Superoxide dismutase mutations of familial amyotrophic lateral sclerosis and the oxidative inactivation of calcineurin

AbstractApproximately 10% of all familial cases of amyotrophic lateral sclerosis (fALS) are linked to mutations in the SOD1 gene, which encodes the copper/zinc superoxide dismutase (CuZnSOD). Recently, wild-type CuZnSOD was shown to protect calcineurin, a calcium/calmodulin-regulated phosphoprotein phosphatase, from inactivation by reactive oxygen species. We asked whether the protective effect of CuZnSOD on calcineurin is affected by mutations associated with fALS. For this, we monitored calcineurin activity in the presence of mutant and wild-type SOD. We found that the degree of protection against inactivation of calcineurin by different SOD mutants correlates with the severity of the phenotype associated with the different mutations, suggesting a potential role for calcineurin–SOD1 interaction in the etiology of fALS

Biophysical characterisation of the recombinant human frataxin precursor

Friedreich's ataxia is a disease caused by a decrease in the levels of expression or loss of functionality of the mitochondrial protein frataxin (FXN). The development of an active and stable recombinant variant of FXN is important for protein replacement therapy. Although valuable data about the mature form FXN81‐210 has been collected, not enough information is available about the conformation of the frataxin precursor (FXN1‐210). We investigated the conformation, stability and function of a recombinant precursor variant (His6‐TAT‐FXN1‐210), which includes a TAT peptide in the N‐terminal region to assist with transport across cell membranes. His6‐TAT‐FXN1‐210 was expressed in Escherichia coli and conditions were found for purifying folded protein free of aggregation, oxidation or degradation, even after freezing and thawing. The protein was found to be stable and monomeric, with the N‐terminal stretch (residues 1–89) mostly unstructured and the C‐terminal domain properly folded. The experimental data suggest a complex picture for the folding process of full‐length frataxin in vitro: the presence of the N‐terminal region increased the tendency of FXN to aggregate at high temperatures but this could be avoided by the addition of low concentrations of GdmCl. The purified precursor was translocated through cell membranes. In addition, immune response against His6‐TAT‐FXN1‐210 was measured, suggesting that the C‐terminal fragment was not immunogenic at the assayed protein concentrations. Finally, the recognition of recombinant FXN by cellular proteins was studied to evaluate its functionality. In this regard, cysteine desulfurase NFS1/ISD11/ISCU was activated in vitro by His6‐TAT‐FXN1‐210. Moreover, the results showed that His6‐TAT‐FXN1‐210 can be ubiquitinated in vitro by the recently identified frataxin E3 ligase RNF126, in a similar way as the FXN1‐210, suggesting that the His6‐TAT extension does not interfere with the ubiquitination machinery.Fil: Castro, Ignacio Hugo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Ferrari, Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Herrera, Maria Georgina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Noguera, Martín Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Maso, Lorenzo. Universita Di Padova. Dipartimento Di Biología; ItaliaFil: Benini, Monica. Fratagene Therapeutics Srl; Italia. University Of Rome Tor Vergata; ItaliaFil: Rufini, Alessandra. University Of Rome Tor Vergata; Italia. Fratagene Therapeutics Srl; ItaliaFil: Testi, Roberto. University Of Rome Tor Vergata; Italia. Fratagene Therapeutics Srl; ItaliaFil: Costantini, Paola. Universita Di Padova. Dipartimento Di Biología; ItaliaFil: Santos, Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentin

Cell‐penetrating peptides: Achievements and challenges in application for cancer treatment

One of the major hurdles to cure cancer lies in the low potency of currently available drugs, which could eventually be solved by using more potent therapeutic macromolecules, such as proteins or genes. However, although these macromolecules possess greater potency inside the cancer cells, the barely permeable cell membrane remains a formidable barrier to exert their efficacy. A widely used strategy is to use cell penetrating peptides (CPPs) to improve their intracellular uptake. Since the discovery of the first CPP, numerous CPPs have been derived from natural or synthesized products. Both in vitro and in vivo studies have demonstrated that those CPPs are highly efficient in transducing cargoes into almost all cell types. Therefore, to date, CPPs have been widely used for intracellular delivery of various cargoes, including peptides, proteins, genes, and even nanoparticles. In addition, recently, based on the successes of CPPs in cellular studies, their applications in vivo have been actively pursued. This review will focus on the advanced applications of CPP‐based in vivo delivery of therapeutics (e.g., small molecule drugs, proteins, and genes). In addition, we will highlight certain updated applications of CPPs for intracellular delivery of nanoparticulate drug carriers, as well as several “smart” strategies for tumor targeted delivery of CPP‐cargoes. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 575–587, 2014.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102051/1/jbma34859.pd

Conjugation of a Ru(II) Arene Complex to Neomycin or to Guanidinoneomycin Leads to Compounds with Differential Cytotoxicities and Accumulation between Cancer and Normal Cells

A straightforward methodology for the synthesis of conjugates between a cytotoxic organometallic ruthenium(II) complex and amino- and guanidinoglycosides, as potential RNA-targeted anticancer compounds, is described. Under microwave irradiation, the imidazole ligand incorporated on the aminoglycoside moiety (neamine or neomycin) was found to replace one triphenylphosphine ligand from the ruthenium precursor [(η6-p-cym)RuCl(PPh3)2]+, allowing the assembly of the target conjugates. The guanidinylated analogue was easily prepared from the neomycin-ruthenium conjugate by reaction with N,N′-di-Boc-N″-triflylguanidine, a powerful guanidinylating reagent that was compatible with the integrity of the metal complex. All conjugates were purified by semipreparative high-performance liquid chromatography (HPLC) and characterized by electrospray ionization (ESI) and matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and NMR spectroscopy. The cytotoxicity of the compounds was tested in MCF-7 (breast) and DU-145 (prostate) human cancer cells, as well as in the normal HEK293 (Human Embryonic Kidney) cell line, revealing a dependence on the nature of the glycoside moiety and the type of cell (cancer or healthy). Indeed, the neomycin-ruthenium conjugate (2) displayed moderate antiproliferative activity in both cancer cell lines (IC50 ≈ 80 μM), whereas the neamine conjugate (4) was inactive (IC50 ≈ 200 μM). However, the guanidinylated analogue of the neomycin-ruthenium conjugate (3) required much lower concentrations than the parent conjugate for equal effect (IC50 = 7.17 μM in DU-145 and IC50 = 11.33 μM in MCF-7). Although the same ranking in antiproliferative activity was found in the nontumorigenic cell line (3 2 > 4), IC50 values indicate that aminoglycoside-containing conjugates are about 2-fold more cytotoxic in normal cells (e.g., IC50 = 49.4 μM for 2) than in cancer cells, whereas an opposite tendency was found with the guanidinylated conjugate, since its cytotoxicity in the normal cell line (IC50 = 12.75 μM for 3) was similar or even lower than that found in MCF-7 and DU-145 cancer cell lines, respectively. Cell uptake studies performed by ICP-MS with conjugates 2 and 3 revealed that guanidinylation of the neomycin moiety had a positive effect on accumulation (about 3-fold higher in DU-145 and 4-fold higher in HEK293), which correlates well with the higher antiproliferative activity of 3. Interestingly, despite the slightly higher accumulation in the normal cell than in the cancer cell line (about 1.4-fold), guanidinoneomycin-ruthenium conjugate (3) was more cytotoxic to cancer cells (about 1.8-fold), whereas the opposite tendency applied for neomycin-ruthenium conjugate (2). Such differences in cytotoxic activity and cellular accumulation between cancer and normal cells open the way to the creation of more selective, less toxic anticancer metallodrugs by conjugating cytotoxic metal-based complexes such as ruthenium(II) arene derivatives to guanidinoglycosides

Mechanisms and strategies for effective delivery of antisense and siRNA oligonucleotides

The potential use of antisense and siRNA oligonucleotides as therapeutic agents has elicited a great deal of interest. However, a major issue for oligonucleotide-based therapeutics involves effective intracellular delivery of the active molecules. In this Survey and Summary, we review recent reports on delivery strategies, including conjugates of oligonucleotides with various ligands, as well as use of nanocarrier approaches. These are discussed in the context of intracellular trafficking pathways and issues regarding in vivo biodistribution of molecules and nanoparticles. Molecular-sized chemical conjugates and supramolecular nanocarriers each display advantages and disadvantages in terms of effective and nontoxic delivery. Thus, choice of an optimal delivery modality will likely depend on the therapeutic context

Typische Aufgaben und Loesungsstrategien bei der Sanierung von bewohnten Altbauten Auswertung der Erfahrungen beim Modellprojekt Bunsenstrasse 15 in Kassel

TIB Hannover: RO 68(14) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEDEGerman

Порівняльна характеристика морфологічних особливостей, типів конституції і моторних якостей дівчат-студенток різних поколінь (Іркутська область, Росія)

Background and Study Aim. The purpose of the study is to give a comparative description of the somatotypes, anatomical components of the body, and motor qualities of female students studied at the university (Irkutsk, Russia) in 2009 and 2019. Material and methods. It was conducted the survey and somatotypological diagnosis of 1226 female students (in 2009 – n = 762; in 2019 – n = 464) on 27 anthropometric parameters. It was measured: Pinier index; the average value of the absolute and percentage of the bone component of the body (BC), the fat component of the body (FC), and muscle component of the body (MC). The following indicators were evaluated by motor tests: speed endurance and agility; speed; speed and strength endurance of the trunk flexor muscles; strength and endurance of the shoulder girdle muscles; dynamic strength of the lower limb muscles; active flexibility of the spine; overall endurance. Results. In 2019, compared to 2009, there was observed: a decrease (by 19.6%) in the number of normosthenic females; an increase in the number of hypersthenics (by 70.9%), and asthenics (by 27.4%); body weight gain; decrease in body length. The values of the following indicators significantly decreased in 2019: the trunk length, upper and lower limbs; chest circumference. There was a decrease in the average values of body circumference; shoulder width. It was observed an increase in pelvic width. In 2019, compared to 2009, the following indicators were determined in females of all somatotypes: a significant increase in body fat content and a decrease in muscle mass (p <0.05). As a result, the strength of the hands' dynamometry decreased. After 10 years, the bone component in the females’ body has not changed. Conclusions. The obtained data indicate a deterioration in all motor skills of females surveyed in 2019, compared to 2009. This is a consequence of the growing hypodynamics of modern youth. The obtained results of surveys expand the database of anthropometric and motor parameters of the young generation of Russia. This data can be used in planning training and coaching activities in educational and sports organizations.Цель. Цель исследования – дать сравнительную характеристику типам конституции, анатомических компонентов тела и двигательных качеств девушек-студенток, обучавшихся в университете (Иркутск, Россия) в 2009 и 2019 гг. Материал и методы. Проведено обследование и соматотипологическая диагностика 1226 девушек-студенток (в 2009 г. - n=762; в 2019 г. - n=464) по 27 антропометрическим параметрам. Рассчитывался: индекс Пинье; среднее значение абсолютного и процентного содержания костного компонента тела (КК), жирового компонента тела (ЖК) и мышечного компонента тела (МК). По моторным тестам оценивали: скоростную выносливость и ловкость; быстроту; скоростно-силовую выносливость мышц сгибателей туловища; силу и силовую выносливость мышц верхнего плечевого пояса; динамическую силу мышц нижних конечностей; активную гибкость позвоночника; общую выносливость. Результаты. В 2019 году, по сравнению с 2009 годом, наблюдается: снижение (на 19,6%) количества девушек-нормостеников; увеличение количества гиперстеников (на 70,9%) и астеников (на 27,4%); увеличение массы тела; уменьшение длины тела. Достоверно уменьшились в 2019 году значения показателей: длины туловища, верхних и нижних конечностей; окружности грудной клетки. Наблюдалось снижение средних значений: обхватных размеров тела; показателей ширины плеч. Наблюдалось увеличения ширины таза. В 2019 году, по сравнению с 2009 годом, установлено у девушек всех типов конституции: достоверное увеличение содержания в теле жировой массы и снижение мышечной массы (р<0,05). В следствие этого, снизились силовые показатели динамометрии кистей обеих рук. Через 10 лет костный компонент в теле девушек практически не изменился. Выводы. Полученные нами данные свидетельствуют об ухудшении всех двигательных качеств девушек, обследованных в 2019 году, по сравнению с 2009 годом. Это является следствием роста гиподинамии современной молодежи. Полученные результаты наблюдений расширяют базу данных антропометрических и двигательных параметров молодого поколения России. Эти данные могут быть использованы при планировании учебных и тренировочных занятий в образовательных и спортивных организациях.Мета. Мета дослідження - дати порівняльну характеристику типам конституції, анатомічних компонентів тіла і рухових якостей дівчат-студенток, які навчалися в університеті (Іркутськ, Росія) в 2009 і 2019 роки.Матеріал і методи. Проведено обстеження та соматотипологічних діагностика тисячу двісті двадцять шість дівчат-студенток (в 2009 році - n = 762; в 2019 г. - n = 464) за 27 антропометричнимb параметрамb. Розраховувався: індекс Пинь'; середнє значення абсолютного і процентного вмісту кісткового компонента тіла (КК), жирового компонента тіла (ЖК) і м'язового компонента тіла (МК). За моторнимb тестfvb оцінювали: швидкісну витривалість і спритність; швидкість; швидкісно-силову витривалість м'язів згиначів тулуба; силу і силову витривалість м'язів верхнього плечового пояса; динамічну силу м'язів нижніх кінцівок; активну гнучкість хребта; загальну витривалість.Результати. У 2019, в порівнянні з 2009 роком, спостерігається: зниження (на 19,6%) кількості дівчат-нормостеников; збільшення кількості гиперстеников (на 70,9%) і астеников (на 27,4%); збільшення маси тіла; зменшення довжини тіла. Достовірно зменшилися в 2019 році значення показників: довжини тулуба, верхніх і нижніх кінцівок; окружності грудної клітини. Спостерігалося зниження середніх значень: обхоплювальної розмірів тіла; показників ширини плечей. Спостерігалося збільшення ширини таза. У 2019, в порівнянні з 2009 роком, встановлено у дівчат всіх типів конституції: достовірне збільшення вмісту в тілі жирової маси і зниження м'язової маси (р <0,05). В наслідок цього, знизилися силові показники динамометрії кистей обох рук. Через 10 років кістковий компонент в тілі дівчат практично не змінився.Висновки. Отримані нами дані свідчать про погіршення всіх рухових якостей дівчат, обстежених в 2019 році, в порівнянні з 2009 роком. Це є наслідком росту гіподинамії сучасної молоді. Отримані результати спостережень розширюють базу даних антропометричних і рухових параметрів молодого покоління Росії. Ці дані можуть бути використані при плануванні навчальних і тренувальних занять в освітніх і спортивних організаціях

Cell Entry of Arginine-rich Peptides Is Independent of Endocytosis*S⃞

Arginine-rich peptides are a subclass of cell-penetrating peptides that are taken up by living cells and can be detected freely diffusing inside the cytoplasm and nucleoplasm. This phenomenon has been attributed to either an endocytic mode of uptake and a subsequent release from vesicles or to direct membrane penetration (transduction). To distinguish between both possibilities, we have blocked endocytic pathways suggested to be involved in uptake of cell-penetrating peptides. We have then monitored by confocal microscopy the uptake and distribution of the cell-penetrating transactivator of transcription (TAT) peptide into living mammalian cells over time. To prevent side effects of chemical inhibitors, we used genetically engineered cells as well as different temperature. We found that a knockdown of clathrin-mediated endocytosis and a knock-out of caveolin-mediated endocytosis did not affect the ability of TAT to enter cells. In addition, the TAT peptide showed the same intracellular distribution throughout the cytoplasm and nucleus as in control cells. Even incubation of cells at 4 °C did not abrogate TAT uptake nor change its intracellular distribution. We therefore conclude that this distribution results from TAT peptide that directly penetrated (transduced) the plasma membrane. The formation of nonselective pores is unlikely, because simultaneously added fluorophores were not taken up together with the TAT peptide. In summary, although the frequency and kinetics of TAT transduction varied between cell types, it was independent of endocytosis