretory effects of AP. PAR-2 expression on dog pancreatic ducts and PDEC was verified by immunofluorescence. Thus, trypsin interacts with basolateral PAR-2 to increase [Ca2+]i and activate ion channels in PDEC. In pancreatitis, when trypsinogen is prematurely activated, PAR-2-mediated ductal secretion may promote clearance of toxins and debris.

Loading of oxidizable transmitters into secretory vesicles permits carbon-fiber amperometry.

Carbon-fiber amperometry detects oxidizable molecules released by exocytosis. We extended this electrochemical technique to cells that do not normally secrete oxidizable transmitters. We incubated AtT-20 cells, pituitary gonadotropes, cultured cerebellar granule cells, and yeast with high concentrations of dopamine (DA) and observed spontaneous and evoked quantal release of DA by amperometry. The rate of detectable spontaneous amperometric events was used as a measure of loading in AtT-20 cells. With 70 mm DA in the bath, loading was complete within 40 min. Cytoplasmic accumulation preceded vesicular loading. Loading decreased proportionally as the bath DA concentration was lowered. Loading rates were similar at 37 and 25 degrees C and much slower at 15 degrees C. Loading was blocked by bafilomycin A(1), a proton pump inhibitor, but not by bupropion, an inhibitor of the plasma membrane DA transporter. Other cells were tested. Spontaneous quantal events became more frequent and evoked events became larger and more frequent when PC12 cells were loaded with DA. Fluid-phase loading of neurons by short stimulation in DA solutions seemed selective for the synaptic vesicles. Thus, many cell types can be loaded with DA to study spontaneous and evoked exocytosis. The amine molecules enter these cells passively and may become concentrated in acidic vesicles by protonation.

Regulation of exocytosis by protein kinases and Ca(2+) in pancreatic duct epithelial cells.

We asked if the mechanisms of exocytosis and its regulation in epithelial cells share features with those in excitable cells. Cultured dog pancreatic duct epithelial cells were loaded with an oxidizable neurotransmitter, dopamine or serotonin, and the subsequent release of these exogenous molecules during exocytosis was detected by carbon-fiber amperometry. Loaded cells displayed spontaneous exocytosis that may represent constitutive membrane transport. The quantal amperometric events induced by fusion of single vesicles had a rapid onset and decay, resembling those in adrenal chromaffin cells and serotonin-secreting leech neurons. Quantal events were frequently preceded by a "foot," assumed to be leak of transmitters through a transient fusion pore, suggesting that those cell types share a common fusion mechanism. As in neurons and endocrine cells, exocytosis in the epithelial cells could be evoked by elevating cytoplasmic Ca(2+) using ionomycin. Unlike in neurons, hyperosmotic solutions decreased exocytosis in the epithelial cells, and giant amperometric events composed of many concurrent quantal events were observed occasionally. Agents known to increase intracellular cAMP in the cells, such as forskolin, epinephrine, vasoactive intestinal peptide, or 8-Br-cAMP, increased the rate of exocytosis. The forskolin effect was inhibited by the Rp-isomer of cAMPS, a specific antagonist of protein kinase A, whereas the Sp-isomer, a specific agonist of PKA, evoked exocytosis. Thus, PKA is a downstream effector of cAMP. Finally, activation of protein kinase C by phorbol-12-myristate-13-acetate also increased exocytosis. The PMA effect was not mimicked by the inactive analogue, 4alpha-phorbol-12,13-didecanoate, and it was blocked by the PKC antagonist, bisindolylmaleimide I. Elevation of intracellular Ca(2+) was not needed for the actions of forskolin or PMA. In summary, exocytosis in epithelial cells can be stimulated directly by Ca(2+), PKA, or PKC, and is mediated by physical mechanisms similar to those in neurons and endocrine cells.

Glucose metabolism and oscillatory behavior of pancreatic islets.

A variety of oscillations are observed in pancreatic islets. We establish a model incorporating two oscillatory systems of different time scales: One is the well-known bursting model in pancreatic cells and the other is the glucose-insulin feedback model which considers direct and indirect feedback of secreted insulin. These two are coupled to interact with each other in the combined model, and two basic assumptions are made on the basis of biological observations: The conductance gK(ATP) for the ATP-dependent potassium current is a decreasing function of the glucose concentration whereas the insulin secretion rate is given by a function of the intracellular calcium concentration. Obtained via extensive numerical simulations are complex oscillations including clusters of bursts, slow and fast calcium oscillations, and so on. We also consider how the intracellular glucose concentration depends upon the extracellular glucose concentration, and examine the inhibitory effects of insulin.

Advances in immunopharmacology of asthma.

Asthma is a chronic inflammatory disease characterized by airway hyperresponsiveness and recurrent reversible airway obstruction. As there appears to be a preponderance of T-helper 2 (Th2) cells over Th1 cells in asthma, more attention has been focused on the role of Th2-derived cytokines such as interleukin (IL)-4 and IL-5 and their corresponding signaling pathways in the pathophysiology of the disease. These complex pathways may involve the activation of signal transducers and activators of transcription (STATs) and nuclear factor-kappaB (NF-kappaB). On the other hand, immunoglobulin (Ig) E-mediated mechanisms and the protein tyrosine kinase signaling cascade are important in triggering the release of mediators from inflammatory cells. In spite of all of these, host regulatory mechanisms exist to limit the inflammation. An increase in the 3', 5'-cyclic adenosine monophosphate (cAMP) level generally suppresses the activities of immune and inflammatory cells, and the level of cAMP is closely regulated by a family of phosphodiesterases (PDEs). Heparin, a glycosaminoglycan released exclusively from mast cells, also is believed to possess anti-inflammatory actions. Many new therapeutic agents have been developed either to attenuate the pro-inflammatory processes in asthma or to augment the host anti-inflammatory mechanisms. In this article, we discuss the immunopharmacology of several of these agents, which include heparin and inhibitors of PDEs, tyrosine kinases, and NF-kappaB, as well as antibodies and soluble receptors directed against IgE, IL-4, and IL-5.

Micronema in man: third fatal infection.

Micronema, normally free-living in soil and humus, rarely invades and reproduces in the central nervous system, kidneys, lungs, maxillae and nasal cavity of equines. Two Micronema infections causing fatal meningoencephalomyelitis in man have been reported from Canada and Texas. Here we report a third infection in a 54-year-old black man, resident of Washington, D.C., who probably acquired the infection from decubitus ulcers. The worms in this patient were in the liver, heart and brain. The Micronema species was not identified.

Rapid fabrication of plastic-insulated carbon-fiber electrodes for micro-amperometry.

Carbon-fiber amperometry and voltammetry are useful techniques to measure secretion of oxidizable neurotransmitters from neurosecretory cells. Recent applications with probes of small geometry permit detection of the exocytosis of single secretory vesicles in individual cells. We have developed a semi-automatic puller and cutter to prepare such plastic-insulated electrodes efficiently with various sizes of carbon fibers. The electrodes are smooth, reproducible, and easy to make.

Activating effect of the flavonoid phloretin on Ca(2+)-activated K+ channels in myelinated nerve fibers of Xenopus laevis [corrected].

The effects of the flavonoid phloretin on K+ channels in amphibian myelinated nerve were studied by patch clamping. The open probability of Ca(2+)-activated K+ channels was greatly increased by external phloretin (10-200 microM) due to a shift of the membrane potential for half-maximal activation, E50, of -63.9 mV (80 microM phloretin). Open times were prolonged and closed times shortened. Channel activation by phloretin developed slowly (tau on = 33.4 s) and its washout was even slower (tau off,1 = 4.7 and tau off,2 = 183.2 s). In contrast, submillimolar phloretin blocked two delayed rectifier K+ channels (I and F) whereas the gating of the ATP-sensitive and the flickering K+ channel were unaffected. Phloretin may directly affect the voltage sensor of K+ channels.

The ageing worker.

In Singapore, the age for retirement has increased from 55 years to 60 years, and will eventually reach 67 years. At the same time, the proportion of workers aged above 45 years will continue to increase. The World Health Organisation has reported that in 1980, 32% of the working population were older than 45 years of age in countries of the Organisation for Economic Co-operation and Development (OECD). This proportion is expected to rise to 35.5% in the year 2000 and 41.3% in the year 2055. What are the implications of the emergence of an ageing workforce? This population represents a special group of individuals in the workforce that have special health, occupational and environmental needs. On the one hand, they have the problem of a reduction in physical work capacity, decreased adaptability, and a generally lower health status. On the other, ageing workers are more experienced and have greater expertise. They are also usually more motivated and may generally have a more positive attitude when compared with younger workers. Society, as well as health care professionals, needs to respond to this issue of an ageing workforce. The response should be three pronged. Firstly, prevention of the premature decline of physical capacities and adaptability of the worker could be addressed by health promotion and continuing job training. Secondly, some measures for adjusting work demands in accordance with functional capacities of the individual are needed. Thirdly, employers and fellow workers should be educated on the strengths of the ageing worker, and the capacity of such workers to continue contributing because of their experience, motivation and skills. If implemented, these measures would ensure a path towards productive ageing. The end results would be that ageing workers would have their functional capacity maintained, the concept of "age-adjusted workload" would be a reality, and ageing workers would not be discriminated against, but instead have their contributions to society maximised.

Impact of tinnitus as measured by the Tinnitus Handicap Inventory among tinnitus sufferers in Singapore.

INTRODUCTION: The effects of tinnitus on quality of life (QOL) have never been extensively studied in Singapore. We describe the characteristics of tinnitus and its impact on QOL as measured by the Tinnitus Handicap Inventory (THI) in a series of ear, nose and throat clinic patients. METHODS: A total of 327 patients who attended a tinnitus counselling clinic completed the THI questionnaire, a self-report measure with 25 items grouped into functional, emotional and catastrophic subscales. RESULTS: The mean age of the 134 female and 193 male patients was 48.9 years. 36.7 percent of these patients had bilateral tinnitus and 64.6 percent had symptoms for less than one year. 270 patients had hearing loss, 74 percent of whom presented with bilateral high frequency hearing loss. Most patients (84.1 percent) perceived only one type of sound. The total THI score distribution was: 107 (33 percent) patients had THI less than 16, 100 (31 percent) had THI 18 to 36, 59 (18 percent) had THI 38 to 56, and 61 (19 percent) had THI more than 58. There were no differences in the overall THI and subscale scores between the patients' gender, those with or without hearing loss, and those with unilateral or bilateral tinnitus. However, significantly higher total THI and all subscale scores were found among patients who were hearing more than one type of tinnitus sound. The areas of concern that were commonly reported by the patients in this series were a lack of control over tinnitus, frustration and stress. CONCLUSION: Tinnitus patients who hear multiple sounds tend to have a higher THI and subscale scores. The management of tinnitus should address common areas of concern, and may include counselling. The THI is a potential screening tool to determine if patients require counselling. A series of THI assessments can be used to chart the progress of treatment.

Epidermal growth factor increases insulin secretion and lowers blood glucose in diabetic mice.

Epidermal growth factor (EGF) is synthesized in the pancreas and diabetic animals have low levels of EGF. However, the role of EGF in regulating the major function of the pancreas, insulin secretion, has not been studied. Here, we show that EGF rapidly increased insulin secretion in mouse pancreatic islets, as well as in a pancreatic beta-cell line. These events were dependent on a Ca(2+) influx and phospholipase D (PLD) activity, particularly PLD2, as determined using pharmacological blockers and molecular manipulations such as over-expression and siRNA of PLD isozymes. In addition, EGF also increased plasma insulin levels and mediated glucose lowering in normal and diabetic mice. Here, for the first time, we provide evidence that EGF is a novel secretagogue that regulates plasma glucose levels and a candidate for the development of therapeutics for diabetes.

A method for rapid exchange of solutions at membrane patches using a 10-microliters microcapsule.

A rapid exchange (less than 2 ms) of the bath solution facing a membrane patch is accomplished by driving the tip of a pipette from the bath through a 100-microns oil layer into a small capsule filled with 10 microliters test solution. The microcapsule method can be applied to both excised patch configurations, inside-out and outside-out patches. On and off reactions of Ca(2+)-activated K+ channel activity have been recorded after changing the intracellular Ca2+ concentration using an inside-out patch. A blockade of these K+ channels by external tetraethylammonium ions is demonstrated with an outside-out patch. The blocking kinetics of delayed-rectifier K+ channels by a purified peptide toxin from snake venom, dendrotoxin, could be measured with our microcapsule method. Using tiny volumes of test solutions this method can be helpful in experiments involving scarce or expensive solutions.

Single-channel analysis of a delayed rectifier K+ channel in Xenopus myelinated nerve.

Single-channel properties of a delayed rectifier voltage-gated K+ channel (I-type) were investigated in peripheral myelinated axons from Xenopus laevis. Channels activated between -60 and -40 mV with a potential of half-maximal activation, E50, at -47.5 mV. Averaged single-channel currents activated with a time delay at all membrane potentials tested. Time to half-maximal activation decreased from 80 to 1.6 msec between -60 and +40 mV. The channel inactivated monoexponentially with a time constant of 10.9 sec at -40 mV. The time constant of deactivation was 126 msec at -80 mV and 16.9 msec at -110 mV. In symmetrical 105 mM K+, the single-channel conductance (gamma) was 22 and 13 pS at negative and positive membrane potentials, respectively, at 13-15 degrees C. In Na+ -rich solution with 2.5 mM extracellular K+ gamma was 7 pS and the reversal potential was negative to -80 mV, indicating a high selectivity for K+ over Na+. gamma depended on extracellular K+ concentration (KD = 19.6 mM) and temperature (Q10 = 1.45). External tetraethylammonium (TEA) reduced the apparent single-channel amplitude at all potentials tested with a half-maximal inhibiting concentration (IC50) of 0.6 mM. Open probability of the channel, but not single-channel current amplitude was decreased by extracellular dendrotoxin (DTX, IC50 = 6.8 nM). mast cell degranulating peptide (MCDP, IC50 = 41.9 microM). In Ringer solution the membrane potential of macroscopic I-channel patches was about -65 mV and depolarized under TEA and DTX. It is concluded that besides their activation during action potentials, I-channels may also stabilize the resting membrane potential.

Modulation by neurotransmitters of catecholamine secretion from sympathetic ganglion neurons detected by amperometry.

Many neuromodulators inhibit N-type Ca2+ currents via G protein-coupled pathways in acutely isolated superior cervical ganglion (SCG) neurons. Less is known about which neuromodulators affect release of norepinephrine (NE) at varicosities and terminals of these neurons. To address this question, we used carbon fiber amperometry to measure catecholamine secretion evoked by electrical stimulation at presumed sites of high terminal density in cultures of SCG neurons. The pharmacological properties of action potential-evoked NE release paralleled those of N-type Ca2+ channels: Release was completely blocked by Cd2+ or omega-conotoxin GVIA, reduced 50% by 10 microM NE or 62% by 2 microM UK-14,304, an alpha2-adrenergic agonist, and reduced 63% by 10 microM oxotremorine M (Oxo-M), a muscarinic agonist. Consistent with action at M2 or M4 receptor subtypes, Oxo-M could be antagonized by 10 microM muscarinic antagonists methoctramine and tropicamide but not by pirenzepine. After overnight incubation with pertussis toxin, inhibition by UK-14,304 and Oxo-M was much reduced. Other neuromodulators known to inhibit Ca2+ channels in these cells, including adenosine, prostaglandin E2, somatostatin, and secretin, also depressed secretion by 34-44%. In cultures treated with omega-conotoxin GVIA, secretion dependent on L-type Ca2+ channels was evoked with long exposure to high K+ Ringer's solution. This secretion was not sensitive to UK-14,304 or Oxo-M. Evidently, many neuromodulators act on the secretory terminals of SCG neurons, and the depression of NE release at terminals closely parallels the membrane-delimited inhibition of N-type Ca2+ currents in the soma.

Na(+)-activated K+ channels localized in the nodal region of myelinated axons of Xenopus.

1. A potassium channel activated by internal Na+ ions (K+Na channel) was identified in peripheral myelinated axons of Xenopus laevis using the cell-attached and excised configurations of the patch clamp technique. 2. The single-channel conductance for the main open state was 88 pS with [K+]o = 105 mM and pS with [K+]o = 2.5 mM ([K+]i = 105 mM). The channel was selectively permeable to K+ over Na+ ions. A characteristic feature of the K+Na channel was the frequent occurrence of subconductance states. 3. The open probability of the channel was strongly dependent on the concentration of Na+ ions at the inner side of the membrane. The half-maximal activating Na+ concentration and the Hill coefficient were 33 mM and 2.9, respectively. The open probability of the channel showed only weak potential dependence. 4. The K+Na channel was relatively insensitive to external tetraethylammonium (TEA+) in comparison with voltage-dependent axonal K+ channels; the half-maximal inhibitory concentration (IC50) was 21.3 mM (at -90 mV). In contrast, the channel was blocked by low concentrations of external Ba2+ and Cs+ ions, with IC50 values of 0.7 and 1.1 mM, respectively (at -90 mV). The block by Ba2+ and Cs+ was more pronounced at negative than at positive membrane potentials. 5. A comparison of the number of K+Na channels in nodal and paranodal patches from the same axon revealed that the channel density was about 10-fold higher at the node of Ranvier than at the paranode. Moreover, a correlation between the number of K+Na channels and voltage-dependent Na+ channels in the same patches was found, suggesting co-localization of both channel types. 6. As weakly potential-dependent ('leakage') channels, axonal K+Na channels may be involved in setting the resting potential of vertebrate axons. Simulations of Na+ ion diffusion suggest two possible mechanisms of activation of K+Na channels: the local increase of Na+ concentration in a cluster of Na+ channels during a single action potential or the accumulation in the intracellular axonal compartment during a train of action potentials.

Block of native Ca(2+)-permeable AMPA receptors in rat brain by intracellular polyamines generates double rectification.

1. The influence of intracellular factors on current rectification of different subtypes of native alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate receptors (AMPARs) was studied in rat brain slices by combining fast application of glutamate with patch pipette perfusion. 2. The peak current-voltage (I-V) relation of the AMPARs expressed in Bergmann glial cells of cerebellum and dentate gyrus (DG) basket cells of hippocampus was weakly rectifying in outside-out patches and nystatin-perforated vesicles, but showed a doubly rectifying shape with a region of reduced slope between 0 and +40 mV in nucleated patches. The I-V relation of AMPARs expressed in hippocampal CA3 pyramidal neurones was linear in all recording configurations. 3. Intracellular application of 25 microM spermine, a naturally occurring polyamine, blocked outward currents in outside-out patches from Bergmann glial cells and DG basket cells in a voltage-dependent manner, generating I-V relations with a doubly rectifying shape which were similar to those recorded in nucleated patches. AMPARs in CA3 pyramidal cell patches were unaffected by 25 microM spermine. 4. The half-maximal blocking concentration of spermine at +40 mV was 0.3 microM in Bergmann glial cell patches and 1.5 microM in DG basket cell patches, whereas it was much higher (>> 100 microM) for CA3 pyramidal cell patches. Spermidine also affected current rectification, but with lower affinity. The block of outward current by polyamines following voltage jumps developed within < 0.5 ms. 5. We conclude that current rectification, rather than being an intrinsic property of the Ca(2+)-permeable AMPAR channel, is generated by polyamine block.