Small Molecule Investigation of KCNQ Potassium Channels: A Dissertation

Voltage-gated K+ channels associate with multiple regulatory proteins to form complexes with diverse gating properties and pharmacological sensitivities. Small molecules which activate or inhibit channel function are valuable tools for dissecting the assembly and function of these macromolecular complexes. My thesis focuses on the discovery and use of small molecules to probe the structure and function of the KCNQ family of voltage-gated K+ channels. One protein that obligatorily assembles with KCNQ channels to mediate proper assembly, trafficking, and gating is the calcium sensor, calmodulin. Although resolution of the crystal structures of calmodulin associated with isolated peptide fragments from other ion channels has provided some insight into how calmodulin interacts with and modulates KCNQ channels, structural information for calmodulin bound to a fully folded ion channel in the membrane is unknown. In Chapter II, I developed an intracellular tethered blocker approach to determine the location of calmodulin binding with respect to the KCNQ ion-conducting pathway. Using distance restraints from a panel of these intracellular tethered blockers we then generated models of the KCNQ-calmodulin complex. Our model places calmodulin close to the gate of KCNQ channels, providing structural insight into how CaM is able to communicate changes in intracellular calcium levels to KCNQ channel complexes. In addition to pore blockers, chemical modification of ion channels has been used to probe ion channel function. During my initial attempt to chemically activate KCNQ channels, I discovered that some boronates modulate KCNQ complexes. In Chapter III, the activating derivative, phenylboronic acid, is characterized. Characterization of activation by phenylboronic acid showed that it targeted the ion conduction pathway of KCNQ channels with some specificity over other voltage-gated K+ channels. The commercial availability of thousands of boronic acid derivatives provides a large class of compounds with which to systematically dissect the mechanisms of KCNQ gating and may lead to the discovery of a potent activator of KCNQ complexes for the treatment of channelopathies. All of the electrophysiological studies presented in this thesis were conducted in Xenopus oocytes. Unexpectedly, during the studies described above, the quality of our Xenopus oocytes declined. The afflicted oocytes developed black foci on their membranes, had negligible electric resting potentials, and poor viability. Culturing the compromised oocytes determined that they were infected with multi-drug resistant Stenotrophomonas maltophilia, Pseudomonas fluorescens and Pseudomonas putida. Antibiotic testing showed that all three species of bacteria were susceptible to amikacin and ciprofloxacin, which when included in the oocyte storage media prevented the appearance of black foci and resulted in oocytes that were usable for electrophysiological recordings. This study provides a solution to a common issue that plagues many electrophysiologists who use Xenopus oocytes. Taken together, these findings provide new insights into activation of KCNQ channel complexes and provide new tools to study the structure-function relationship of voltage-gated K+ channels

Hanna Kupś, Maciej Szatkowski, Michał Dahl (Eds.). 70 lat Chińskiej Republiki Ludowej w ujęciu interdyscyplinarnym, Warszawa 2021, pp. 309

Review of Hanna Kupś, Maciej Szatkowski, Michał Dahl (Eds.). 70 lat Chińskiej Republiki Ludowej w ujęciu interdyscyplinarnym, Warszawa 2021, pp. 309

„Jak wierna córka, która troszczy się o to, co pozostało po jej matce, świętej Jerozolimie”. Akka jako ośrodek praktyk pątniczych w XII i XIII stuleciu

In the history of Acre, located on the Syrian coast, the 12th and 13th centu­ries were of particular importance. Under the rule of the Crusaders, the city experienced a period of rapid demographic, economic, cultural, and religious growth. As the main port of the Kingdom of Jerusalem—and in the 13th cen­tury its capital as well—it was an important stop on the route of Latin pil­grims. Nevertheless, it was mentioned extremely rarely in the pilgrimage writ­ings of that period, where information about the sites of worship in the city is scarce. This problem was noticed by Aryeh Graboïs and David Jacoby, but their attempts to explain this state of affairs need to be partly reexamined. The most important reason for the “silence of pilgrimage sources” about the city and its religious life seems to be the marginal presence of Acre in the pages of the Bible and its negligible place in the history of salvation.W dziejach położonej na wybrzeżu syryjskim Akki okres XII i XIII stulecia miał znaczenie szczególne. Pod panowaniem krzyżowców miasto przeżywało szybki rozwój demograficzny, gospodarczy, kulturalny i religijny. Jako główny port, a w XIII wieku także stolica Królestwa Jerozolimskiego było ważnym punktem na szlaku ówczesnych łacińskich pielgrzymów. Mimo to w piśmien­nictwie pielgrzymkowym tego okresu wspominano o nim niezwykle rzadko, a informacje o znajdujących się w mieście ośrodkach kultu mają charakter marginalny. Problem ten został zauważony przez A. Graboïsa i D. Jacoby’ego, ale przedstawione przez obu badaczy próby wyjaśnienia przyczyn takiego stanu rzeczy wymagają częściowej rewizji. Najistotniejszym powodem „mil­czenia źródeł” pielgrzymkowych na temat miasta i jego życia religijnego wydaje się marginalna obecność Akki na kartach Biblii i jej niewielkie znacze­nie w historii zbawienia

Experimental and theoretical study of shuttle lee-side heat transfer rates

The experimental program which was conducted in the Calspan 96-inch hypersonic shock tunnel to investigate what effect the windward surface temperature had on the heat transfer to the leeward surface of the space shuttle orbiter is discussed. Heat-transfer distributions, surface-pressure distributions, and schlieren photographs were obtained for an 0.01-scale model of the 139 configuration space shuttle orbiter at angles-of-attack of 30 and 40 deg. Similar data were obtained for an 0.01 scale wingless model of the 139 configuration at angles-of-attack of 30 and 90 deg. Data were obtained for Mach numbers from Reynolds numbers, and surface temperatures and compared with theoretical results

Eksklamacje (wykrzyknienia) o funkcji emotywnej w dawnych i współczesnych tekstach nabożeństwa drogi krzyżowej

The article presents a comparison of older and more contemporary texts in respect of the functions and frequency of exclamations. The analysis is based on about 100 Polish texts used in Catholic ceremonies of the Stations of the Cross, published before the Second Vatican Council and 100 contemporary texts of the same devotional practice. It appears that the number of exclamations in contemporary texts has decreased radically. In the older material exclamations are numerous and have a variety of structures. In contemporary texts we find far fewer, usually in special contexts, especially in highly stylised and ironic ones. One can see the influence of other discourses and genres and the communicative situation as well. The paper also deals with indicators of exclamations in spoken and written texts.Artykuł przedstawia porównanie dawnych i współczesnych tekstów pod względem funkcji i częstości występowania eksklamacji. Analiza opiera się 100 tekstach nabożeństwa drogi krzyżowej wydanych przed soborem watykańskim II i 100 współczesnych tekstach. Okazuje się, że liczba wykrzyknień znacząco spada w ciągu ostatniego stulecia. W dawnym nabożeństwie eksklamacje są liczne i urozmaicone pod względem struktury. Obecnie pojawiają się pojedynczo i w specjalnych kontekstach, zwłaszcza w stylizacji i w użyciach ironicznych. Widoczny jest wpływ innych dyskursów i gatunków oraz sytuacji komunikacyjnej. Artykuł porusza także problem wyznaczników eksklamacji w tekstach pisanych i mówionych.

Drug transporters in spermatogenesis: A re-evaluation of recent data on P-glycoprotein

Drug transporters are integral membrane proteins expressed by a variety of organs, including the liver, kidney, small intestine and testis, and they are generally known to mediate drug or xenobiotic transport into and out of cells. Previous studies have also reported the presence of several drug transporters at blood-tissue barriers where they are thought to protect organs from harmful agents. In this editorial, we briefly discuss and re-evaluate recent findings that show P-glycoprotein, an efflux pump, to function at the blood-testis barrier. We also put forth a mechanistic model, hoping this information will form a strong basis for future studies

Succeeding at Your Internship: A Handbook Written for and with Students

There are several textbooks for students whose majors include internships in human services, broadly defi­­­ned, such as case management, counseling, criminal justice, and social work. Most of these books are written in an academic format. Typically, it involves an introduction to a theoretical orientation that concerns working with others followed by a series of chapters devoted to learning professional skills associated with a given discipline. This approach is fine, as far as it goes, but also has two drawbacks. One is that the texts are usually sold by main stream publishers, which means they are expensive. Another is that they seldom address what might be described as the experiential dimension of the internship that most beginners face on their own. This new book addresses both concerns. The fact that it is offered as a free text addresses the first issue, of course, but the second one requires a new approach. It began with asking students to talk about what they experienced when going through their first internship and what they would tell others about how to make it a successful one. That work led to a structured narrative about basic practical topics, such as finding an internship, getting started there, making effective use of supervision, understanding ethics, appreciating cultural diversity, becoming competent, and completing the internship. The text includes descriptions, suggestions, and exercises. It may be used as either a primary course text or, due to its relative brevity, a supplemental one. Although the lead editor is an experienced clinician and professor who has supervised internships for a variety of human services majors over many years, the book was written with and for students to make it more readable and more useful.https://scholarworks.bgsu.edu/oer/1000/thumbnail.jp

The adhering junction dynamics in the testis are regulated by an interplay of beta 1-integrin, focal adhesion kinase (FAK) and the focal adhesion complex (FAC)-associated proteins

published_or_final_versio

Regulation of blood-testis barrier dynamics: An in vivo study

An in vivo model was used to investigate the regulation of tight junction (TJ) dynamics in the testis when adult rats were treated with CdCl2. It was shown that the CdCl2-induced disruption of the blood-testis barrier (BTB) associated with a transient induction in testicular TGF-β2 and TGF-β3 (but not TGF-β1 and the phosphorylated p38 mitogen activated protein (MAP) kinase, concomitant with a loss of occludin and zonula occludens-1 (ZO-1) from the BTB site in the seminiferous epithelium. These results suggest that BTB dynamics in vivo are regulated by TGF-β2/-β3 via the p38 MAP kinase pathway. Indeed, SB202190, a specific p38 MAP kinase inhibitor, blocked the CdCl2-induced occludin and ZO-1 loss from the BTB. This result clearly illustrates that CdCl2 mediates its BTB disruptive effects via the TGF-β3/p38 MAP kinase signaling pathway. Besides, this CdCl2-induced occludin and ZO-1 loss from the BTB also associated with a significant loss of the cadherin/catenin and the nectin/afadin protein complexes at the site of cell-cell actin-based adherens junctions (AJs). An induction of α2-macroglobulin (a non-specific protease inhibitor) was also observed during BTB damage and when the seminiferous epithelium was being depleted of germ cells. These data illustrate that a primary disruption of the BTB can lead to a secondary loss of cell adhesion function at the site of AJs, concomitant with an induction in protease inhibitor, which apparently is used to protect the epithelium from unwanted proteolysis. α2-Macroglobulin was also shown to associate physically with TGF-β3, afadin and nectin 3, but not occludin, E-cadherin or N-cadherin, indicating its possible role in junction restructuring in vivo. Additionally, the use of SB202190 to block the TGF-β3/p-38 MAP kinase pathway also prevented the CdCl2-induced loss of cadherin/catenin and nectin/afadin protein complexes from the AJ sites, yet it had no apparent effect on α2-macroglobulin. These results demonstrate for the first time that the TGF-β3/p38 MAP kinase signaling pathway is being used to regulate both TJ and AJ dynamics in the testis, mediated by the effects of TGF-β3 on TJ- and AJ-integral membrane proteins and adaptors, but not protease inhibitors.published_or_final_versio

Differential interactions between transforming growth factor-β3/ TβR1, TAB1, and CD2AP disrupt blood-testis barrier and sertoli-germ cell adhesion

The biochemical basis that regulates the timely and selective opening of the blood-testis barrier (BTB) to migrating preleptotene/leptotene spermatocytes at stage VIII of the epithelial cycle in adult rat testes is virtually unknown. Recent studies have shown that cytokines (e.g. transforming growth factor (TGF)-β3) may play a crucial role in this event. However, much of this information relies on the use of toxicants (e.g. CdCl 2), making it difficult to relay these findings to normal testicular physiology. Here we report that overexpression of TGF-β3 in primary Sertoli cells cultured in vitro indeed perturbed the tight junction (TJ) barrier with a concomitant decline in the production of BTB constituent proteins as follows: occludin, N-cadherin, and ZO-1. Additionally, local administration of TGF-β3 to testes in vivo was shown to reversibly perturb the BTB integrity and Sertoli-germ cell adhesion via the p38 MAPK and ERK signaling pathways. Most importantly, the simultaneous activation of p38 and ERK signaling pathways is dependent on the association of the TGF-β3-TβR1 complex with adaptors TAB1 and CD2AP because if TβR1 was associated preferentially with CD2AP, only Sertoli-germ cell adhesion was perturbed without compromising the BTB. Collectively, these data illustrate that local production of TGF-β3, and perhaps other TGF-βs and cytokines, by Sertoli and germ cells into the microenvironment at the BTB during spermatogenesis transiently perturbs the BTB and Sertoli-germ cell adhesion to facilitate germ cell migration when the activated TβRI interacts with adaptors TAB1 and CD2AP. However, TGF-β3 selectively disrupts Sertoli-germ cell adhesion in the seminiferous epithelium to facilitate germ cell migration without compromising BTB when TβRI interacts only with adaptor CD2AP. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.postprin