48 research outputs found
Funktionelle Kooperation der Transmembransegmente S3 und S4 beim Schaltverhalten von TRPM8
No full textTRP channels are polymodal receptors that are involved in many substantial physiological processes. TRPM8 is a sensor for cold temperatures, but can additionally be activated by substances that mediate a cold feeling such as menthol. Furthermore, the TRPM8 channel is voltage-dependent, whereby the voltage sensitivity can be positively modulated both by, cold as well as by various TRPM8 channel agonist. In contrast to the well studied "classical" voltage-dependent cation channels, there is no generally accepted model for TRPM8, which describes the gating mechanism in detail so far. Based on a recently published computer simulated model for TRPM8 gating, we used site directed mutagenesis to systematically analyze potential interactions between transmembrane domain S3 and S4, which has been demonstrated to be essential for voltage sensitivity. Furthermore, the structural and functional importance of S3 and S4 for gating and folding of TRPM8 was analyzed. In the present work, the evidence of a functional cooperation between the transmembrane segment S3 and S4 in TRPM8 was proofed. The charge distribution in the central region of these protein domains plays a crucial role for a proper folding and for posttranslational maturation (glycosylation) of the channel and therefor is essential for its functionality. Furthermore, the highly conserved sequence motif N-x-x-D located in S3, is not only for TRPM8 but also for the closely related TRPM2 channel of functional relevance. In addition, new insights could be gained about the voltage dependence of TRPM8 by systematic changes of charged amino acids within the S4 segment, which question the so far favored model of an autonomous mobile S4 voltage sensor in TRPM8. Taken together, the results of this study indicate a protein domain corporately formed by S3 and S4 in TRPM8. This domain conduces i.a. the interaction of the channel with different TRPM8 agonists and additionally is of crucial importance for the voltage sensitivity of the channel, possibly through an interaction with the negatively charged phospholipid PIP2
The Cell Adhesion Molecules Roughest, Hibris, Kin of Irre and Sticks and Stones Are Required for Long Range Spacing of the Drosophila Wing Disc Sensory Sensilla.
No full textMost animal tissues and organ systems are comprised of highly ordered arrays of varying cell types. The development of external sensory organs requires complex cell-cell communication in order to give each cell a specific identity and to ensure a regular distributed pattern of the sensory bristles. This involves both long and short range signaling mediated by either diffusible or cell anchored factors. In a variety of processes the heterophilic Irre Cell Recognition Module, consisting of the Neph-like proteins: Roughest, Kin of irre and of the Nephrin-like proteins: Sticks and Stones, Hibris, plays key roles in the recognition events of different cell types throughout development. In the present study these proteins are apically expressed in the adhesive belt of epithelial cells participating in sense organ development in a partially exclusive and asymmetric manner. Using mutant analysis the GAL4/UAS system, RNAi and gain of function we found an involvement of all four Irre Cell Recognition Module-proteins in the development of a highly structured array of sensory organs in the wing disc. The proteins secure the regular spacing of sensory organs showing partial redundancy and may function in early lateral inhibition events as well as in cell sorting processes. Comparisons with other systems suggest that the Irre Cell Recognition module is a key organizer of highly repetitive structures
The match between current national assessment initiatives in reading and validated dimensions of authentic assessment
No full text"The current emphasis on national standards and accountability in education is accompanied by a burgeoning concern for educating individuals who are able to competently perform complex tasks necessary for success in ""real world"" situations. The ability to perform flexibly in ""real world"" situations implies movement toward authentic assessment of high-level thinking skills that match criterion performance settings and transcend traditional subject matter divisions. Recognizing the cognitive complexity of reading and the need for critical and evaluative reading in diverse situations, assessment and standards initiatives have strived to provide a more authentic assessment of reading processes than that which is found in previous standardized tests."This study examines the match between authentic assessment tasks associated with two major national assessment initiatives in reading (The NAEP Trial State Assessment in reading and The New Standards Project reading) assessment and validated dimensions of authentic assessment.A panel expert reviewers in the areas of reading, educational measurement, and assessment were involved in a multistage validation process which culminated in the identification of 31 important dimensions of authentic reading assessment. These dimensions describe content and format, scoring and interpretation, and technical aspects of assessment. Task, items, and texts; assessment frameworks; and administration, scoring, and interpretation procedures of The NAEP Trial State Assessment in reading and The New Standards Project reading assessment were examined to determine the fit between characteristics of the assessments and each validated dimension. Profiles are provided which offer a graphic illustration of the relationships between each of the two initiatives and dimensions of authentic assessment. Where fit does not exist between assessment characteristics and specific dimensions of authentic assessment, feasibility of compromise between the goals of high-stakes assessment and authentic assessment are explored. Implications will be discussed for developing assessment systems that incorporate local curricular decisions and national concerns for efficiency, comparability, and reliability.Made available in DSpace on 2011-05-07T13:24:08Z (GMT). No. of bitstreams: 2
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Modulation of activation and inactivation by Ca2+ and 2-APB in the pore of an archetypal TRPM channel from Nematostella vectensis
No full textAbstractThe archetypal TRPM2-like channel of the sea anemone Nematostella vectensis is gated by ADPR like its human orthologue but additionally exhibits properties of other vertebrate TRPM channels. Thus it can help towards an understanding of gating and regulation of the whole subfamily. To elucidate further the role of Ca2+ as a co-factor of ADPR, we exploited 2-aminoethyl diphenylborinate (2-APB), previously shown to exert either inhibitory or stimulatory effects on diverse TRPM channels, or both in a concentration-dependent manner. 2-APB in high concentrations (1 mM) induced large, non-inactivating currents through nvTRPM2. In lower concentrations (≤0.5 mM), it prevented the fast current inactivation typical for nvTRPM2 stimulated with ADPR. Both these effects were rapidly reversed after wash-out of 2-APB, in contrast to a considerable lag time of their onset. A detailed analysis of nvTRPM2 mutants with modified selectivity filter or reduced ADP-ribose sensitivity revealed that the actions of 2-APB depend on its access to the pore which is enhanced by channel opening. Moreover, access of Ca2+ to the pore is decisive which again depends on the open state of the channel. We conclude that separate regulatory processes by Ca2+ on the pore can be discriminated with the aid of 2-APB.</jats:p
FW 9
No full textThe four fables presented and illustrated in this volume are: The Fisherman and the Fish; The Fox and The Food; The Snobbish Monk; and The Wolf and the Lion. Surprisingly, the story after which the booklet is named comes second, not first. This version of The Fisherman and the Fish offers an explanation for the usually non-motivated expectation of the fisherman that fish would respond to his flute-playing. When he played, friends and neighbors came together to enjoy his music. He expected that fish would do the same. The moral for this version is There is no free lunch in the world. The Fox and The Food starts with this unfortunate sentence: A fix didn't found anything to eat after a long day (5). This fox soon found a hole curiously placed beside a tree and crawled into it. Where did this hole lead? Usually in the fable it leads into a tree, where a shepherd has stored his lunch. The third story is not well told but is nonetheless a fine fable. A poor man visits a temple and is snubbed by a monk. While he is there, a rich man comes and the monk fawns all over him. Accosted then by the poor man for showing favor to the rich, the monk lies, saying that he inwardly respects the poor man. The latter hits him on the head with his cane saying Good. And now I hit you because I respect you, too (12)! As you make your bed, so you must lie on it. The wolf in the fourth fable is impressed by the length of his own shadow and challenges the lion, who beats him soundly. Empty vessels make the most sound (16). The best illustration in this volume may be that of the monk winking while fawning on the rich man (10-11).Language note: Bilingual: English/Mandarin ChinesePaul Skjervol
Rst acts cooperatively with Kirre to secure the bristle pattern in the anterior wing margin.
No full text<p>(A-L) Projection views of IRM-protein immunoreactivity in late third instar larvae. Rst is shown in red (A, E, and I), Hbs in green (B, F and J), Kirre in blue (C, G and K) and SNS in yellow (D, H and L). (A-D) The <i>rst</i> allele <i>rst</i><sup><i>1R34</i></sup> shows no detectable Rst staining (A). (B) Hbs staining is reduced in the membranes surrounding the SOPs and is mainly detected in SOP membranes. Kirre (C) and SNS (D) show no significant pattern change. (E-H) <i>MZ1369-GAL4</i>><i>UAS-kirre-RNAi</i> shows no significant changes of the Rst (E) and Hbs pattern (F). Kirre immunoreactivity is hardly detectable (G) while SNS (H) is mildly reduced. (I-L) In the <i>rst</i>, <i>kirre</i> double RNAi hardly any Rst (I) and Kirre (K) can be detected. Enrichment of Hbs (J) around SOPs is reduced and the SOP arrangement as seen with SNS (L) is severely disrupted. (M) In the adult <i>MZ1369>rst-RNAi</i> shows a mild spacing phenotype with spacing ranging from 2 to 5 intervening bristles. (N) <i>MZ1369>kirre-RNAi</i> shows a mild spacing phenotype with spacing ranging from 1 to 7 (similar data was obtained for the mutant <i>rst</i><sup><i>1R34</i></sup>, data not shown). (O) <i>MZ1369>rst-RNAi</i>, <i>kirre-RNAi</i> shows a significant disturbance of the spacing of recurved bristles with 0 to 8 intervening cells. (P) <i>MZ1369-GAL4</i> misexpression of <i>rst</i> has a strong impact on the spacing of recurved bristles with spacing ranging from 0 to 13 intervening cells. Clustered recurved bristles are frequently observed and similarly long areas without any chemosensory bristles are seen. (Q) shows the quantitative analysis of the recurved bristle spacing as measured by the number of slender bristles between the recurved bristles. The distribution of <i>MZ1369>GFP</i> differs significantly from <i>MZ1369>rst-RNAi</i>, <i>kirre-RNAi</i> in the following spacing values: < = 1: p-value = 0.034, 5: p-value = 0.041. <i>MZ1369>rst</i> differs significantly in the following spacing values: < = 1: p-value = 0.0002, 3: p-value = 0.0002, > = 6: p-value = 0.0001. Scale bars correspond to 10μm in all images.</p
Model of IRM-protein interactions in the <i>Drosophila</i> anterior wing margin.
No full text<p>(A-E) Illustration of IRM-protein functions in the anterior wing margin. In black are epithelial cells shown, while SOPs are shown in orange. Protein interactions are shown in different sizes according to the strength of the interaction. Red represents Rst, green Hbs, blue Kirre and SNS is shown in yellow. In the wild type (A) preferred adhesion was observed between the SOPs and the surrounding epithelial cells. Epithelial cells are additionally stable connected through the Hbs, Rst and Hbs, Kirre interaction. In <i>rst</i><sup><i>1R34</i></sup> (B), as an example for Neph-like loss of function, preferential adhesion can still be observed between the SOPs and epithelial cells through the SNS, Kirre and Hbs Kirre interaction. Only in the case of Rst and Kirre loss, the adhesive properties of the wing margin is changed leading to bristle clusters (C). Loss of Hbs prevents heterophilic interaction between the non-SOP cells resulting in mild disturbances of the SOP pattern (D). Loss of Hbs and SNS results in total loss of heterophilic interaction between all cell types in the presumptive anterior wing margin (E). This results in strong disturbances of the SOP and later the bristle pattern (F) Summary of the inductive and competitive interactions between the IRM-proteins <i>in trans</i> and <i>in cis</i>. In the interaction between two cells <i>in trans</i> several inductive events were observed, if these events represent inductions of gene expression or stabilization of proteins in the adhesive belt by heterophilic interactions is currently unknown. Inside cells <i>in cis</i> several competitive interactions were observed, resulting in degradation of proteins in vesicles. Altogether, these interactions allow a precise regulation of IRM-protein abundance and function. (G) Chain model of preferential adhesion of IRM-proteins in the wing disc. The IRM-proteins in the wing disc secure a strong adhesive chain in the distal growing wing. Preferential adhesion around the SOPs secures a constant high number of cells between the SOPs. Growth in distal directions explains the lower number of cells between precursors compared to the adult sensory organs.</p
Surface expression and channel function of TRPM8 are cooperatively controlled by transmembrane segments S3 and S4
No full textThe Neph-like proteins Rst and Kirre affect the localization of other IRM members in <i>cis</i> and <i>trans</i>.
No full text<p>(A-M, O, Q-X) Projection views of IRM immunoreactivity in third instar larvae. Rst is shown in red (A, E, I, M-Q and U-V), Hbs in green (B, F, J, M-P, R and U-V), Kirre in blue (C, G, K, S and W-Z) and SNS in yellow (D, H, L, T and W-Z). (A-D) Misexpression of <i>rst</i> using <i>MZ1369-GAL4</i> leads to ubiquitous Rst staining (A) in the entire wing disc. Hbs (B) and Kirre (C) are significantly reduced. SNS (D) staining is unaffected in strength, but the localization is not limited to the apical contact zone of the SOPs. Instead it is found in the entire cell. Additionally, the order of the SOPs is severely disturbed. (E-H) Misexpression of <i>kirre</i> via <i>MZ1369-GAL4</i> leads to wider stripes of Rst (E) and Hbs (F) staining. Kirre (G) can be ubiquitously detected in the entire wing disc. SNS (H) staining is strong on all membranes in contact with Kirre positive membranes. Spacing of SOPs is already disrupted at this developmental stage. (I-L) Misexpression of <i>rst</i> using <i>neur-GAL4</i> leads to strongly stained Rst (I) positive SOPs. Hbs (J) is found only around the SOPs and staining of membranes not in contact to the SOPs is reduced. (K) Kirre staining is further enriched around the SOPs. (L) SNS is relocated and it is not specifically located at the adherens junction any more. (M) Magnification of three SOPs of a wild type control stained for Rst and Hbs. The dashed line shows the approximate area of cut for the Z-projection in (N). Hbs can be detected inside the SOPs (arrowhead) and also in the basal appendix (arrow). (O) Magnification of three SOPs in <i>neur-GAL4</i>><i>UAS-rst</i>. The approximate area of cut for the Z-projection in (P) is given with a dashed line. Arrowheads mark the immunopositive interior of the SOPs showing strong Rst staining. Arrows marks the basal appendix of the SOPs. Asterisk mark the co-localization of Rst and Hbs immunoreactivity at the Border of SOPs. (Q-T) Misexpression of <i>kirre</i> using <i>neur-GAL4</i> leads to strong Rst staining around the SOPs. Hbs (R) is found much stronger around or in the SOPs and is strongly reduced on the membranes not in contact with any SOPs. (S) Kirre staining is strongly found in all membranes of the SOPs, showing no apical-basal polarity. (T) SNS localization inside the SOPs is disrupted and a possible degradation product can be found in vesicles basal of the adherens junction. (U) Magnification of three SOPs of a wild type control stained for Rst and Hbs. (V) Magnification of three SOPs in <i>neur-GAL4</i>><i>UAS-kirre</i>. Strong accumulation of Hbs immunoreactivity is evident around the SOPs. (W) Magnification of three SOPs of a wild type control stained for Kirre and SNS. (X) The magnification of three SOPs of <i>neur-GAL4</i>><i>UAS-kirre</i> shows the strong Kirre staining in the entire SOP and the mislocalization of SNS. A dotted line in (W and X) shows the approximate area of a Z-cut that is shown in (Y and Z). In wild type (Y) both proteins interact with each other only in a defined apical contact zone. The distribution of the proteins is clearly visible. SNS is inside the SOPs and Kirre in the surrounding cells. In (Z) the distribution of Kirre and SNS is shown in the mutant situation. Mislocalization and vesicular degradation products of SNS can be seen basal of the adherens junction in this z-axis view. Scale bars correspond to 10μm in all images.</p
The IRM-proteins Rst, Hbs, Kirre and SNS are expressed in distinct patterns during the development of sensory bristles in the anterior wing margin.
No full text<p>(A) The <i>Drosophila</i> IRM-proteins consist of the Neph-like proteins Rst and Kirre and of the Nephrin-like proteins Hbs and SNS. (B) Representative image showing the three bristle rows of the anterior wing margin [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128490#pone.0128490.ref005" target="_blank">5</a>]. The dorsal row consists of recurved bristles marked with an arrow. The medial row consists of mechanosensory stout bristles. The ventral row is composed of mechanosensory slender bristles and recurved bristles marked with an arrow. (C) Representative image of the presumptive wing margin of a late third instar larvae. Kirre in blue can be found in the presumptive posterior wing margin (pwm), anterior wing margin (awm) and the wing veins L3, L4 and L5. SNS in yellow can be only found in the SOPs of the awm. Six example SOPs are marked with an arrow. The ventral (v) side faces up and dorsal (d) faces down. (D-E) The apical localization of the IRM-protein Rst (red) is shown in third instar larvae of the genotype <i>neur-GAL4</i>><i>UAS-mCD8-GFP</i> (green). The SOPs are specifically marked by GFP. The images show a 3D reconstruction in (D) and a lateral view (E). (F-I) Localization of the IRM-proteins in the awm of third instar larvae. Rst (F) in red is localized in two adhesive belts in the awm and is enriched at the border to the SOPs of the recurved bristles. <i>neur-GAL4</i>><i>UAS-rst-RNAi</i> shows no effect on the enrichment of staining around the SOPs (F’). A similar staining pattern in green can be seen for Hbs in wild type (G), but the SOP specific RNAi shows a reduction of staining around the SOP borders (G’), indicating Hbs expression inside the SOPs. Kirre in blue (H) shows a similar pattern as Rst and SOP specific RNAi has no effect (H’). SNS (I) in yellow can only be found at the border of the SOPs. <i>neur-GAL4</i>><i>UAS-sns-RNAi</i> reduces SNS in the SOPs (I’). Scale bars correspond to 10μm in all images.</p
