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P12: Living Lab as academic practice partnership to improve care for people with dementia
- A. Bieber, F. Bühler, A. Leinen, J.P.H. Hamers, S. Wilm, H. Schneider-Schelte, S. Köpke, G. Meyer, J.H.J. Urlings, M.N. Dichter
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- Journal:
- International Psychogeriatrics / Volume 35 / Issue S1 / December 2023
- Published online by Cambridge University Press:
- 02 February 2024, pp. 234-235
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Objective:
Bridging the gap between clinical practice and research in health care is a challenging task. Living Labs are academic practice partnerships to stress the ambition to start up a longstanding collaboration, which have been developed and implemented in the Netherlands (Verbeek et al., 2020). The “PraWiDem” (“Living Lab Dementia”) project aims to adapt the Maastricht Living Lab approach to long-term dementia care in different regions in Germany.
Methods:A mixed methods approach was used to guide the adaptation of the Maastricht Living Lab. A focus group study investigated perspectives of people with dementia, informal carers and professionals on expectations and experiences concerning collaboration and networking between research and practice. A scoping review mapped international experiences in knowledge transfer practices and collaboration approaches in nursing care. Experts from the Maastricht Living Lab supported the research team in adapting the approach to the German national context. Parts of the German “Living Lab Dementia” concept were discussed with members of a recently formed research participation group of people with dementia (“experts by experience”).
Results:In total, 10 focus groups and 5 individual interviews were conducted. Key themes include researchers’ and professionals’ skills, participation of people with dementia and informal carers, and multi-professional requirements. The scoping review identified 17 different approaches of knowledge translation and collaboration. Few approaches address the common development and implementation of knowledge and networking. Dutch experts recommend the early development of long-lasting strategies for collaboration. Experts by experience wish to participate, but traditional research methods may need to be adapted to allow their participation.
Conclusion:The “Living Lab Dementia” is currently under investigation in collaboration with institutional and community care services in three regions in Germany.
Genome-wide association study of pathological gambling
- M. Lang, T. Leménager, F. Streit, M. Fauth-Bühler, J. Frank, D. Juraeva, S.H. Witt, F. Degenhardt, A. Hofmann, S. Heilmann-Heimbach, F. Kiefer, B. Brors, H.-J. Grabe, U. John, A. Bischof, G. Bischof, U. Völker, G. Homuth, M. Beutel, P.A. Lind, S.E. Medland, W.S. Slutske, N.G. Martin, H. Völzke, M.M. Nöthen, C. Meyer, H.-J. Rumpf, F.M. Wurst, M. Rietschel, K.F. Mann
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- Journal:
- European Psychiatry / Volume 36 / August 2016
- Published online by Cambridge University Press:
- 23 March 2020, pp. 38-46
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Background
Pathological gambling is a behavioural addiction with negative economic, social, and psychological consequences. Identification of contributing genes and pathways may improve understanding of aetiology and facilitate therapy and prevention. Here, we report the first genome-wide association study of pathological gambling. Our aims were to identify pathways involved in pathological gambling, and examine whether there is a genetic overlap between pathological gambling and alcohol dependence.
MethodsFour hundred and forty-five individuals with a diagnosis of pathological gambling according to the Diagnostic and Statistical Manual of Mental Disorders were recruited in Germany, and 986 controls were drawn from a German general population sample. A genome-wide association study of pathological gambling comprising single marker, gene-based, and pathway analyses, was performed. Polygenic risk scores were generated using data from a German genome-wide association study of alcohol dependence.
ResultsNo genome-wide significant association with pathological gambling was found for single markers or genes. Pathways for Huntington's disease (P-value = 6.63 × 10−3); 5′-adenosine monophosphate-activated protein kinase signalling (P-value = 9.57 × 10−3); and apoptosis (P-value = 1.75 × 10−2) were significant. Polygenic risk score analysis of the alcohol dependence dataset yielded a one-sided nominal significant P-value in subjects with pathological gambling, irrespective of comorbid alcohol dependence status.
ConclusionsThe present results accord with previous quantitative formal genetic studies which showed genetic overlap between non-substance- and substance-related addictions. Furthermore, pathway analysis suggests shared pathology between Huntington's disease and pathological gambling. This finding is consistent with previous imaging studies.
Implications of weed seedbank dynamics to weed management
- Douglas D. Buhler, Robert G. Hartzler, Frank Forcella
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- Weed Science / Volume 45 / Issue 3 / June 1997
- Published online by Cambridge University Press:
- 12 June 2017, pp. 329-336
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The species composition and density of weed seed in the soil vary greatly and are closely linked to the cropping history of the land. Altering tillage practices changes weed seed depth in the soil, which plays a role in weed species shifts and affects efficacy of control practices. Crop rotation and weed control practices also affect the weed seedbank. Information on the influence of cropping practices on the weed seedbank should be a useful tool for integrated weed management. Decision aid models use information on the weed seedbank to estimate weed populations, crop yield loss, and recommend weed control tactics. Understanding the light requirements of weed seed may provide new approaches to weed management. Improving and applying our understanding of weed seedbank dynamics is essential to developing improved weed management systems. The principles of plant ecology must be integrated with the science of weed management to develop strategies that take advantage of basic plant responses in weed management systems for agronomic crops.
Weed seed bank emergence across the Corn Belt
- Frank Forcella, Robert G. Wilson, Jack Dekker, Robert J. Kremer, John Cardina, Randy L. Anderson, David Alm, Karen A. Renner, R. Gordon Harvey, Sharon Clay, Douglas D. Buhler
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- Weed Science / Volume 45 / Issue 1 / February 1997
- Published online by Cambridge University Press:
- 12 June 2017, pp. 67-76
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Field experiments, conducted from 1991 to 1994, generated information on weed seedbank emergence for 22 site-years from Ohio to Colorado and Minnesota to Missouri. Early spring seedbank densities were estimated through direct extraction of viable seeds from soil cores. Emerged seedlings were recorded periodically, as were daily values for air and soil temperature, and precipitation. Percentages of weed seedbanks that emerged as seedlings were calculated from seedbank and seedling data for each species, and relationships between seedbank emergence and microclimatic variables were sought. Fifteen species were found in 3 or more site-years. Average emergence percentages (and coefficients of variation) of these species were as follows: giant foxtail, 31.2 (84%); velvetleaf, 28.2 (66); kochia, 25.7 (79); Pennsylvania smartweed, 25.1 (65); common purslane, 15.4 (135); common ragweed, 15.0 (110); green foxtail, 8.5 (72); wild proso millet, 6.6 (104); hairy nightshade, 5.2 (62); common sunflower, 5.0 (26); yellow foxtail, 3.4 (67); pigweed species, 3.3 (103); common lambsquarters, 2.7 (111); wild buckwheat, 2.5 (63), and prostrate knotweed, 0.6 (79). Variation among site-years, for some species, could be attributed to microclimate variables thought to induce secondary dormancy in spring. For example, total seasonal emergence percentage of giant foxtail was related positively to the 1st date at which average daily soil temperature at 5 to 10 cm soil depth reached 16 C. Thus, if soil warmed before mid April, secondary dormancy was induced and few seedlings emerged, whereas many seedlings emerged if soil remained cool until June.
Emergence characteristics of four annual weed species
- Robert G. Hartzler, Douglas D. Buhler, David E. Stoltenberg
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- Weed Science / Volume 47 / Issue 5 / October 1999
- Published online by Cambridge University Press:
- 12 June 2017, pp. 578-584
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Emergence characteristics, including initial time of emergence, magnitude of emergence, and mean time of emergence, of Amaranthus rudis Sauer, Setaria faberi Herrm., Eriochloa villosa (Thunb.) Kunth, and Abutilon theophrasti Medik. were studied in central Iowa during the first 3 yr following burial of seed collected and buried in the fall of 1994 or 1995. Although the initial emergence date varied among years, the emergence sequence among species was consistent. Eriochloa villosa and A. theophrasti were the first species to emerge, with initial emergence occurring between April 28 and May 10. Initial emergence dates for these species were the same, except for 1995 when A. theophrasti emerged 4 d prior to E. villosa. Amaranthus rudis was the last species to emerge, with initial emergence ranging from 5 to 25 d after A. theophrasti. First-year emergence in 1995 was 8% for A. theophrasti, 7% for A. rudis, 41% for E. villosa, and 33% for S. faberi, based on the number of buried seed. Proportional emergence during the first year following burial in 1996 was similar to 1995 for A. theophrasti and S. faberi, but greater emergence was observed in 1996 for E. villosa and A. rudis. During the 3 yr of both studies, cumulative emergence of the two grass species (43 to 71%) was higher than for the broadleaf species (13 to 35%). A high percentage of the total annual emergence of E. villosa occurred within the first 2 wk of initial emergence, whereas a high percentage of A. rudis emergence occurred late in its emergence period. Emergence characteristics of the four species were consistent among years and seed sources.
Weed Seedbanks of the U.S. Corn Belt: Magnitude, Variation, Emergence, and Application
- Frank Forcella, Robert G. Wilson, Karen A. Renner, Jack Dekker, Robert G. Harvey, David A. Alm, Douglas D. Buhler, John Cardina
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- Weed Science / Volume 40 / Issue 4 / December 1992
- Published online by Cambridge University Press:
- 12 June 2017, pp. 636-644
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Seedbanks and seedling emergence of annual weeds were examined in arable fields at eight locations in the Corn Belt. Seed densities were estimated by direct seed extraction from each of several soil cores in each sampled plot. Average total seedbank densities ranged from 600 to 162 000 viable seed m-2 among locations. Coefficients of variation (CV) typically exceeded 50%. CV for seed densities of individual species usually exceeded 100%, indicating strongly aggregated distributions. CV were lower for species with dense seed populations than those with sparse seed populations. Variance of total seedbank densities was unstable when < 10 cores were examined per plot, but stabilized at all locations when ≥ 15 cores were analyzed, despite a 12-fold difference in plot size and 270-fold difference in seed density among locations. Percentage viable seed that emerged as seedlings in field plots ranged from < 1% for yellow rocket to 30% for giant foxtail. Redroot pigweed and common lambsquarters were the most frequently encountered species. Emergence percentages of these species were related inversely to rainfall or air temperatures in April or May, presumably because anoxia and/or high temperatures induced secondary dormancy in nondormant seed. From 50 to 90% of total seed in the seedbank were dead. This information can be employed by bioeconomic weed management models, which currently use coarse estimates of emergence percentages to customize recommendations for weed control.
Anisotropic Helmholtz and wave–vortex decomposition of one-dimensional spectra
- Oliver Bühler, Max Kuang, Esteban G. Tabak
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- Journal of Fluid Mechanics / Volume 815 / 25 March 2017
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- 21 February 2017, pp. 361-387
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We present an extension to anisotropic flows of the recently developed Helmholtz and wave–vortex decomposition method for one-dimensional spectra measured along ship or aircraft tracks in Bühler et al. (J. Fluid Mech., vol. 756, 2014, pp. 1007–1026). Here, anisotropy refers to the statistical properties of the underlying flow field, which in the original method was assumed to be homogeneous and isotropic in the horizontal plane. Now, the flow is allowed to have a simple kind of horizontal anisotropy that is chosen in a self-consistent manner and can be deduced from the one-dimensional power spectra of the horizontal velocity fields and their cross-correlation. The key result is that an exact and robust Helmholtz decomposition of the horizontal kinetic energy spectrum can be achieved in this anisotropic flow setting, which then also allows the subsequent wave–vortex decomposition step. The anisotropic method is as easy to use as its isotropic counterpart and it robustly converges back to it if the observed anisotropy tends to zero. As a by-product of our analysis we also found a simple test for statistical correlation between rotational and divergent flow components. The new method is developed theoretically and tested with encouraging results on challenging synthetic data as well as on ocean data from the Gulf Stream.
Predicting Emergence of 23 Summer Annual Weed Species
- Rodrigo Werle, Lowell D. Sandell, Douglas D. Buhler, Robert G. Hartzler, John L. Lindquist
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- Weed Science / Volume 62 / Issue 2 / June 2014
- Published online by Cambridge University Press:
- 20 January 2017, pp. 267-279
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First- and second-year seedbank emergence of 23 summer annual weed species common to U.S. corn production systems was studied. Field experiments were conducted between 1996 and 1999 at the Iowa State University Johnson Farm in Story County, Iowa. In the fall of 1996 and again in 1997, 1,000 seeds for most species were planted in plastic crates. Seedling emergence was counted weekly for a 2-yr period following seed burial (starting in early spring). Soil temperature at 2 cm depth was estimated using soil temperature and moisture model software (STM2). The Weibull function was fit to cumulative emergence (%) on cumulative thermal time (TT), hydrothermal time (HTT), and day of year (DOY). To identify optimum base temperature (Tbase) and base matric potential (ψbase) for calculating TT or HTT, Tbase and ψbase values ranging from 2 to 17 C and −33 to −1,500 kPa, respectively, were evaluated for each species. The search for the optimal model for each species was based on the Akaike's Information Criterion (AIC), whereas an extra penalty cost was added to HTT models. In general, fewer seedlings emerged during the first year of the first experimental run (approximately 18% across all species) than during the second experimental run (approximately 30%). However, second-year seedbank emergence was similar for both experimental runs (approximately 6%). Environmental effects may be the cause of differences in total seedling emergence among years. Based on the AIC criterion, for 17 species, the best fit of the model occurred using Tbase ranging from 2 to 15 C with four species also responding to ψbase = −750 kPa. For six species, a simple model using DOY resulted in the best fit. Adding penalty costs to AIC calculation allowed us to compare TT and HTT when both models behaved similarly. Using a constant Tbase, species were plotted and classified as early-, middle-, and late-emerging species, resulting in a practical tool for forecasting time of emergence. The results of this research provide robust information on the prediction of the time of summer annual weed emergence, which can be used to schedule weed and crop management.
Emergence and persistence of seed of velvetleaf, common waterhemp, woolly cupgrass, and giant foxtail
- Douglas D. Buhler, Robert G. Hartzler
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- Weed Science / Volume 49 / Issue 2 / April 2001
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- 20 January 2017, pp. 230-235
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Annual emergence and seed persistence of common waterhemp, velvetleaf, woolly cupgrass, and giant foxtail were characterized in central Iowa for 4 yr following burial of seeds collected and buried in autumn 1994. First-year emergence as a percentage of the original seed bank ranged from 5 to 40%, and the relative order was common waterhemp < velvetleaf < giant foxtail < woolly cupgrass. During the second and third years, there were no differences in percent emergence among species, with emergence percentages ranging from 1 to 9% of the original seed bank. During the fourth year, seedlings continued to emerge from only the velvetleaf and common waterhemp seed banks. A greater percentage of common waterhemp seed persisted each year and 12% of the original seed was recovered after 4 yr of burial. Five percent of the velvetleaf was recovered at the end of the fourth year. No woolly cupgrass and giant foxtail seed was recovered after the third and fourth years. The proportion of the seed that was accounted for from year to year through emergence and seed recovery varied by species and year. Total recovery of velvetleaf ranged from 61 to 87%, common waterhemp from 50 to 81%, woolly cupgrass from 29 to 79%, and giant foxtail from 23 to 79%. Based on the results of this research, velvetleaf and common waterhemp form more persistent seed banks than woolly cupgrass and giant foxtail. Therefore, woolly cupgrass and giant foxtail should be more amenable to management through seed bank depletion than velvetleaf and common waterhemp.
Effect of guaianolides in the meiosis reinitiation of amphibian oocytes
- J. Zapata-Martínez, G. Sánchez-Toranzo, F. Chaín, C.A.N. Catalán, M.I. Bühler
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Sesquiterpene lactones (STLs) are a large and structurally diverse group of plant metabolites generally found in the Asteraceae family. STLs exhibit a wide spectrum of biological activities and it is generally accepted that their major mechanism of action is the alkylation of the thiol groups of biological molecules. The guaianolides is one of various groups of STLs. Anti-tumour and anti-migraine effects, an allergenic agent, an inhibitor of smooth muscle cells and of meristematic cell proliferation are only a few of the most commonly reported activities of STLs. In amphibians, fully grown ovarian oocytes are arrested at the beginning of meiosis I. Under stimulus with progesterone, this meiotic arrest is released and meiosis progresses to metaphase II, a process known as oocyte maturation. There are previous records of the inhibitory effect of dehydroleucodin (DhL), a guaianolide lactone, on the progression of meiosis. It has been also shown that DhL and its 11,13-dihydroderivative (2H-DhL; a mixture of epimers at C-11) act as blockers of the resumption of meiosis in fully grown ovarian oocytes from the amphibian Rhinella arenarum (formerly classified as Bufo arenarum). The aim of this study was to analyze the effect of four closely related guaianolides, i.e., DhL, achillin, desacetoxymatricarin and estafietin as possible inhibitors of meiosis in oocytes of amphibians in vitro and discuss some structure–activity relationships. It was found that the inhibitory effect on meiosis resumption is greater when the lactone has two potentially reactive centres, either a α,β–α′,β′-diunsaturated cyclopentanone moiety or an epoxide group plus an exo-methylene-γ-lactone function.
Participation of PLA2 and PLC in DhL-induced activation of Rhinella arenarum oocytes
- J. Zapata-Martínez, M.F. Medina, M.C. Gramajo-Bühler, G. Sánchez-Toranzo
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Rhinella arenarum oocytes can be artificially activated, a process known as parthenogenesis, by a sesquiterpenic lactone of the guaianolide group, dehydroleucodine (DhL). Transient increases in the concentration of cytosolic Ca2+ are essential to trigger egg activation events. In this sense, the 1-4-5 inositol triphosphate receptors (IP3R) seem to be involved in the Ca2+ transient release induced by DhL in this species. We analyzed the involvement of phosphoinositide metabolism, especially the participation of phospholipase A2 (PLA2) and phospholipase C (PLC) in DhL-induced activation. Different doses of quinacrine, aristolochic acid (ATA) (PLA2 inhibitors) or neomycin, an antibiotic that binds to PIP2, thus preventing its hydrolysis, were used in mature Rhinella arenarum oocytes. In order to assay the participation of PI-PLC and PC- PLC we used U73122, a competitive inhibitor of PI-PLC dependent events and D609, an inhibitor of PC-PLC. We found that PLA2 inhibits quinacrine more effectively than ATA. This difference could be explained by the fact that quinacrine is not a specific inhibitor for PLA2 while ATA is specific for this enzyme. With respect to the participation of PLC, a higher decrease in oocyte activation was detected when cells were exposed to neomycin. Inhibition of PC-PLC with D609 and IP-PLC with U73122 indicated that the last PLC has a significant participation in the effect of DhL-induced activation. Results would indicate that DhL induces activation of in vitro matured oocytes of Rhinella arenarum by activation of IP-PLC, which in turn may induce IP3 formation which produces Ca2+ release.
Histological and morphometric study of the epididymus of Chinchilla lanigera Grey under controlled conditions in captivity
- M.C. Gramajo-Bühler, F.J. Pucci, G. Sanchez-Toranzo
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Chinchilla lanigera, native to the Andean Mountains of Perú, Chile, Bolivia and Argentina, is a specimen of great economic importance because of its fur. In mammals, spermatozoa originate in testes and are transported to the epididymis, where they undergo morphological and biochemical modifications known as sperm maturation, a basic step in the acquisition of their fertilizing ability. The aim of this work is the macroscopic and microscopic analysis of the epididymis of Chinchilla lanigera Grey and its sectorization based on a histomorphological study. The epididymis presents a clear segmentation into four regions: initial segment, caput, corpus and cauda. The epithelium lining the seminiferous tubules is pseudostratified, with principal cells with stereocilia and basal, clear, apical, narrow and halo cells. The histological analysis showed that principal and basal cells are the prevailing populations in all regions, also revealing narrow cells and the absence of clear cells in the initial segment. Each segment presents its different histological and morphometric characteristics, which supports the idea of the specific behaviour of each region, giving a segment-specific character to the process of sperm maturation in this species. No significant differences were found in the morphometric measurements or in the histological evaluation of the epididymis of samples collected in April and October. The fact that no differences were found between the samples collected during the two periods when the reproductive ability in nature is different suggests the importance of external factors in the control of the reproductive cycle of Chinchilla lanigera.
Chemical activation in Rhinella arenarum oocytes: effect of dehydroleucodine (DhL) and its hydrogenated derivative (2H-DhL)
- M.F. Medina, M.I. Bühler, G. Sánchez-Toranzo
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Mature oocytes are arrested in metaphase II due to the presence of high levels of active maturation promoting factor (MPF). After fertilization, active MPF levels decline abruptly, enabling oocytes to complete meiosis II. One of the first and universal events of oocyte activation is an increase in cytosolic Ca2+ that would be responsible for MPF inactivation. Mature oocytes can also be activated by parthenogenetic activation. The aims of this work are to test the ability of dehydroleucodine (DhL) and its hydrogenated derivative 11,13-dihydro-dehydroleucodine (2H-DhL) to induce chemical activation in amphibian oocytes and to study the participation of calcium in the process. Results indicated that DhL and 2H-DhL induced oocyte activation in a dose-dependent manner. After 90 min of treatment, DhL 36 μM was able to induce 95% activation, while 2H-DhL 36 μM was less active, with only 40% activation. Our results suggest that DhL induced the inhibition of MPF activity, probably by an increase in intracellular Ca2+ concentration. Extracellular Ca2+ would not be significant, although Ca2+ release from intracellular stores is critical. In this sense, IP3Rs and RyRs were involved in the Ca2+ transient induced by lactones. In this species, RyRs appears to be the largest contributor to Ca2+ release in DhL-induced activation. Although more studies are needed on the mechanism of action through which these lactones induce oocyte activation in Rhinella arenarum, the results of this research provide interesting perspectives for the use of these lactones as chemical activators in in vitro fertilization and cloning.
Effect of different types of sesquiterpene lactones on the maturation of Rhinella arenarum oocytes
- G. Sánchez-Toranzo, J. Zapata-Martínez, C. Catalán, M.I. Bühler
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The sesquiterpene lactones (STLs) are a large class of plant secondary metabolites that are generally found in the Asteraceae family and that have high diversity with respect to chemical structure as well as biological activity. STLs have been classified into different groups, such as guaianolides, germacranolides, and melampolides etc., based on their carboxylic skeleton. In amphibians, fully grown ovarian oocytes are arrested at the beginning of meiosis I. Under the stimulus of progesterone, this meiotic arrest is released and meiosis progresses to metaphase II, a process known as oocyte maturation. The purpose of this work was to determine whether sesquiterpene lactones from the germacranolide and melampolide groups act as inhibitor agents on the meiosis of amphibian oocytes in vitro. Results for germacranolides indicated that the addition of deoxyelephantopins caused a high degree of inhibition and that minimolide showed a moderate inhibitory effect, whereas glaucolide A was inactive. Furthermore, the addition of melampolides (uvedalin, enhydrin, polymatin A and polymatin B) showed inhibitory effects. For enhydrin and uvedalin, inhibitory effects were observed at the higher concentrations assayed. The results of this study suggest that the inhibitory activity of the tested sesquiterpene lactones on the meiosis of Rhinella arenarum oocytes is not dependent on the group to which they belong, i.e. not on the carboxylic skeleton, but probably due to the arrangement and type of function groups present in the molecules. All assayed lactones in the germacranolide group showed low toxicity. In contrast, important differences in toxicity were observed for lactones from the melampolide group: enhydrin and uvedalin showed low toxicity, but polymatin A and B were highly toxic.
Participation of IP3R, RyR and L-type Ca2+ channel in the nuclear maturation of Rhinella arenarum oocytes
- G. Sánchez Toranzo, M.C. Gramajo Bühler, M.I. Bühler
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During meiosis resumption, oocytes undergo a series of nuclear and cytosolic changes that prepare them for fertilization and that are referred to as oocyte maturation. These events are characterized by germinal vesicle breakdown (GVBD), chromatin condensation and spindle formation and, among cytosolic changes, organelle redistribution and maturation of Ca2+-release mechanisms. The progression of the meiotic cell cycle is regulated by M phase/maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK). Changes in the levels of intracellular free Ca2+ ion have also been implicated strongly in the triggering of the initiation of the M phase. Ca2+ signals can be generated by Ca2+ release from intracellular Ca2+ stores (endoplasmic reticulum; ER) or by Ca2+ influx from the extracellular space. In this sense, the L-type Ca2+ channel plays an important role in the incorporation of Ca2+ from the extracellular space. Two types of intracellular Ca2+ receptor/channels are known to mediate the intracellular Ca2+ release from the ER lumen. The most abundant, the inositol 1,4,5-trisphosphate receptor (IP3R), and the other Ca2+ channel, the ryanodine receptor (RyR), have also been reported to mediate Ca2+ release in several oocytes. In amphibians, MPF and MAPK play a central role during oocyte maturation, controlling several events. However, no definitive relationships have been identified between Ca2+ and MPF or MAPK. We investigated the participation of Ca2+ in the spontaneous and progesterone-induced nuclear maturation in Rhinella arenarum oocytes and the effect of different pharmacological agents known to produce modifications in the Ca2+ channels. We demonstrated that loading competent and incompetent oocytes with the intracellular calcium chelator BAPTA/AM produced suppression of spontaneous and progesterone-induced GVBD. In our results, the capacity of progesterone to trigger meiosis reinitiation in Rhinella in the presence of L-type Ca2+ channel blockers (nifedipine and lanthane) indicated that spontaneous and progesterone-induced maturation would be independent of extracellular calcium influx, but would be sensitive to intracellular Ca2+ deprivation. As demonstrated by the effect of thimerosal and heparin in Rhinella arenarum, the intracellular increase in Ca2+ during maturation is also mediated mainly by IP3R. In addition, our results using caffeine, an agonist of the RyR, could suggest that Ca2+ release from ryanodine-sensitive stores is not essential for oocyte maturation in Rhinella. The decrease in MPF activity with NaVO3 negatively affected the percentage of thimerosal-induced GVBD. This finding suggests that Ca2+ release through the IP3R could be involved in the signalling pathway that induces MPF activation. However, the inhibition of MAP/ERK kinase (MEK) by PD98128 or P90 by geldanamycin produced a significant decrease in the percentages of GVBD induced by thimerosal. This finding suggests that Ca2+ release per se cannot bypass the inhibition of the MAPK activity.
Effect on sperm quality of different cryoprotectants in sperm of Chinchilla lanigera
- M.C. Gramajo-Bühler, F. Pucci-Alcaide, G. Sánchez-Toranzo
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Chinchilla lanigera is an endangered species therefore the development of cryopreservation protocols for its gametes is a useful tool in the application of assisted reproduction techniques. A study of the functionality of the spermatozoa punctured from the cauda epididymis was performed on fresh or frozen–thawed samples with three cryoprotective media (test-yolk buffer, sucrose and glycerol). The effect that these media had on sperm physiology during the freezing, storage and later thawing process was analysed. A decrease in the percentages of viability, motility, membrane integrity and capacity to undergo the induced acrosome reaction was found with all the media assayed, an increase in the percentages of DNA fragmentation was also observed. The comparative analysis of the effect of the different cryoprotectants assayed showed that the best medium to use to cryopreserve epididymal sperm in this species is test-yolk buffer. This medium had the least effect on the abovementioned physiological parameters, especially at the level of genetic material.
Participation of MAPK, PKA and PP2A in the regulation of MPF activity in Bufo arenarum oocytes
- G. Sánchez Toranzo, F. Bonilla, M.C. Gramajo Bühler, M.I. Bühler
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The objectives of the present paper were to study the involvement and possible interactions of both cAMP-PKA and protein phosphatases in Bufo arenarum oocyte maturation and to determine if these pathways are independent or not of the MAP kinase (MAPK) cascade. Our results indicated that the inhibition of PKA by treatment with H-89, an inhibitor of the catalytic subunit of PKA, was capable of inducing GVBD in a dose-dependent manner by a pathway in which Cdc25 phosphatase but not the MAPK cascade is involved. The injection of 50 nl of H-89 10 μM produced GVBD percentages similar to those obtained with treatment with progesterone. In addition, the assays with okadaic acid (OA), a PP2A inhibitor, significantly enhanced the percentage of oocytes that resumed meiosis by a signal transducing pathway in which the activation of the MEK–MAPK pathway is necessary, but in which Cdc25 phosphatase was not involved. Treatment with H-89, was able to overcome the inhibitory effect of PKA on GVBD; however, the inhibition of Cdc25 activity with NaVO3 was able to overcome the induction of GVBD by H-89. Although the connections between PKA and other signalling molecules that regulate oocytes maturation are still unclear, our results suggest that phosphatase Cdc25 may be the direct substrate of PKA. In Xenopus oocytes it was proposed that PP2A, a major Ser/Thr phosphatase present, is a negative regulator of Cdc2 activation. However, in Bufo arenarum oocytes, inhibition of Cdc25 with NaVO3 did not inhibit OA-induced maturation, suggesting that the target of PP2A was not the Cdc25 phosphatase. MAPK activation has been reported to be essential in Xenopus oocytes GVBD. In B. arenarum oocytes we demonstrated that the inhibition of MAPK by PD 98059 prevented the activation of MPF induced by OA, suggesting that the activation of the MAPK cascade produced an inhibition of Myt1 and, in consequence, the activation of MPF without participation of the Cdc25 phosphatase. Our results suggest that in incompetent oocytes of B. arenarum two signal transduction pathways may be involved in the control of MPF activation: (1) the inhibition of phosphatase 2A that through the MEK–MAPK pathway regulates the activity of the Myt1; and (2) the inhibition of AMPc–PKA, which affects the activity of the Cdc25 phosphatase.
Involvement of the dehydroleucodine alpha-methylene-gamma-lactone function in GVBD inhibition in Bufo arenarum oocytes
- G. Sánchez Toranzo, L.A. López, J. Zapata Martínez, M.C. Gramajo Bühler, M.I. Bühler
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Dehydroleucodine (DhL), a sesquiterpenic lactone, was isolated and purified from aerial parts of Artemisia douglasiana Besser, a medicinal herb used in Argentina. DhL is an alpha-methylene butyro-gamma-lactone ring connected to a seven-membered ring fused to an exocyclic alpha,beta-unsaturated cyclopentenone ring
It has been previously shown that DhL selectively induces a dose-dependent transient arrest in G2 of both meristematic cells and vascular smooth muscle cells. Treatment with DhL induces an inhibition of spontaneous and progesterone-induced maturation in a dose-dependent manner in Bufo arenarum fully grown oocytes arrested at G2, at the beginning of meiosis I. However, the nature of the mechanisms involved in the process is still unknown.
The aim of this work was to analyse whether DhL's alpha-methylene-gamma-lactone function is responsible for the inhibition effect on meiosis reinitiation of Bufo arenarum oocytes as well as some of the transduction pathways that could be involved in this effect using a derivative of DhL inactivated for alpha-methylenelactone, the 11,13-dihydro-dehydroleucodine (2H-DhL).
The use of 2H-DhL in the maturation promoting factor (MPF) amplification experiments by injection of both cytoplasm with active MPF and of germinal vesicle content showed results similar to the ones obtained with DhL, suggesting that the hydrogenated derivative would act in a similar way to DhL.
Pretreatment with DhL or 2H-DhL did not affect the percentage of germinal vesicle breakdown (GVBD) induced by H89, a protein kinase A (PKA) inhibitor, which suggests that these lactones would act on another step of the signalling pathway that induces MPF activation. The fact that both DhL and 2H-Dhl inhibit GVBD induced by okadaic acid microinjection suggests that they could act on the activity of the Myt1 kinase. This idea is supported by the experiments of injection of GV contents in which an inhibitory effect of these lactones on GVBD was also observed.
Our results indicate that the inhibitory effect on meiosis progression of DhL does not depend only on the activity of the alpha-methylenelactone function, as its hydrogenated derivative, 2H-DhL, in which this function has been inactivated, causes similar effects on amphibian oocytes. However, 2H-DhL was less active than DhL as higher doses were required to obtain a significant inhibition. On the other hand, the analysis of the participation of certain mediators in some of the signalling pathways leading to MPF activation suggests that the Myt1 kinase could be a target of these lactones, while cdc25 phosphatase would not be affected. Besides, the PKA inhibition assays indicate that these lactones would act earlier in the signalling pathways.
Activation of amphibian oocytes by sperm extracts
- F. Bonilla, M. T. Ajmat, G. Sánchez Toranzo, L. Zelarayán, J. Oterino, M. I. Bühler
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In the fertilization of most animals, egg activation is accompanied by an increase in cytoplasmatic Ca2+; however, the mechanism through which the fertilizing sperm induce this phenomenon is still controversial. An increase in intracellular free Ca2+ is required to trigger egg activation events, a process that includes cortical granule exocytosis, resumption and completion of meiosis and DNA replication, and culminates in the first mitotic cleavage. In this work, we investigated the effect of microinjection and incubation of different fractions of homologous sperm extract on the activation of Bufo arenarum oocytes matured in vitro. Two heat treatment-sensitive fractions obtained by chromatography were able to induce oocyte activation. The sperm fraction, which contained a 24 kDa protein, induced 90% activation when it was microinjected into the oocytes. Whilst the sperm fraction, which contained a 36 kDa protein, was able to induce about 70% activation only when it was applied on the oocyte surface.
Polyspermy in Bufo arenarum oocytes matured in vitro
- J. Oterino, G. Sánchez Toranzo, L. Zelarayán, M.I. Bühler
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Full-grown ovarian oocytes of the amphibian Bufo arenarum were induced to mature in vitro by removing the follicular layers (spontaneous maturation) or by treatment with progesterone (hormone-induced maturation). These oocytes were then treated with trypsin and inseminated with homologous spermatozoa. Oocytes matured in vivo that had not undergone any influence of the oviducts (coelomic oocytes), inseminated under the same experimental conditions, were used as controls. The results show that oocytes induced to mature in vitro and exhibiting apparently normal signs of activation were polyspermic. In fact, 2 h after insemination numerous functioning pronuclei could be observed in the animal hemisphere. These results suggest that even though the oocytes which matured in vitro were able to undergo activation after insemination, they were unable to establish an effective block to polyspermy.
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