A TRP homolog in Saccharomyces cerevisiae forms an intracellular Ca2+-permeable channel in the yeast vacuolar membrane

The molecular identification of ion channels in internal membranes has made scant progress compared with the study of plasma membrane ion channels. We investigated a prominent voltage-dependent, cation-selective, and calcium-activated vacuolar ion conductance of 320 pS (yeast vacuolar conductance, YVC1) in Saccharomyces cerevisiae. Here we report on a gene, the deduced product of which possesses significant homology to the ion channel of the transient receptor potential (TRP) family. By using a combination of gene deletion and re-expression with direct patch clamping of the yeast vacuolar membrane, we show that this yeast TRP-like gene is necessary for the YVC1 conductance. In physiological conditions, tens of micromolar cytoplasmic Ca2+ activates the YVC1 current carried by cations including Ca2+ across the vacuolar membrane. Immunodetection of a tagged YVC1 gene product indicates that YVC1 is primarily localized in the vacuole and not other intracellular membranes. Thus we have identified the YVC1 vacuolar/lysosomal cation-channel gene. This report has implications for the function of TRP channels in other organisms and the possible molecular identification of vacuolar/lysosomal ion channels in other eukaryotes.

Identification of a protein that confers calcitonin gene-related peptide responsiveness to oocytes by using a cystic fibrosis transmembrane conductance regulator assay.

An expression-cloning strategy was used to isolate a cDNA that encodes a protein that confers calcitonin gene-related peptide (CGRP) responsiveness to Xenopus laevis oocytes. A guinea pig organ of Corti (the mammalian hearing organ) cDNA library was screened by using an assay based on the cystic fibrosis transmembrane conductance regulator (CFTR). The CFTR is a chloride channel that is activated upon phosphorylation; this channel activity was used as a sensor for CGRP-induced activation of intracellular kinases. A cDNA library from guinea pig organ of Corti was screened by using this oocyte-CFTR assay. A cDNA was identified that contained an open reading frame coding for a small hydrophilic protein that is presumed to be either a CGRP receptor or a component of a CGRP receptor complex. This CGRP receptor component protein confers CGRP-specific activation to the CFTR assay, as no activation was detected upon application of calcitonin, amylin, neuropeptide Y, vasoactive intestinal peptide, or beta-endorphin. In situ hybridization demonstrated that the CGRP receptor component protein is expressed in outer hair cells of the organ of Corti and is colocalized with CGRP-containing efferent nerve terminals.

Identification of geostationary satellites using polarization data from unresolved images

In order to protect critical military and commercial space assets, the United States Space Surveillance Network must have the ability to positively identify and characterize all space objects. Unfortunately, positive identification and characterization of space objects is a manual and labor intensive process today since even large telescopes cannot provide resolved images of most space objects. Since resolved images of geosynchronous satellites are not technically feasible with current technology, another method of distinguishing space objects was explored that exploits the polarization signature from unresolved images. The objective of this study was to collect and analyze visible-spectrum polarization data from unresolved images of geosynchronous satellites taken over various solar phase angles. Different collection geometries were used to evaluate the polarization contribution of solar arrays, thermal control materials, antennas, and the satellite bus as the solar phase angle changed. Since materials on space objects age due to the space environment, it was postulated that their polarization signature may change enough to allow discrimination of identical satellites launched at different times. The instrumentation used in this experiment was a United States Air Force Academy (USAFA) Department of Physics system that consists of a 20-inch Ritchey-Chrétien telescope and a dual focal plane optical train fed with a polarizing beam splitter. A rigorous calibration of the system was performed that included corrections for pixel bias, dark current, and response. Additionally, the two channel polarimeter was calibrated by experimentally determining the Mueller matrix for the system and relating image intensity at the two cameras to Stokes parameters S0 and S1. After the system calibration, polarization data was collected during three nights on eight geosynchronous satellites built by various manufacturers and launched several years apart. Three pairs of the eight satellites were identical buses to determine if identical buses could be correctly differentiated. When Stokes parameters were plotted against time and solar phase angle, the data indicates that there were distinguishing features in S0 (total intensity) and S1 (linear polarization) that may lead to positive identification or classification of each satellite.

Mutations within the selectivity filter of the NMDA receptor-channel influence voltage dependent block by 5-hydroxytryptamine

Background and purpose: Voltage-dependent block by Mg2+ is a cardinal feature of NMDA receptors which acts as a coincidence detector to prevent the receptor from over-activation. Inhibition of NMDA receptor currents by 5-hydroxytryptamine (5-HT) indicated that 5-HT, similar to Mg2+, binds within the membrane electric field. In the present study, we assessed whether point mutations of critical asparagine residues located within the selectivity filter of NR1 and NR2A subunits of NMDA receptor-channel affect voltage-dependent block by 5-HT. Experimental approach: The mode of action of 5-HT and Mg2+ on wild-type and mutated NMDA receptor-channels expressed in Xenopus oocytes was investigated using the two-electrode voltage clamp recording technique. Key results: The mutation within the NR1 subunit NR1(N0S or N0Q) strongly reduced the voltage dependent block by 5-HT and increased the IC50. The corresponding mutations within the NR2 subunits NR2A(N0Q or N + 1Q) reduced the block by 5-HT to a lesser extent. This is in contrast to the block produced by external Mg2+ where a substitution at the NR2A(N0) and NR2A(N + 1) sites but not at the NR1(N0) site significantly reduced Mg2+ block. Conclusion and implications: The block of NMDA receptor-channels by 5-HT depends on the NR1-subunit asparagine residue and to a lesser extent on the NR2A-subunit asparagine residues. These data suggest that the interaction of 5-HT with functionally important residues in a narrow constriction of the pore of the NMDA receptor-channel provides a significant barrier to ionic fluxes through the open channel due to energetic factors governed by chemical properties of the binding site and the electric field.

Identification of residues controlling transport through the yeast aquaglyceroporin Fps1 using a genetic screen

Aquaporins and aquaglyceroporins mediate the transport of water and solutes across biological membranes. Saccharomyces cerevisiae Fps1 is an aquaglyceroporin that mediates controlled glycerol export during osmoregulation. The transport function of Fps1 is rapidly regulated by osmotic changes in an apparently unique way and distinct regions within the long N- and C-terminal extensions are needed for this regulation. In order to learn more about the mechanisms that control Fps1 we have set up a genetic screen for hyperactive Fps1 and isolated mutations in 14 distinct residues, all facing the inside of the cell. Five of the residues lie within the previously characterized N-terminal regulatory domain and two mutations are located within the approach to the first transmembrane domain. Three mutations cause truncation of the C-terminus, confirming previous studies on the importance of this region for channel control. Furthermore, the novel mutations identify two conserved residues in the channel-forming B-loop as critical for channel control. Structural modelling-based rationalization of the observed mutations supports the notion that the N-terminal regulatory domain and the B-loop could interact in channel control. Our findings provide a framework for further genetic and structural analysis to better understand the mechanism that controls Fps1 function by osmotic changes.

Chronotopographical analysis of the pattern onset visual evoked magnetic response (VEMR):Implications for waveform peak identification

The topography of the visual evoked magnetic response (VEMR) to a pattern onset stimulus was studied in five normal subjects using a single channel BTi magnetometer. Topographic distributions were analysed at regular intervals following stimulus onset (chronotopograpby). Two distinct field distributions were observed with half field stimulation: (1) activity corresponding to the C11 m which remains stable for an average of 34 msec and (2) activity corresponding to the C111 m which remains stable for about 50 msec. However, the full field topography of the largest peak within the first 130 msec does not have a predictable latency or topography in different subjects. The data suggest that the appearance of this peak is dependent on the amplitude, latency and duration of the half field C11 m peaks and the efficiency of half field summation. Hence, topographic mapping is essential to correctly identify the C11 m peak in a full field response as waveform morphology, peak latency and polarity are not reliable indicators. © 1993.

Manometry-Based Cough Identification Algorithm

Gastroesophageal reflux disease (GERD) is a common cause of chronic cough. For the diagnosis and treatment of GERD, it is desirable to quantify the temporal correlation between cough and reflux events. Cough episodes can be identified on esophageal manometric recordings as short-duration, rapid pressure rises. The present study aims at facilitating the detection of coughs by proposing an algorithm for the classification of cough events using manometric recordings. The algorithm detects cough episodes based on digital filtering, slope and amplitude analysis, and duration of the event. The algorithm has been tested on in vivo data acquired using a single-channel intra-esophageal manometric probe that comprises a miniature white-light interferometric fiber optic pressure sensor. Experimental results demonstrate the feasibility of using the proposed algorithm for identifying cough episodes based on real-time recordings using a single channel pressure catheter. The presented work can be integrated with commercial reflux pH/impedance probes to facilitate simultaneous 24-hour ambulatory monitoring of cough and reflux events, with the ultimate goal of quantifying the temporal correlation between the two types of events.

Entropy feature extraction of EEG signals for automatic person identification

The use of human brain electroencephalography (EEG) signals for automatic person identi cation has been investigated for a decade. It has been found that the performance of an EEG-based person identication system highly depends on what feature to be extracted from multi-channel EEG signals. Linear methods such as Power Spectral Density and Autoregressive Model have been used to extract EEG features. However these methods assumed that EEG signals are stationary. In fact, EEG signals are complex, non-linear, non-stationary, and random in nature. In addition, other factors such as brain condition or human characteristics may have impacts on the performance, however these factors have not been investigated and evaluated in previous studies. It has been found in the literature that entropy is used to measure the randomness of non-linear time series data. Entropy is also used to measure the level of chaos of braincomputer interface systems. Therefore, this thesis proposes to study the role of entropy in non-linear analysis of EEG signals to discover new features for EEG-based person identi- cation. Five dierent entropy methods including Shannon Entropy, Approximate Entropy, Sample Entropy, Spectral Entropy, and Conditional Entropy have been proposed to extract entropy features that are used to evaluate the performance of EEG-based person identication systems and the impacts of epilepsy, alcohol, age and gender characteristics on these systems. Experiments were performed on the Australian EEG and Alcoholism datasets. Experimental results have shown that, in most cases, the proposed entropy features yield very fast person identication, yet with compatible accuracy because the feature dimension is low. In real life security operation, timely response is critical. The experimental results have also shown that epilepsy, alcohol, age and gender characteristics have impacts on the EEG-based person identication systems.

Radio-Frequency Interference Identification Over Oceans for C- and X-Band AMSR2 Channels

A new method for radio-frequency interference (RFI) contamination identification over open oceans for the two C-subbands and X-band of Advanced Microwave Scanning Radiometer 2 (AMSR2) channel measurements is suggested. The method is based both on the AMSR2 brightness temperature (T-B) modeling and on the analysis of AMSR2 measurements over oceans. The joint analysis of T-B spectral differences allowed to identify the relations between them and the limits of their variability, which are ensured by the changes in the environmental conditions. It was found that the constraints, based on the ratio of spectral differences, are more regionally and seasonally independent than the spectral differences themselves. Although not all possible RFI combinations are considered, the developed simple criteria can be used to detect most RFI-contaminated pixels over the World Ocean for AMSR2 measurements in two C-subbands and the X-band.

Changes In The Bacterial Community Of Soil From A Neutral Mine Drainage Channel.

Mine drainage is an important environmental disturbance that affects the chemical and biological components in natural resources. However, little is known about the effects of neutral mine drainage on the soil bacteria community. Here, a high-throughput 16S rDNA pyrosequencing approach was used to evaluate differences in composition, structure, and diversity of bacteria communities in samples from a neutral drainage channel, and soil next to the channel, at the Sossego copper mine in Brazil. Advanced statistical analyses were used to explore the relationships between the biological and chemical data. The results showed that the neutral mine drainage caused changes in the composition and structure of the microbial community, but not in its diversity. The Deinococcus/Thermus phylum, especially the Meiothermus genus, was in large part responsible for the differences between the communities, and was positively associated with the presence of copper and other heavy metals in the environmental samples. Other important parameters that influenced the bacterial diversity and composition were the elements potassium, sodium, nickel, and zinc, as well as pH. The findings contribute to the understanding of bacterial diversity in soils impacted by neutral mine drainage, and demonstrate that heavy metals play an important role in shaping the microbial population in mine environments.

Taurine Supplementation Increases K(atp) Channel Protein Content, Improving Ca2+ Handling And Insulin Secretion In Islets From Malnourished Mice Fed On A High-fat Diet.

Pancreatic β-cells are highly sensitive to suboptimal or excess nutrients, as occurs in protein-malnutrition and obesity. Taurine (Tau) improves insulin secretion in response to nutrients and depolarizing agents. Here, we assessed the expression and function of Cav and KATP channels in islets from malnourished mice fed on a high-fat diet (HFD) and supplemented with Tau. Weaned mice received a normal (C) or a low-protein diet (R) for 6 weeks. Half of each group were fed a HFD for 8 weeks without (CH, RH) or with 5% Tau since weaning (CHT, RHT). Isolated islets from R mice showed lower insulin release with glucose and depolarizing stimuli. In CH islets, insulin secretion was increased and this was associated with enhanced KATP inhibition and Cav activity. RH islets secreted less insulin at high K(+) concentration and showed enhanced KATP activity. Tau supplementation normalized K(+)-induced secretion and enhanced glucose-induced Ca(2+) influx in RHT islets. R islets presented lower Ca(2+) influx in response to tolbutamide, and higher protein content and activity of the Kir6.2 subunit of the KATP. Tau increased the protein content of the α1.2 subunit of the Cav channels and the SNARE proteins SNAP-25 and Synt-1 in CHT islets, whereas in RHT, Kir6.2 and Synt-1 proteins were increased. In conclusion, impaired islet function in R islets is related to higher content and activity of the KATP channels. Tau treatment enhanced RHT islet secretory capacity by improving the protein expression and inhibition of the KATP channels and enhancing Synt-1 islet content.

Mucopolysaccharidosis Type Ii: Identification Of 30 Novel Mutations Among Latin American Patients.

In this study, 103 unrelated South-American patients with mucopolysaccharidosis type II (MPS II) were investigated aiming at the identification of iduronate-2-sulfatase (IDS) disease causing mutations and the possibility of some insights on the genotype-phenotype correlation The strategy used for genotyping involved the identification of the previously reported inversion/disruption of the IDS gene by PCR and screening for other mutations by PCR/SSCP. The exons with altered mobility on SSCP were sequenced, as well as all the exons of patients with no SSCP alteration. By using this strategy, we were able to find the pathogenic mutation in all patients. Alterations such as inversion/disruption and partial/total deletions of the IDS gene were found in 20/103 (19%) patients. Small insertions/deletions/indels (<22 bp) and point mutations were identified in 83/103 (88%) patients, including 30 novel mutations; except for a higher frequency of small duplications in relation to small deletions, the frequencies of major and minor alterations found in our sample are in accordance with those described in the literature.

Identification Of Target Genes Using Gene Expression Profile Of Granulocytes From Patients With Chronic Myeloid Leukemia Treated With Tyrosine Kinase Inhibitors.

Differential gene expression analysis by suppression subtractive hybridization with correlation to the metabolic pathways involved in chronic myeloid leukemia (CML) may provide a new insight into the pathogenesis of CML. Among the overexpressed genes found in CML at diagnosis are SEPT5, RUNX1, MIER1, KPNA6 and FLT3, while PAN3, TOB1 and ITCH were decreased when compared to healthy volunteers. Some genes were identified and involved in CML for the first time, including TOB1, which showed a low expression in patients with CML during tyrosine kinase inhibitor treatment with no complete cytogenetic response. In agreement, reduced expression of TOB1 was also observed in resistant patients with CML compared to responsive patients. This might be related to the deregulation of apoptosis and the signaling pathway leading to resistance. Most of the identified genes were related to the regulation of nuclear factor κB (NF-κB), AKT, interferon and interleukin-4 (IL-4) in healthy cells. The results of this study combined with literature data show specific gene pathways that might be explored as markers to assess the evolution and prognosis of CML as well as identify new therapeutic targets.

The Analgesic Effect Of Dipyrone In Peripheral Tissue Involves Two Different Mechanisms: Neuronal K(atp) Channel Opening And Cb(1) Receptor Activation.

Dipyrone (metamizole) is an analgesic pro-drug used to control moderate pain. It is metabolized in two major bioactive metabolites: 4-methylaminoantipyrine (4-MAA) and 4-aminoantipyrine (4-AA). The aim of this study was to investigate the participation of peripheral CB1 and CB2 cannabinoid receptors activation in the anti-hyperalgesic effect of dipyrone, 4-MAA or 4-AA. PGE2 (100ng/50µL/paw) was locally administered in the hindpaw of male Wistar rats, and the mechanical nociceptive threshold was quantified by electronic von Frey test, before and 3h after its injection. Dipyrone, 4-MAA or 4-AA was administered 30min before the von Frey test. The selective CB1 receptor antagonist AM251, CB2 receptor antagonist AM630, cGMP inhibitor ODQ or KATP channel blocker glibenclamide were administered 30min before dipyrone, 4-MAA or 4-AA. The antisense-ODN against CB1 receptor expression was intrathecally administered once a day during four consecutive days. PGE2-induced mechanical hyperalgesia was inhibited by dipyrone, 4-MAA, and 4-AA in a dose-response manner. AM251 or ODN anti-sense against neuronal CB1 receptor, but not AM630, reversed the anti-hyperalgesic effect mediated by 4-AA, but not by dipyrone or 4-MAA. On the other hand, the anti-hyperalgesic effect of dipyrone or 4-MAA was reversed by glibenclamide or ODQ. These results suggest that the activation of neuronal CB1, but not CB2 receptor, in peripheral tissue is involved in the anti-hyperalgesic effect of 4-aminoantipyrine. In addition, 4-methylaminoantipyrine mediates the anti-hyperalgesic effect by cGMP activation and KATP opening.

High-throughput Analysis By Sp-ldi-ms For Fast Identification Of Adulterations In Commercial Balsamic Vinegars.

Balsamic vinegar (BV) is a typical and valuable Italian product, worldwide appreciated thanks to its characteristic flavors and potential health benefits. Several studies have been conducted to assess physicochemical and microbial compositions of BV, as well as its beneficial properties. Due to highly-disseminated claims of antioxidant, antihypertensive and antiglycemic properties, BV is a known target for frauds and adulterations. For that matter, product authentication, certifying its origin (region or country) and thus the processing conditions, is becoming a growing concern. Striving for fraud reduction as well as quality and safety assurance, reliable analytical strategies to rapidly evaluate BV quality are very interesting, also from an economical point of view. This work employs silica plate laser desorption/ionization mass spectrometry (SP-LDI-MS) for fast chemical profiling of commercial BV samples with protected geographical indication (PGI) and identification of its adulterated samples with low-priced vinegars, namely apple, alcohol and red/white wines.