bluuebunny/crossref_metadata_2025 huggingface dataset:
The Effect of Iron Oxide Magnetic Nanoparticles on Smooth Muscle Cells
Authors: Zhang Song , Chen Xiangjian , Gu Chunrong , Zhang Yu , Xu Jindan , Bian Zhiping , Yang Di , Gu Ning
Published: December 2008

Recently, magnetic nanoparticles of iron oxide (Fe3O4, γ-Fe2O3) have shown an increasing number of applications in the field of biomedicine, but some questions have been raised about the potential impact of these nanoparticles on the environment and human health. In this work, the three types of magnetic nanoparticles (DMSA-Fe2O3, APTS-Fe2O3, and GLU-Fe2O3) with the same crystal structure, magnetic properties, and size distribution was designed, prepared, and characterized by transmission electronic microscopy, powder X-ray diffraction, zeta potential analyzer, vibrating sample magnetometer, and Fourier transform Infrared spectroscopy. Then, we have investigated the effect of the three types of magnetic nanoparticles (DMSA-Fe2O3, APTS-Fe2O3, and GLU-Fe2O3) on smooth muscle cells (SMCs). Cellular uptake of nanoparticles by SMC displays the dose, the incubation time and surface property dependent patterns. Through the thin section TEM images, we observe that DMSA-Fe2O3is incorporated into the lysosome of SMCs. The magnetic nanoparticles have no inflammation impact, but decrease the viability of SMCs. The other questions about metabolism and other impacts will be the next subject of further studies.
Inhibitory Effect of Conjugated α‐Linolenic Acid from Bifidobacteria of Intestinal Origin on SW480 Cancer Cells
Authors: Coakley Mairéad , Banni Sebastiano , Johnson Mark C. , Mills Susan , Devery Rosaleen , Fitzgerald Gerald , Paul Ross R. , Stanton Catherine
Published: December 2008

In this study, we assessed the ability of six strains of bifidobacteria (previously shown by us to possess the ability to convert linoleic acid to c9, t11‐conjugated linoleic acid (CLA) to grow in the presence of α‐linolenic acid and to generate conjugated isomers of the fatty acid substrate during fermentation for 42 h. The six strains of bifidobacteria were grown in modified MRS (mMRS) containing α‐linolenic acid for 42 h at 37 °C, after which the fatty acid composition of the growth medium was assessed by gas liquid chromatography (GLC). Indeed, following fermentation of one of the strains, namely Bifidobacterium breve NCIMB 702258, in the presence of 0.41 mg/ml α‐linolenic acid, 79.1% was converted to the conjugated isomer, C18:3 c9, t11, c15 conjugated α‐linolenic acid (CALA). To examine the inhibitory effect of the fermented oils produced, SW480 colon cancer cells were cultured in the presence of the extracted fermented oil (10–50 μg/ml) for 5 days. The data indicate an inhibitory effect on cell growth (p ≤ 0.001) of CALA, with cell numbers reduced by 85% at a concentration of 180 μM, compared with a reduction of only 50% with α‐linolenic acid (p ≤ 0.01).
Identification of Acylglycerols Containing Dihydroxy Fatty Acids in Castor Oil by Mass Spectrometry
Authors: Lin Jiann‐Tsyh , Arcinas Arthur , Harden Leslie A.
Published: December 2008

Ricinoleate, a monohydroxy fatty acid, in castor oil has many industrial uses. Dihydroxy fatty acids can also be used in industry. The C18 HPLC fractions of castor oil were analyzed by electrospray ionization mass spectrometry of lithium adducts to identify the acylglycerols containing dihydroxy fatty acids and the dihydroxy fatty acids. Four diacylglycerols identified were diOH18:1‐diOH18:1, diOH18:2‐OH18:1, diOH18:1‐OH18:1 and diOH18:0‐OH18:1. Eight triacylglycerols identified were diOH18:1‐diOH18:1‐diOH18:1, diOH18:1‐diOH18:1‐diOH18:0, diOH18:2‐diOH18:1‐OH18:1, diOH18:1‐diOH18:1‐OH18:1, diOH18:1‐diOH18:0‐OH18:1, diOH18:2‐OH18:1‐OH18:1, diOH18:1‐OH18:1‐OH18:1 and diOH18:0‐OH18:1‐OH18:1. The locations of fatty acids on the glycerol backbone were not determined. The structures of these three newly identified dihydroxy fatty acids were proposed as 11,12‐dihydroxy‐9‐octadecenoic acid, 11,12‐dihydroxy‐9,13‐octadecadienoic acid and 11,12‐dihydroxyoctadecanoic acid. These individual acylglycerols were at the levels of about 0.5% or less in castor oil and can be isolated from castor oil or overproduced in a transgenic oil seed plant for future industrial uses.
Molekulare Karyotypisierung in der klinischen Diagnostik
Authors: Rauch A.
Published: December 2008

Molekulare Karyotypisierung bezeichnet die genomweite Analyse bezüglich genetischer Kopienzahlveränderungen mit Hilfe von Arrays, welche das Genom mehr oder weniger dicht mit molekularen Markern abdecken. Die Hauptanwendung in der klinischen Diagnostik liegt derzeit bei der Analyse von Patienten mit geistiger Behinderung und multiplen Anomalien unbekannter Ursache. Bei ihnen lassen sich nach Ausschluss von konventionell-zytogenetisch sichtbaren Aberrationen, kryptischen subtelomerischen Aberrationen und klinisch gut erkennbaren, häufigen Mikrodeletionssyndromen in rund 10% der Fälle mittels molekularer Karyotypisierung chromosomale Mikroaberrationen nachweisen. Hierdurch konnten in den letzten Jahren zahlreiche neue Mikrodeletions- und -duplikationssyndrome charakterisiert werden, jedoch wurde auch ersichtlich, dass die Interpretation von Einzelbefunden aufgrund der großen Anzahl an genomischen Kopienzahlpolymorphismen nicht trivial ist.
Tubulohelical membrane arrays: Novel association of helical structures with intracellular membranes
Authors: Reipert Siegfried , Kotisch Harald , Wysoudil Bhuma , Neumüller Josef
Published: December 2008

A novel organelle‐like membrane specialization has been found in an epithelial cell line. Characteristically, the helical membrane arrays (referred to hereafter as TUHMAs) are organized around tubular, proteinaceous electron‐dense cores of 80 nm in diameter. Depending on the cell status, up to 8 of these cores provide the basis for an intermingled membrane scaffold of an overall length of 3–5 μm. TUHMAs exist as single organelles in transient association with the nucleus, the rough endoplasmic reticulum, patches of annulate lamellae, and the Golgi complex. While most of the constituents are still unknown, evidence for an involvement of nucleoporins in TUHMA organization is presented, as shown by fluorescence immunochemistry. This should make TUHMAs more easily accessible for future studies on their structure and function.
Amniotic fluid index versus single deepest vertical pocket: A meta‐analysis of randomized controlled trials
Authors: Nabhan Ashraf F. , Abdelmoula Yasser A.
Published: December 2008

Objective

To compare the use of the amniotic fluid index with the single deepest vertical pocket measurement, during antepartum fetal surveillance, in preventing adverse pregnancy outcome.

Search strategy

We searched the Cochrane Pregnancy and Childbirth Group's Trials Register (January 2008), the Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library 2008, Issue 2), MEDLINE (1966 to May 2008), and the metaRegister of Controlled Trials (May 2008). We handsearched the citation lists of relevant publications, review articles, and included studies.

Selection criteria

Randomized controlled trials involving women with a singleton pregnancy, whether at low or high risk, undergoing ultrasound measurement of amniotic fluid volume as part of antepartum assessment of fetal well‐being that compared the amniotic fluid index and the single deepest vertical pocket measurement.

Data collection and analysis

Both authors independently assessed eligibility and quality, and extracted the data.

Results

Four trials (3125 women) met the inclusion criteria. When the amniotic fluid index was used, significantly more cases of oligohydramnios were diagnosed (risk ratio (RR) 2.33, 95% CI 1.67–3.24), and more women had inductions of labor (RR 2.10, 95% CI 1.60–2.76) and cesarean delivery for fetal distress (RR 1.45, 95% CI 1.07–1.97). There is no evidence that one method is superior to the other in the prevention of poor peripartum outcomes, including: admission to a neonatal intensive care unit; an umbilical artery pH of less than 7.1; the presence of meconium; an Apgar score of less than 7 at 5 minutes; or cesarean delivery.

Conclusion

Single deepest vertical pocket measurement is the method of choice for the assessment of amniotic fluid volume.
Elliptic symbols, elliptic operators and Poincaré duality on conical pseudomanifolds
Authors: Lescure Jean-Marie
Published: December 2008

In [7], a notion of noncommutative tangent space is associated with a conical pseudomanifold and Poincaré duality inK-theory is proved between this space and the pseudomanifold. The present paper continues this line of work. We show that an appropriate presentation of the notion of symbol on a manifold generalizes right away to conical pseudomanifolds and that it enables us to interpret Poincaré duality in the singular setting as a noncommutative symbol map.
Exercise in improving health v. performance
Authors: Thompson Janice L.
Published: December 2008

Regular and goal-appropriate exercise is critical to improving and maintaining both health and performance. However, the frequency, intensity, duration and type of activities needed to optimise health or achieve successful sports performance will differ considerably depending on an individual's goals and capabilities. Although sport is one of many forms of exercise that can be counted towards daily physical activity, participation in sport is not necessary to meet current physical activity recommendations. The current consensus is that the minimum amount of physical activity needed to improve and maintain good health is 30 min moderate-intensity activity/d on ≥5 d/week. The evidence supporting this consensus is based on predominantly observational evidence that performing regular aerobic (endurance)-type physical activity is associated with reduced morbidity and premature mortality from CVD, CHD, stroke and colo-rectal cancer. The exact dose needed to improve health and the slope of the dose–response gradient between physical activity and mortality for various diseases are not known, and one major limitation of the existing evidence is the lack of objective measurement of physical activity. Limited evidence indicates that a much higher dose of activity (45–90 min each day on ≥5 d/week) may be needed to prevent overweight and obesity and to avoid weight regain in previously overweight and obese individuals. The role of resistance training and heavy domestic work in reducing morbidity and premature mortality for various diseases is unclear. As most adults do not meet current recommendations there is a critical need for innovative approaches to increase physical activity across large-scale populations.
Nutritional therapies to improve health: lessons from companion animals
Authors: Hill Richard C.
Published: December 2008

Companion animals represent an under-utilised resource. The present paper is designed to encourage collaborative studies. Dogs and cats are out-bred animals that are willing to consume a consistent diet for long periods, so are ideal candidates for prospective studies of naturally-occurring disease. In some studies the effect of diet on survival has been substantial. Food restriction, for example, slows the development of osteoarthritis and increases the lifespan of Labrador retrievers by 2 years, protein and P restriction more than doubles the median survival time of dogs and cats with chronic kidney disease and addingn-3 fats and arginine to the diet of dogs with stage 3 lymphoma improves median survival time by one-quarter. Obesity is also very common in both dogs and cats and is also associated with disease as in human subjects. When interpreting these results, however, it is essential to take into account pathophysiological differences among species. Dogs and cats do not display all the characteristics of metabolic disease in human subjects, they metabolise fat well and atherosclerosis and cardiac infarction are uncommon. Such differences should not, however, preclude further study because differences among species often clarify knowledge. Monitoring of disease in companion animals may also provide a surveillance system for the safety of the food supply, as illustrated by recent outbreaks of acute renal failure and liver failure in cats and dogs in the USA caused respectively by melamine and mycotoxin contamination of pet foods.
Management of cancerisation of the lobules with pre-invasive or invasive breast carcinoma: a case series
Authors: Ampil Federico , Abreo Fleurette , Li Benjamin , Baluna Roxana , Burton Gary , Chu Quyen
Published: December 2008

Cancerisation of the lobules (CL), the intra-luminal extension of carcinoma from ducts and ductules into the breast lobules, is not often encountered in clinical practice. A retrospective study of eight individuals diagnosed with CL and ductal carcinoma in situ (DCIS) or cancer of the breast between 1999 and 2006 was undertaken to determine the management of the disease and patient outcomes. In most cases, breast conservation surgery with postoperative radiotherapy or mastectomy and systemic therapy were the chosen methods of treatment. None of the patients experienced tumour relapse or progression during a mean follow-up period of 40.6 (range 8–90) months. The management philosophy for this particular neoplastic condition should be based on the understanding that CL, when detected with DCIS or invasive breast cancer, may represent more extensive disease. Moreover, the finding of such histopathology underlines the need for meticulous attention to the resection margins and additional treatment as indicated.
Zukünftige Hausärztinnen und Hausärzte – Gründe für die Berufswahl und berufliche Kernkompetenzen
Authors: Hasler L. B. , Stamm M. , Buddeberg-Fischer B.
Published: December 2008

Hintergrund: Ziel der vorliegenden Studie ist es zu untersuchen, aus welchen Motiven junge Ärztinnen und Ärzte eine Tätigkeit in der Grundversorgung anstreben und welche Kenntnisse, Fähigkeiten und Fertigkeiten ihnen während der Weiterbildungszeit vermittelt werden sollten. Methodik: Im Rahmen einer seit 2001 laufenden prospektiven Studie zu Determinanten der Karriereentwicklung junger Ärztinnen und Ärzte wurden 84 zukünftige Schweizer Hausärztinnen und Hausärzte am Ende ihrer Weiterbildung zu Motiven ihrer Facharztwahl und zu hausärztlichen Kernkompetenzen befragt. Die mittels offener Fragen im Rahmen einer umfassenden Fragebogenerhebung gewonnenen Angaben wurden inhaltsanalytisch ausgewertet und induktiv gebildeten Kategorien zugeordnet. Ergebnisse: Die Nennungen zu motivierenden Faktoren (254 Antworten) für die Facharztwahl Allgemeinmedizin bzw. Allgemeine Innere Medizin sowie zu den Kernkompetenzen (375 Antworten) einer Hausärztin/eines Hausarztes wurden je elf Kategorien zugeordnet. Als Motive wurden am häufigsten die Kategorien «Selbstständigkeit», «breites Patienten- und Krankheitsspektrum», «Langzeitbetreuung», «Vielseitigkeit der ärztlichen Tätigkeit» und «Arzt-Patient-Beziehung» genannt. Als zu erwerbende Kernkompetenzen wurden am häufigsten «betriebswirtschaftliche, gesundheitsökonomische und (versicherungs-)rechtliche Kompetenzen», «breites medizinisches Grundwissen» und «Sozialkompetenz» erwähnt. Schlussfolgerung: Die Motive für eine Tätigkeit in der ärztlichen Grundversorgung und die zu erwerbenden Kernkompetenzen sind Ausdruck eines am Patienten orientierten Arztbildes. Sie zielen auf Eigenverantwortlichkeit und Gestaltungsfreiheit in der ärztlichen Tätigkeit.
Kaiser, Komet und Katastrophe – die «Justinianische» Pest und das Ende der Antike
Authors: Leven K.-H.
Published: December 2008

Die komplexen Phänomene des Untergangs der griechisch-römischen Kultur und der Umgestaltung der Mittelmeerwelt sind seit jeher Gegenstand der Geschichtswissenschaft und in ihrer Deutung umstritten. Die vorliegende Arbeit skizziert unter Konzentration auf medizinhistorische Aspekte einige neuere Theorien und Forschungsansätze, die auch naturwissenschaftliche Befunde in die Argumentation einführen.
47-jähriger Patient mit Sehstörungen, Polydipsie und Polyurie
Authors: Chatterjee B. , Berger D. , Joost Ch. , Stucki A.
Published: December 2008

Das Bronchuskarzinom ist die häufigste Todesursache bei Tumorpatienten; der primäre Risikofaktor ist bei Männern in 90% und bei Frauen in 80% der Nikotinkonsum. Vielfach besteht bei Diagnosestellung bereits ein fortgeschrittenes, respektive inoperables Stadium. Wir präsentieren einen Patienten (Nie-Raucher), der sich aufgrund einer Polydipsie, Sehstörungen und Polyurie in der medizinischen Poliklinik vorstellte. Laborchemisch wurde ein Diabetes insipidus diagnostiziert; die MRT des Schädels zeigte multiple Raumforderungen. Die Staginguntersuchung zeigte weitere Raumforderungen pulmonal, mediastinal, intrahepatisch, vertebral sowie in der Nebenniere. Die Histologie bestätigte ein Bronchuskarzinom vom nichtkleinzelligen Typ.
Summertime Taklimakan dust structure
Authors: Hara Y. , Uno I. , Yumimoto K. , Tanaka M. , Shimizu A. , Sugimoto N. , Liu Z.
Published: December 2008

Vertical dust structures and meteorological conditions over the Taklimakan Desert during the summertime periods (July–September) in 2006 and 2007 were investigated using satellite data including the CALIPSO lidar (CALIOP) measurements and the Weather Research and Forecasting (WRF) model simulation. Summertime convective velocity simulated by WRF was greater than 3 m/s, and the convective time scale was about 1000 s (ca. 15 min). We examined 42 CALIPSO paths (24 daytime and 18 nighttime paths) of data acquired under convective dusty conditions. The dust layer thickness derived from the CALIOP measurements reaches 3000–4000 m, which is approximately equal to the Tarim Basin depth. This thickness shows a good correlation with the WRF simulated depth of the convective boundary layer (BL). The dust remains suspended during both daytime and nighttime; the CALIOP average dust extinction coefficients in the BL are respectively 0.151 ± 0.102 km−1 and 0.128 ± 0.079 km−1 for daytime and nighttime. Finally, we estimated the dust amount transported from the BL to the free atmosphere. Typically, 10–20 Gg/day of dust (assuming area of 600 km × 300 km as a main part of the Taklimakan Desert) is transported from the Taklimakan Desert by vertical mixing. The daily horizontal dust flux above the BL was estimated to be 40–50 Gg/day over the Taklimakan Desert.
Satellite evidence of wintertime atmospheric boundary layer responses to multiple SST fronts in the Japan Sea
Authors: Shimada Teruhisa , Kawamura Hiroshi
Published: December 2008

We present satellite evidence of wintertime atmospheric boundary layer responses to multiple sea surface temperature (SST) fronts in the Japan Sea under cold‐air outbreak during 22–28 December 2005. It is found that the lower atmosphere adheres to the stepwise SST variations, focusing on a wind trajectory from the Eurasian continent to the northern Japan, which goes through the meandering Polar Front and the Tsushima Warm Current. Wind speed anomaly and wind divergence are linearly related to the SST anomaly and downwind SST gradient, respectively. Vertically uniform layers of potential temperature are accomplished when reaching the Polar Front and Tsushima Warm Current. Variations of water vapor mixing ratio and the resulting surface turbulent heat fluxes show also stepwise increases. Little wind vertical shear on the Japanese coast suggests that well‐mixed boundary layer is eventually formed.
The dynamics of zonal current variations in the central equatorial Indian Ocean
Authors: Nagura Motoki , McPhaden Michael J.
Published: December 2008

This paper examines the depth integrated zonal momentum balance in the central equatorial Indian Ocean (0°, 80.5°E) for two periods in 1993–1994 and 2004–2006 using a combination of in‐situ and satellite observations. One‐ to two‐year record length mean balances are approximately linear and steady state. Monthly to seasonal time scale variations are also governed by linear dynamics, with imbalances between the pressure gradient force and surface wind stress leading to zonal mass transport variations along the equator. Interannual variations in zonal transports along the equator associated with Indian Ocean Dipole events in 1994 and 2006 are likewise consistent with linear dynamics. These results provide a valuable benchmark for evaluating models of equatorial Indian Ocean circulation on seasonal time scales.
Relative age of interior layered deposits in southwest Candor Chasma based on high‐resolution structural mapping
Authors: Okubo Chris H. , Lewis Kevin W. , McEwen Alfred S. , Kirk Randolph L.
Published: December 2008

High‐resolution topography generated from stereo HiRISE (High‐Resolution Imaging Science Experiment) imagery reveals the meter‐scale structure of interior layered deposits (ILD) in southwest Candor Chasma. This study seeks to determine the age of the local ILD relative to any normal faults that can be attributed to chasma formation. The study area is located near the contact of these ILD and the wall rock and is in an area where chasma‐forming normal faults have been proposed. We find that while normal faults are found in the study area, these faults are not sufficiently large nor appropriately located or oriented to accommodate the roughly northeast‐southwest extension that is required for normal faults that can be attributed to chasma formation. Additionally, bedding exposed in the local ILD generally dips toward the center of Candor Chasma, consistent with sediment deposition in a preexisting basin. Further, pit craters of Tithonia Catena, presumed to predate or be contemporaneous with the formation of west Candor Chasma, do not cut into the ILD within the study area. These independent lines of evidence support a postchasma age for the ILD exposed within the study area. Chasma‐related normal faults may exist within these ILD at depth but are not exposed at the surface. Approximately 2 km of conformable stratigraphy is exposed in the study area, and therefore at least several kilometers of the local ILD were deposited subsequent to any chasma‐related normal faulting that may have occurred in this part of Candor Chasma.
Correction of electronic record for weighing bucket precipitation gauge measurements
Authors: Nayak Anurag , Chandler David G. , Marks Danny , McNamara James P. , Seyfried Mark
Published: December 2008

Electronic sensors generate valuable streams of forcing and validation data for hydrologic models but are often subject to noise which must be removed as part of model input and testing database development. We developed an automated precipitation correction program (APCP) for weighing bucket precipitation gauge records, which are subject to several types of mechanical and electronic noise and discontinuities, including gauge maintenance, missing data, wind vibration, and sensor drift. Corrected cumulative water year precipitation from APCP did not exhibit an error bias and matched measured water year total precipitation within 2.1% for 58 station years tested. Removal of low‐amplitude periodic noise was especially important for developing accurate instantaneous precipitation records at subdaily time steps. Model flexibility for use with other data types is demonstrated through application to time domain reflectometry soil moisture content data, which are also frequently subject to substantial noise.
Mammalian cells contain a second nucleocytoplasmic hexosaminidase
Authors: Gutternigg Martin , Rendić Dubravko , Voglauer Regina , Iskratsch Thomas , Wilson Iain B. H.
Published: December 2008

Some thirty years ago, work on mammalian tissues suggested the presence of two cytosolic hexosaminidases in mammalian cells; one of these has been more recently characterized in a recombinant form and has an important role in cellular function due to its ability to cleave β-N-acetylglucosamine residues from a variety of nuclear and cytoplasmic proteins. However, the molecular nature of the second cytosolic hexosaminidase, named hexosaminidase D, has remained obscure. In the present study, we molecularly characterize for the first time the human and murine recombinant forms of enzymes, encoded by HEXDC genes, which appear to correspond to hexosaminidase D in terms of substrate specificity, pH dependency and temperature stability. Furthermore, a Myc-tagged form of this novel hexosaminidase displays a nucleocytoplasmic localization. Transcripts of the corresponding gene are expressed in a number of murine tissues. On the basis of its sequence, this enzyme represents, along with the lysosomal hexosaminidase subunits encoded by the HEXA and HEXB genes, the third class 20 glycosidase to be identified from mammalian sources.
Solving the Correspondence Problem within the Ternus Display: The Differential-Activation Theory
Authors: Odic Darko , Pratt Jay
Published: December 2008

The Ternus display produces a bistable illusion of motion: at very short interstimulus intervals (ISIs; < 30 ms) observers perceive element motion while at longer ISIs (> 30 ms) observers perceive group motion. In experiment 1, however, we find that, when the Ternus display's ISI contains an occluding box, group motion is mostly eliminated. These results do not fit the predictions made by the short-range/long-range two-process theory [Braddick and Adlard, 1978, in Visual Psychophysics and Psychology (New York: Academic Press)]. We propose that the differential-activation theory (Gilroy et al, 2001 Perception & Psychophysics63 847–861) accounts for our results. We then extend the differential-activation theory as an explanatory mechanism for the Ternus display in experiment 2 by selectively placing an occluder over the first, second, or third Ternus display element. As predicted by the differential-activation theory, the occlusion of the far-left element produced a normal distribution of group motion increasing with ISI, while the occlusion of the other two elements produced an illusion of occluded elements remaining stationary throughout the display. Furthermore, as predicted by the differential-activation theory, each moving element was assigned to its nearest neighbour, producing, in the case of second and third element occlusion, a novel Ternus display motion illusion where only two out of three elements are perceived as moving.
Feeling Pain in the Rubber Hand: Integration of Visual, Proprioceptive, and Painful Stimuli
Authors: Capelari Edla D P , Uribe Carlos , Brasil-Neto Joaquim P
Published: December 2008

The visual capture phenomenon has recently been explored, especially in the context of the rubber-hand illusion (RHI)—an illusion in which tactile sensations are referred to an illusory limb. We have induced the RHI with the difference that tactile-painful stimuli were added in order to verify the interaction between vision, touch, proprioception, and pain. Thirty volunteers were used. We found that tactile-painful stimuli could cause the same illusion as purely tactile stimuli. This result suggests that localisation of pain may also be distorted by spurious visual cues. The implications of this finding for distorted human proprioception (as in amputees with phantom pain or limbs) are discussed.
Multiple autophosphorylation sites are dispensable for murine ATM activation in vivo
Authors: Daniel Jeremy A. , Pellegrini Manuela , Lee Ji-Hoon , Paull Tanya T. , Feigenbaum Lionel , Nussenzweig André
Published: December 2008

Cellular responses to both physiological and pathological DNA double-strand breaks are initiated through activation of the evolutionarily conserved ataxia telangiectasia mutated (ATM) kinase. Upon DNA damage, an activation mechanism involving autophosphorylation has been reported to allow ATM to phosphorylate downstream targets important for cell cycle checkpoints and DNA repair. In humans, serine residues 367, 1893, and 1981 have been shown to be autophosphorylation sites that are individually required for ATM activation. To test the physiological importance of these sites, we generated a transgenic mouse model in which all three conserved ATM serine autophosphorylation sites (S367/1899/1987) have been replaced with alanine. In this study, we show that ATM-dependent responses at both cellular and organismal levels are functional in mice that express a triple serine mutant form of ATM as their sole ATM species. These results lend further support to the notion that ATM autophosphorylation correlates with the DNA damage–induced activation of the kinase but is not required for ATM function in vivo.
Genome-wide analysis reveals a cell cycle–dependent mechanism controlling centromere propagation
Authors: Erhardt Sylvia , Mellone Barbara G. , Betts Craig M. , Zhang Weiguo , Karpen Gary H. , Straight Aaron F.
Published: December 2008

Centromeres are the structural and functional foundation for kinetochore formation, spindle attachment, and chromosome segregation. In this study, we isolated factors required for centromere propagation using genome-wide RNA interference screening for defects in centromere protein A (CENP-A; centromere identifier [CID]) localization in Drosophila melanogaster. We identified the proteins CAL1 and CENP-C as essential factors for CID assembly at the centromere. CID, CAL1, and CENP-C coimmunoprecipitate and are mutually dependent for centromere localization and function. We also identified the mitotic cyclin A (CYCA) and the anaphase-promoting complex (APC) inhibitor RCA1/Emi1 as regulators of centromere propagation. We show that CYCA is centromere localized and that CYCA and RCA1/Emi1 couple centromere assembly to the cell cycle through regulation of the fizzy-related/CDH1 subunit of the APC. Our findings identify essential components of the epigenetic machinery that ensures proper specification and propagation of the centromere and suggest a mechanism for coordinating centromere inheritance with cell division.
μ2 adaptin facilitates but is not essential for synaptic vesicle recycling inCaenorhabditis elegans
Authors: Gu Mingyu , Schuske Kim , Watanabe Shigeki , Liu Qiang , Baum Paul , Garriga Gian , Jorgensen Erik M.
Published: December 2008

Synaptic vesicles must be recycled to sustain neurotransmission, in large part via clathrin-mediated endocytosis. Clathrin is recruited to endocytic sites on the plasma membrane by the AP2 adaptor complex. The medium subunit (μ2) of AP2 binds to cargo proteins and phosphatidylinositol-4,5-bisphosphate on the cell surface. Here, we characterize the apm-2 gene (also called dpy-23), which encodes the only μ2 subunit in the nematode Caenorhabditis elegans. APM-2 is highly expressed in the nervous system and is localized to synapses; yet specific loss of APM-2 in neurons does not affect locomotion. In apm-2 mutants, clathrin is mislocalized at synapses, and synaptic vesicle numbers and evoked responses are reduced to 60 and 65%, respectively. Collectively, these data suggest AP2 μ2 facilitates but is not essential for synaptic vesicle recycling.
Recruitment of RNA polymerase II cofactor PC4 to DNA damage sites
Authors: Mortusewicz Oliver , Roth Wera , Li Na , Cardoso M. Cristina , Meisterernst Michael , Leonhardt Heinrich
Published: December 2008

The multifunctional nuclear protein positive cofactor 4 (PC4) is involved in various cellular processes including transcription, replication, and chromatin organization. Recently, PC4 has been identified as a suppressor of oxidative mutagenesis in Escherichia coli and Saccharomyces cerevisiae. To investigate a potential role of PC4 in mammalian DNA repair, we used a combination of live cell microscopy, microirradiation, and fluorescence recovery after photobleaching analysis. We found a clear accumulation of endogenous PC4 at DNA damage sites introduced by either chemical agents or laser microirradiation. Using fluorescent fusion proteins and specific mutants, we demonstrated that the rapid recruitment of PC4 to laser-induced DNA damage sites is independent of poly(ADP-ribosyl)ation and γH2AX but depends on its single strand binding capacity. Furthermore, PC4 showed a high turnover at DNA damages sites compared with the repair factors replication protein A and proliferating cell nuclear antigen. We propose that PC4 plays a role in the early response to DNA damage by recognizing single-stranded DNA and may thus initiate or facilitate the subsequent steps of DNA repair.
The Two-Spotted Cricket Gryllus bimaculatus: An Emerging Model for Developmental and Regeneration Studies
Authors: Mito Taro , Noji Sumihare
Published: December 2008

INTRODUCTION

The two-spotted cricket Gryllus bimaculatus De Geer (Orthoptera: Gryllidae), which is one of the most abundant cricket species, inhabits the tropical and subtropical regions of Asia, Africa, and Europe. G. bimaculatus can be easily bred in the laboratory and has been widely used to study insect physiology and neurobiology. Recently, this species has become established as a model animal for studies on molecular mechanisms of development and regeneration because its mode of development is more typical of arthropods than that of Drosophila melanogaster, and the cricket is probably ancestral for this phylum. Moreover, the cricket is a hemimetabolous insect, in which nymphs possess functional legs with a remarkable capacity for regeneration after damage. Because RNA interference (RNAi) works effectively in this species, the elucidation of mechanisms of development and regeneration has been expedited through loss-of-function analyses of genes. Furthermore, because RNAi-based techniques for analyzing gene functions can be combined with assay systems in other research areas (such as behavioral analyses), G. bimaculatus is expected to become a model organism in various fields of biology. Thus, it may be possible to establish the cricket as a simple model system for exploring more complex organisms such as humans.
Gene Silencing by Injection and Electroporation of dsRNA in Avian Embryos
Authors: Mauti Olivier , Baeriswyl Thomas , Stoeckli Esther T.
Published: December 2008

INTRODUCTION

In ovo RNA interference (RNAi) is a method for silencing a gene of interest using a combination of in ovo injection and electroporation in avian embryos. Here we describe gene silencing in the developing spinal cord, but the procedure can easily be adapted to other parts of the nervous system. Double-stranded RNA (dsRNA) derived from the gene of interest is injected into the developing spinal cord of the chicken embryo, and is followed by electroporation to allow for the uptake of the dsRNA. With this method, temporal as well as spatial control of gene silencing is possible. The time point of injection should be chosen according to the expression profile of the gene or the half-life of the protein. Proteins with slow turnover may require RNAi at earlier stages, ideally before the onset of gene expression. The electroporation parameters can be adjusted such that only a specific population of neurons is targeted in the spinal cord.
Analysis of Cell Movement in Amphimedon Embryos by Injection of Fluorescent Tracers
Authors: Adamska Maja , Degnan Bernard M.
Published: December 2008

INTRODUCTION

Although attempts to culture prepigmentation-stage embryos (i.e., blastulas and early gastrulas) outside of brood chambers have so far been unsuccessful in Amphimedon, it is possible to manipulate embryos within the brood chamber and follow their development under laboratory conditions. This protocol describes microinjection of lipophilic tracers such as 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) into embryos embedded in their native brood chamber. DiI does not appear to perturb embryonic development and is relatively resistant to photobleaching. As long as care is taken not to damage the fragile embryos during observation and photography, the same embryo can be photographed multiple times, permitting its development to be tracked (up to 4 wk) from early cleavage stages to hatching of free-swimming parenchymella larvae. The embryos or larvae also can be fixed without loss of fluorescence. This method also can be used to deliver other types of solutions to embryos or individual cells of early embryos.
Genotyping Individual Amphimedon Embryos, Larvae, and Adults
Authors: Degnan Sandie M. , Craigie Alina , Degnan Bernard M.
Published: December 2008

INTRODUCTION

The distribution of Amphimedon queenslandica is patchy on coral reefs in the Great Barrier Reef, with small, localized populations detected in shallow, still water reef-flat environments. A. queenslandica is a spermcast spawner, in which fertilization occurs internally. Sperm presumably originate from neighboring reproductive individuals within the population. The ability to genotype individual embryos within a single brood chamber has the potential to shed light on the fertilization biology and generation/maintenance of genetic diversity in this sessile invertebrate. Here, we describe a protocol for rapidly genotyping individuals using polymorphic microsatellite loci. The loci are amplified by PCR using a pair of primers specifically designed for the region of interest with a fluorescent dye attached to the 5′-end to enable easy detection of the amplified product. An advantage of this procedure is that fluorescently labeled PCR products can be combined (i.e., multiplexed) to reduce time and cost when using the genotyping machine. The dye label and size of the product must be taken into consideration when multiplexing. For example, three differently labeled PCR products can be multiplexed, or PCR products with the same label can be multiplexed as long as the allelic size ranges do not overlap. The amount of each cleaned, labeled PCR product added to the multiplex must be optimized depending on the dye and the PCR efficiency.
Growth and Maintenance of Dictyostelium Cells
Authors: Gaudet Pascale , Fey Petra , Chisholm Rex
Published: December 2008

INTRODUCTION

Dictyostelium discoideum is a unicellular eukaryote often referred to as a “social ameba” because it can form a multicellular structure when nutrient conditions are limiting. General principles for cell-to-cell communication, intracellular signaling, and cytoskeletal organization during cell motility have been derived from cellular and molecular studies of Dictyostelium and have been found to be conserved across all eukaryotes. Dictyostelium also provides an excellent model system for the study of phagocytosis, the molecular basis of various human diseases, and the mechanisms of drug action. The availability of a complete genome database and stocks of wild-type and mutant strains make D. discoideum an accessible and powerful model organism. Most Dictyostelium strains used in the laboratory can be grown either with bacteria or in axenic medium. When grown in the presence of bacteria, cells double approximately every 4 h, whereas axenically grown cells double more slowly, every 8–12 h. The cells can be grown in a standard microbiology incubator or on the laboratory bench, provided the room temperature is consistently ∼22°C. This protocol describes a variety of methods for growing and maintaining Dictyostelium.
Multicellular Development of Dictyostelium
Authors: Gaudet Pascale , Fey Petra , Chisholm Rex
Published: December 2008

INTRODUCTION

Dictyostelium discoideum is a unicellular eukaryote often referred to as a “social ameba” because it can form a multicellular structure when nutrients are depleted from the immediate environment of the cells. In the laboratory, this is accomplished simply by replacing the growth medium with a buffer solution. For best results, it is important to harvest the cells while they are still in exponential growth (1–4 × 106 cells/mL). At high cell density, many of the cells in a culture will have initiated development, thus yielding asynchronous development. Dictyostelium cells can be developed on solid media, either on filter paper or on KK2 plates. If only the early stages of development are important (e.g., to study chemotaxis), cells can be developed in suspension. Under these conditions, cells will only progress through the first 6–8 h of development. Addition of cAMP pulses to the starved suspension culture will allow development to progress up to the 12-h stage, corresponding to the beginning of culmination.
Making Permanent Stocks of Dictyostelium
Authors: Gaudet Pascale , Fey Petra , Chisholm Rex
Published: December 2008

INTRODUCTION

Dictyostelium discoideum is a unicellular eukaryote often referred to as a social ameba because it can form a multicellular structure when nutrients are depleted from the immediate environment of the cells. Dictyostelium can be grown axenically or in the presence of bacteria, either on agar plates or in suspension. Because Dictyostelium growth rates are relatively slow compared to those of bacteria or yeast, laboratories commonly maintain stocks of growing cultures in order to start experiments rapidly. However, it is important to remember that the genome of Dictyostelium, like that of any living organism, is subject to genetic modification. It is well documented that cell lines that are kept in culture for an extended period of time exhibit undesirable changes that yield unreliable experimental results (Hughes et al. 2007). Dictyostelium strains from different laboratories are known to contain various large genome duplications, presumably due to clone selection. Thus, good handling of the cells is essential. To obtain consistent results, new cultures must be started every 2–4 wk, and cultures should never be allowed to grow beyond 4 × 106 cells/mL. If overgrowth occurs, a new culture should be started. This protocol describes two methods for preparing long-term stocks of Dictyostelium, either as frozen cells or as spores.
Transformation of Dictyostelium with Plasmid DNA by Calcium Phosphate Precipitation
Authors: Gaudet Pascale , Fey Petra , Chisholm Rex
Published: December 2008

INTRODUCTION

Dictyostelium discoideum is a unicellular eukaryote often referred to as a social ameba because it can form a multicellular structure when nutrient conditions are limiting. General principles for cell-to-cell communication, intracellular signaling, and cytoskeletal organization during cell motility have been derived from cellular and molecular studies of Dictyostelium and have been found to be conserved across all eukaryotes. The availability of a complete genome database and stocks of wild-type and mutant strains make D. discoideum an accessible and powerful model organism. Dictyostelium is amenable to genetic manipulations that require the introduction of DNA into cells, such as gene knockout, overexpression, antisense RNA expression, RNA interference (RNAi)-mediated gene knockdown, and restriction-enzyme-mediated mutagenesis. Calcium phosphate precipitation is a commonly used method for DNA-mediated transformation in Dictyostelium. Calcium phosphate precipitation produces high-copy-number transformants and is often used for overexpression experiments in conjunction with the G418 resistance gene, which needs to be present at high levels to produce efficient selection.
Transformation of Dictyostelium with Plasmid DNA by Electroporation
Authors: Gaudet Pascale , Fey Petra , Chisholm Rex
Published: December 2008

INTRODUCTION

Dictyostelium discoideum is a unicellular eukaryote often referred to as a social ameba because it can form a multicellular structure when nutrient conditions are limiting. General principles for cell-to-cell communication, intracellular signaling, and cytoskeletal organization during cell motility have been derived from cellular and molecular studies of Dictyostelium and have been found to be conserved across all eukaryotes. The availability of a complete genome database and stocks of wild-type and mutant strains make D. discoideum an accessible and powerful model organism. Dictyostelium is amenable to genetic manipulations that require the introduction of DNA into cells, such as gene knockout, overexpression, antisense RNA expression, RNA interference (RNAi)-mediated gene knockdown, and restriction-enzyme-mediated mutagenesis. This protocol describes the use of electroporation for DNA-mediated transformation in Dictyostelium.
Extraction of DNA from Dictyostelium
Authors: Gaudet Pascale , Fey Petra , Chisholm Rex
Published: December 2008

INTRODUCTION

Dictyostelium discoideum is a unicellular eukaryote often referred to as a social ameba because it can form a multicellular structure when nutrient conditions are limiting. General principles for cell-to-cell communication, intracellular signaling, and cytoskeletal organization during cell motility have been derived from cellular and molecular studies of Dictyostelium and have been found to be conserved across all eukaryotes. The availability of a complete genome database and stocks of wild-type and mutant strains make D. discoideum an accessible and powerful model organism. Dictyostelium is amenable to genetic manipulations that require the introduction of DNA into cells, such as gene knockout, overexpression, antisense RNA expression, RNA interference (RNAi)-mediated gene knockdown, and restriction-enzyme-mediated mutagenesis. Extraction of genomic DNA is used to clone gene fragments and for analysis of mutants to determine the site of vector integration. Because Dictyostelium cells contain relatively high levels of carbohydrate and nucleases, commercially available DNA preparation kits are not very successful. The DNA isolated according to the following protocol is suitable for digestion by restriction enzymes, amplification by polymerase chain reaction (PCR), and Southern blotting.
Extraction of RNA from Dictyostelium
Authors: Gaudet Pascale , Fey Petra , Chisholm Rex
Published: December 2008

INTRODUCTION

Dictyostelium discoideum is a unicellular eukaryote often referred to as a social ameba because it can form a multicellular structure when nutrient conditions are limiting. General principles for cell-to-cell communication, intracellular signaling, and cytoskeletal organization during cell motility have been derived from cellular and molecular studies of Dictyostelium and have been found to be conserved across all eukaryotes. The availability of a complete genome database and stocks of wild-type and mutant strains make D. discoideum an accessible and powerful model organism. Dictyostelium is amenable to genetic manipulations that require the introduction of DNA into cells, such as gene knockout, overexpression, antisense RNA expression, RNA interference (RNAi)-mediated gene knockdown, and restriction-enzyme-mediated mutagenesis. The extraction of RNA from Dictyostelium is relatively easy because RNA levels are very high in comparison to DNA levels (i.e., ∼40 times higher). Certain commercially available kits, such as Trizol (Invitrogen) and RNeasy (QIAGEN) have been used successfully, although lysis conditions need to be adjusted. RNA samples are stable for several years at −80°C in diethylpyrocarbonate (DEPC)-treated H2O. For longer-term storage, the RNA pellet can be stored in 100% ethanol at −80°C. Such samples are suitable for Northern blots, reverse transcriptase-polymerase chain reaction (RT-PCR), and microarray analysis of gene expression.
Collection, Cryopreservation, Storage, and Revitalization of Transgenic Mouse Embryos
Authors: Schwab Anna , Schenkel Johannes
Published: December 2008

INTRODUCTION

Transgenic mice possess enormous scientific potential. However, the need to maintain breeding stocks to prevent the loss of unique mutants (particularly if they are not in current use for experiments) is a major challenge. Cryopreservation of spermatozoa or preimplantation embryos is a valuable tool to address this issue, with cryopreservation of embryos in the two- to eight-cell stage being the most common method. Cryopreserved samples can be stored indefinitely in liquid nitrogen, and cryopreserved embryos can be shipped easily. Colonies of a transgenic line need not be maintained if a sufficient stock of embryos has been cryopreserved. After revitalizing preserved samples, embryo transfer must be used to re-establish the line. However, considerable effort is often needed to obtain a sufficient number of embryos. This protocol describes the methods necessary to collect and cryopreserve eight-cell embryos and to handle cryopreserved samples, as well as subsequent procedures needed to revitalize and re-derive transgenic lines.