Evolution of Vertebrate Transient Receptor Potential Vanilloid 3 Channels: Opposite Temperature Sensitivity between Mammals and Western Clawed Frogs

Transient Receptor Potential (TRP) channels serve as temperature receptors in a wide variety of animals and must have played crucial roles in thermal adaptation. The TRP vanilloid (TRPV) subfamily contains several temperature receptors with different temperature sensitivities. The TRPV3 channel is known to be highly expressed in skin, where it is activated by warm temperatures and serves as a sensor to detect ambient temperatures near the body temperature of homeothermic animals such as mammals. Here we performed comprehensive comparative analyses of the TRPV subfamily in order to understand the evolutionary process; we identified novel TRPV genes and also characterized the evolutionary flexibility of TRPV3 during vertebrate evolution. We cloned the TRPV3 channel from the western clawed frog Xenopus tropicalis to understand the functional evolution of the TRPV3 channel. The amino acid sequences of the N- and C-terminal regions of the TRPV3 channel were highly diversified from those of other terrestrial vertebrate TRPV3 channels, although central portions were well conserved. In a heterologous expression system, several mammalian TRPV3 agonists did not activate the TRPV3 channel of the western clawed frog. Moreover, the frog TRPV3 channel did not respond to heat stimuli, instead it was activated by cold temperatures. Temperature thresholds for activation were about 16 °C, slightly below the lower temperature limit for the western clawed frog. Given that the TRPV3 channel is expressed in skin, its likely role is to detect noxious cold temperatures. Thus, the western clawed frog and mammals acquired opposite temperature sensitivity of the TRPV3 channel in order to detect environmental temperatures suitable for their respective species, indicating that temperature receptors can dynamically change properties to adapt to different thermal environments during evolution

Triad pattern algorithm for predicting strong promoter candidates in bacterial genomes

Abstract Background Bacterial promoters, which increase the efficiency of gene expression, differ from other promoters by several characteristics. This difference, not yet widely exploited in bioinformatics, looks promising for the development of relevant computational tools to search for strong promoters in bacterial genomes. Results We describe a new triad pattern algorithm that predicts strong promoter candidates in annotated bacterial genomes by matching specific patterns for the group I σ70 factors of Escherichia coli RNA polymerase. It detects promoter-specific motifs by consecutively matching three patterns, consisting of an UP-element, required for interaction with the α subunit, and then optimally-separated patterns of -35 and -10 boxes, required for interaction with the σ70 subunit of RNA polymerase. Analysis of 43 bacterial genomes revealed that the frequency of candidate sequences depends on the A+T content of the DNA under examination. The accuracy of in silico prediction was experimentally validated for the genome of a hyperthermophilic bacterium, Thermotoga maritima, by applying a cell-free expression assay using the predicted strong promoters. In this organism, the strong promoters govern genes for translation, energy metabolism, transport, cell movement, and other as-yet unidentified functions. Conclusion The triad pattern algorithm developed for predicting strong bacterial promoters is well suited for analyzing bacterial genomes with an A+T content of less than 62%. This computational tool opens new prospects for investigating global gene expression, and individual strong promoters in bacteria of medical and/or economic significance.</p

Acid-evoked Ca2+ signalling in rat sensory neurones: effects of anoxia and aglycaemia

Ischaemia excites sensory neurones (generating pain) and promotes calcitonin gene-related peptide release from nerve endings. Acidosis is thought to play a key role in mediating excitation via the activation of proton-sensitive cation channels. In this study, we investigated the effects of acidosis upon Ca2+ signalling in sensory neurones from rat dorsal root ganglia. Both hypercapnic (pHo 6.8) and metabolic–hypercapnic (pHo 6.2) acidosis caused a biphasic increase in cytosolic calcium concentration ([Ca2+]i). This comprised a brief Ca2+ transient (half-time approximately 30 s) caused by Ca2+ influx followed by a sustained rise in [Ca2+]i due to Ca2+ release from caffeine and cyclopiazonic acid-sensitive internal stores. Acid-evoked Ca2+ influx was unaffected by voltage-gated Ca2+-channel inhibition with nickel and acid sensing ion channel (ASIC) inhibition with amiloride but was blocked by inhibition of transient receptor potential vanilloid receptors (TRPV1) with (E)-3-(4-t-butylphenyl)-N-(2,3-dihydrobenzo[b][1,4] dioxin-6-yl)acrylamide (AMG 9810; 1 μM) and N-(4-tertiarybutylphenyl)-4-(3-cholorphyridin-2-yl) tetrahydropryazine-1(2H)-carbox-amide (BCTC; 1 μM). Combining acidosis with anoxia and aglycaemia increased the amplitude of both phases of Ca2+ elevation and prolonged the Ca2+ transient. The Ca2+ transient evoked by combined acidosis, aglycaemia and anoxia was also substantially blocked by AMG 9810 and BCTC and, to a lesser extent, by amiloride. In summary, the principle mechanisms mediating increase in [Ca2+]i in response to acidosis are a brief Ca2+ influx through TRPV1 followed by sustained Ca2+ release from internal stores. These effects are potentiated by anoxia and aglycaemia, conditions also prevalent in ischaemia. The effects of anoxia and aglycaemia are suggested to be largely due to the inhibition of Ca2+-clearance mechanisms and possible increase in the role of ASICs

Genetic mapping studies of 40 loci and 23 cosmids in chromosome 11p13-11q13, and exclusion of mu-calpain as the multiple endocrine neoplasia type 1 gene.

Forty loci (16 polymorphic and 24 non-polymorphic) together with 23 cosmids isolated from a chromosome 11-specific library were used to construct a detailed genetic map of 11p13-11q13. The map was constructed by using a panel of 13 somatic cell hybrids that sub-divided this region into 19 intervals, a meiotic mapping panel of 33 multiple endocrine neoplasia type 1 (MEN1) families (134 affected and 269 unaffected members) and a mitotic mapping panel that was used to identify loss of heterozygosity in 38 MEN1-associated tumours. The results defined the most likely order of the 16 loci as being: 11pter-D11S871-(D11S288, D11S149)-11cen-CNTF-PGA-ROM1-D11S480-PYGM- SEA-D11S913-D11S970-D11S97- D11S146-INT2-D11S971-D11S533-11qter. The meiotic mapping studies indicated that the most likely location of the MEN1 gene was in the interval flanked by PYGM and D11S97, and the results of mitotic mapping suggested a possible location of the MEN1 gene telomeric to SEA. Mapping studies of the gene encoding mu-calpain (CAPN1) located CAPN1 to 11q13 and in the vicinity of the MEN1 locus. However, mutational analysis studies did not detect any germ-line CAPN1 DNA sequence abnormalities in 47 unrelated MEN1 patients and the results therefore exclude CAPN1 as the MEN1 gene. The detailed genetic map that has been constructed of the 11p13-11q13 region should facilitate the construction of a physical map and the identification of candidate genes for disease loci mapped to this region

Ammattiosaamisen näytöt yrityksen ja ammattiopiston yhteistyössä

Opinnäytetyön aiheena oli selvittää, miten ammattiosaamisen näytöt toimivat paikallisen osuuskaupan ja ammattiopiston yhteistyössä koskien ravintolan salipuolen tarjoilijoita. Ammattiosaamisen näytöt ovat olleet käytössä työssäoppijoiden arvioinnissa viisi vuotta. Tavoitteena oli saada tietoa näyttöjen hyödyistä työantajanäkökulmasta katsottuna sekä löytää mahdollisia kehittämiskohteita. Opinnäytetyön teoreettisessa osassa selvennettiin työssäoppimisen ja ammattiosaamisen näytön käsitteitä, mikä on niiden merkitys ammattikoulutuksessa ja toisaalta niiden merkitys työelämälle. Lisäksi selvitettiin yritysten rekrytointikäytäntöjä ja sitä, mitä on työelämän ja oppilaitosten yhteistyö. Tutkimuksen empiirisessä osassa käytettiin puolistrukturoitua teemahaastattelua. Haastateltavat olivat paikallisen osuuskaupan ravintoloiden esimiehiä, ja haastattelut tehtiin kuudelle henkilölle. Haastattelut nauhoitettiin ja litteroitiin sanasta sanaan tietokoneelle jatkoanalyysia varten. Tutkimuksen tuloksena vahvistui se, että ammattiosaamisen näytöt ovat kaikille selkeä asia ja niistä on hyötyä sekä työnantajalle että opiskelijalle. Yhteistyö osuuskaupan ja ammattiopiton kanssa toimii hyvin, eivätkä näyttötilanteet ole aiheuttaneet suurempia ongelmia. Ainoastaan työssäoppijan osaamisen arviointi koetaan hankalana nykyisellä asteikolla. Alan ammattiopistotasoista koulutusta tarvitaan edelleen Joensuun alueella, ja tutkimuksen mukaan sitä pitäisi jopa lisätä. Koulutetun työvoiman puute ei vielä ole maakunnassa suuri ongelma verrattuna pääkaupunkiseutuun, mutta uhkakuvia on jo nyt havaittavissa. Tähän tulisi yhteistyökumppaneiden kiinnittää huomiota, jotta jatkossa Joensuun alueella koulutetaan riittävästi työvoimaa alalle.The purpose of the thesis was to find out how the collaboration between a company and vocational college works concerning vocational skills demonstrations of restaurant waiters. In addition, the task was to examine if there is any benefit of skills demonstrations for the company, and how to develop co-operation. Partners for the study are a local hotel and restaurant company and a local vocational college. The information was gathered from literature, journals, the Internet and by interviewing. In the theoretical part of the study the main issue was to explain what on-the-job learning is and what vocational skills demonstrations are, and what their consequence is for vocational education and for work life. Also how companies carry out their recruitment was clarified. Data for this study were collected by interviewing six supervisors in partner restaurants. The results of the study show that vocational skills demonstrations are perceived well without any complexities. Rather the results indicate more utilities for students, for the company and for the vocational school. The final result of this thesis was that it is important to keep on vocational education in Joensuu and even to enhance it. Now the lack of trained waiters is a fact in southern Finland. A vision is that it will be a threat also in eastern Finland in the future. To ensure professional labour for hotel and restaurant business, companies support for vocational education is essential