bluuebunny/crossref_metadata_2025 huggingface dataset:
Distribution and function of splash, an achaete‐scute homolog in the adult olfactory organ of the Caribbean spiny lobster Panulirus argus
Authors: Tadesse Tizeta , Schmidt Manfred , Walthall William W. , Tai Phang C. , Derby Charles D.
Published: December 2010

achaete‐scute complex (ASC) genes, which encode basic helix‐loop‐helix transcription factors, regulate embryonic and adult neurogenesis in many animals. In adult arthropods, including crustaceans, ASC homologs have been identified but rarely functionally characterized. We took advantage of the recently identified crustacean homolog, splash (spiny lobster achaete scute homolog), in the olfactory organ of the Caribbean spiny lobster Panulirus argus to examine its role in adult neurogenesis. We tested the hypothesis that splash is associated with but not restricted to sensory neuron formation in the olfactory organ, the antennular lateral flagellum (LF), of adult spiny lobsters. We demonstrated splash labeling in epithelial cells across LF developmental zones (i.e., proliferation and mature zones), in auxiliary cells surrounding dendrites of olfactory receptor neurons (ORNs), and in immature and mature ORNs, but not in granulocytes or chromatophores. Since ORN proliferation varies with molt stage, we examined splash expression across molt stages and found that molt stage affected splash expression in the ORN mature zone but not in the proliferation zone. In vivo incorporation of bromodeoxyuridine (BrdU) showed no correlation in the cellular pattern of splash expression and BrdU labeling. The intensity of splash labeling was dramatically enhanced in the proliferation zones following LF damage, suggesting enhanced splash expression during repair and/or regeneration. We conclude that splash is not closely associated with the formation of sensory neurons under normal physiological conditions, and we propose that splash is involved in repair and regeneration. We also propose that splash has additional roles other than neurogenesis in adult crustaceans. © Wiley Periodicals, Inc. Develop Neurobiol 71: 316–335, 2011
Synthesis of a Small Library of Estradiol‐Based Glycosteroid Mimics Containing a Modified D‐Ring
Authors: Ekholm Filip S. , Schneider Gyula , Wölfling János , Leino Reko
Published: December 2010

Glycosylated steroids, such as cardenolides and saponins, are widely distributed in nature and have been shown to possess various types of biological activities. In the present study, carbohydrate donors were evaluated for the glycosylation of steroids; the results show that benzoylated imidate donors were superior in terms of efficiency and operational simplicity. These donors were then applied to the synthesis of a limited library of estradiol‐based glycosteroid mimics containing a modified D‐ring. The deprotected molecules contained three types of variations in the steroid backbone and four types of variations in the sugar moiety. The synthesized structures will be screened for biological activity and for their potential use in drug delivery systems.
Reversible Aminal Formation: Controlling the Evaporation of Bioactive Volatiles by Dynamic Combinatorial/Covalent Chemistry
Authors: Buchs (née Levrand) Barbara , Godin Guillaume , Trachsel Alain , de Saint Laumer Jean‐Yves , Lehn Jean‐Marie , Herrmann Andreas
Published: December 2010

Dynamic mixtures generated by reversible aminal formation efficiently prolong the duration of evaporation of bioactive volatile aldehydes. Secondary diamines used for the generation of dynamic mixtures are obtained by treatment of primary diamines with carbonyl compounds and reduction of the diimines with NaBH4. The reversibilities of the reactions were demonstrated by NMR measurements in buffered aqueous solutions. Kinetic rate constants and equilibrium constants for the formation and hydrolysis of aminals were determined. The performance of dynamic mixtures as delivery systems for perfumery ingredients was tested after deposition onto cotton, and the long‐lastingness of fragrance evaporation was investigated by dynamic headspace analysis against a reference sample. The simplicity of the concept together with its excellent performance makes this delivery system highly interesting for applied perfumery. Reversible aminal formation might also be successfully applicable to dynamic combinatorial/covalent chemistry for screening of pharmaceutically or catalytically active ligands and receptors.
Optimal conditions for stability of photoemission and freeze drying of two luminescent bacteria for use in a biosensor
Authors: Camanzi Laura , Bolelli Luca , Maiolini Elisabetta , Girotti Stefano , Matteuzzi Diego
Published: December 2010

Bioluminescent bacteria have been used for many years for biotoxicological analysis. One of the main concerns with this microorganism is the low experimental repeatability when subjected to external factors. The aim of the present study was to obtain accurate, sensitive, and repeatable measurements with stable signals (during the detection and over days) for application in a water-analysis device for the detection of pollutants. Growth conditions were tested and optimized. An optimal freeze-drying procedure for the constitutive bioluminescent bacteria Vibrio fischeri and Photobacterium phosphoreum was developed. The luminescence stability after rehydration was also investigated. Freeze drying was found to be a critical process in survival and signal stability of luminescent bacteria; for this reason, different suspension fluids and various bacterial pellet/suspension fluid ratios (g/ml) were evaluated. The toxicity of heavy metals and organic compounds in water was determined to investigate the applicability of a test based on bacteria obtained in this way, comparing the data with legal limits. A scale-up process was developed with industrial technology: freeze-dried bacteria that emitted a stable luminous signal after rehydration were obtained. Moreover, the median effective concentration (EC50) was calculated with these bacteria. Environ. Toxicol. Chem. 2011; 30:801–805. © 2010 SETAC
Influence of water hardness and sulfate on the acute toxicity of chloride to sensitive freshwater invertebrates
Authors: Soucek David J , Linton Tyler K , Tarr Christopher D , Dickinson Amy , Wickramanayake Nilesh , Delos Charles G , Cruz Luis A
Published: December 2010

Total dissolved solids (TDS) represent the sum of all common ions (e.g., Na, K, Ca, Mg, chloride, sulfate, and bicarbonate) in freshwater. Currently, no federal water quality criteria exist for the protection of aquatic life for TDS, but because the constituents that constitute TDS are variable, the development of aquatic life criteria for specific ions is more practical than development of aquatic life criteria for TDS. Chloride is one such ion for which aquatic life criteria exist; however, the current aquatic life criteria dataset for chloride is more than 20 years old. Therefore, additional toxicity tests were conducted in the current study to confirm the acute toxicity of chloride to several potentially sensitive invertebrates: water flea (Ceriodaphnia dubia), fingernail clams (Sphaerium simile and Musculium transversum), snail (Gyraulus parvus), and worm (Tubifex tubifex), and determine the extent to which hardness and sulfate modify chloride toxicity. The results indicated a significant ameliorating effect of water hardness (calcium and magnesium) on chloride toxicity for all species tested except the snail; for example, the 48-h chloride median lethal concentration (LC50) for C. dubia at 50 mg/L hardness (977 mg Cl−/L) was half that at 800 mg/L hardness (1,836 mg Cl−/L). Conversely, sulfate over the range of 25 to 600 mg/L exerted a negligible effect on chloride toxicity to C. dubia. Rank order of LC50 values for chloride at a given water hardness was in the order (lowest to highest): S. simile < C. dubia < M. transversum < G. parvus < T. tubifex. Results of the current study support the contention that the specific conductivity or TDS concentration of a water body alone is not a sufficient predictor of acute toxicity and that knowledge of the specific ion composition is critical. Environ. Toxicol. Chem. 2011; 30:930–938. © 2010 SETAC
An egg injection technique to evaluate the effect of polychlorinated biphenyls on the hatching success of the snapping turtle (Chelydra serpentina serpentina)
Authors: Schnars Jeanette L , Voss Margaret A , Stauffer Jay R
Published: December 2010

Embryos of oviparous organisms are exposed to contaminants by two pathways: contaminant uptake from the surrounding environment, and the transfer from female to offspring (maternal transfer). The initial source of contaminant exposure for most embryos is likely to be maternal transfer; therefore, maternal transfer studies are critical in determining the effects of contaminants on future populations. Injection of contaminants directly into eggs is one route of experimental contaminant exposure that permits controlled doses and potential reliable replication. This technique, however, has been used in the past with little success in reptiles. The objective of the present study was to evaluate egg injection as a means of mimicking maternal transfer of polychlorinated biphenyls (PCBs) to snapping turtle eggs. Eggs from several clutches were injected with a PCB solution and incubated at several temperatures and moisture levels to measure interactive effects of injection, environmental condition, and contaminant load on hatching success. The injection technique allowed for application of consistent and specific doses among replicates. Overall hatching success in this study was 61% and was as high as 71% within specific treatments. Hatching success was much higher in this study than in other studies using egg injections to mimic maternal transfer in chelonians and crocodilians. Environ. Toxicol. Chem. 2011; 30:915–919. © 2010 SETAC
Long memory and structural breaks in commodity futures markets
Authors: Coakley Jerry , Dollery Jian , Kellard Neil
Published: December 2010

This study employs daily data for 14 commodities and three financial assets 1990–2009 to explore the impact of the time series properties of the futures‐spot basis and the cost of carry on forward market unbiasedness. The main result is that the basis of 16 assets exhibits both long memory and structural breaks. The long memory in the basis is robust even to the use of break‐adjusted data. It implies that the cost‐of‐carry has long memory which the empirical results confirm using the interest cost as a proxy. These new findings suggest that the forecast error has long memory and are inconsistent with unbiasedness. They could be consistent with a weaker version of market efficiency in the presence of a fractionally integrated, time‐varying risk premium but they could also be rationalized by priced noise trader risk with limits to arbitrage in less than fully efficient markets. © 2010 Wiley Periodicals, Inc. Jrl Fut Mark
Willingness‐to‐pay for predictive tests with no immediate treatment implications: a survey of US residents
Authors: Neumann Peter J. , Cohen Joshua T. , Hammitt James K. , Concannon Thomas W. , Auerbach Hannah R. , Fang ChiHui , Kent David M.
Published: December 2010

We assessed how much, if anything, people would pay for a laboratory test that predicted their future disease status. A questionnaire was administered via an internet‐based survey to a random sample of adult US respondents. Each respondent answered questions about two different scenarios, each of which specified: one of four randomly selected diseases (Alzheimer's, arthritis, breast cancer, or prostate cancer); an ex ante risk of developing the disease (randomly designated 10 or 25%); and test accuracy (randomly designated perfect or ‘not perfectly accurate’). Willingness‐to‐pay (WTP) was elicited with a double‐bounded, dichotomous‐choice approach. Of 1463 respondents who completed the survey, most (70‐88%, depending on the scenario) were inclined to take the test. Inclination to take the test was lower for Alzheimer's and higher for prostate cancer compared with arthritis, and rose somewhat with disease prevalence and for the perfect versus imperfect test [Correction made here after initial online publication.]. Median WTP varied from $109 for the imperfect arthritis test to $263 for the perfect prostate cancer test. Respondents' preferences for predictive testing, even in the absence of direct treatment consequences, reflected health and non‐health related factors, and suggests that conventional cost‐effectiveness analyses may underestimate the value of testing. Copyright © 2010 John Wiley & Sons, Ltd.
Adipofascial perforator flaps for “aesthetic” head and neck reconstruction
Authors: Hanasono Matthew M. , Skoracki Roman J. , Silva Amanda K. , Yu Peirong
Published: December 2010

Background

Most head and neck reconstructions are performed for wound closure or functional rehabilitation with aesthetic restoration being an important but secondary consideration.

Methods

Contour deformities in 40 patients undergoing head and neck resections were reconstructed immediately with adipofascial perforator flaps, including 37 anterolateral thigh and 3 deep inferior epigastric perforator flaps.

Results

Reconstructions could be grouped into 1 of 3 defect areas: temporal fossa (n = 3), malar cheek (n = 13), and parotid‐mastoid (n = 24). The mean time needed for reconstruction in excess of the oncologic resection was 3.1 hours. There were no flap losses. In no case did a complication delay adjuvant therapy and no patient experienced a decrease in level of activity related to donor site morbidity.

Conclusion

Immediate restoration of facial contour with adipofascial perforator flaps is warranted in head and neck oncologic patients to help improve self‐image and maintain quality of life. Head Neck, 2010
Association of p16INK4a overexpression with improved outcomes in young patients with squamous cell cancers of the oral tongue
Authors: Harris Stephen L. , Thorne Leigh B. , Seaman William T. , Neil Hayes D. , Couch Marion E. , Kimple Randall J.
Published: December 2010

Background

The aim of this study was to examine biomolecular profiles in a cohort of young adults with squamous cell cancers (SCCs) of the oral tongue.

Methods

We identified all patients aged 18 to 39 years diagnosed with SCC of the oral tongue at our institution. Immunohistochemical (IHC) staining was performed for p16INK4a, epidermal growth factor receptor (EGFR), phosphorylated‐EGFR (pEGFR), p53, and ERCC1. Human papillomavirus (HPV) testing was performed using in situ hybridization (ISH) and polymerase chain reaction (PCR). Biomarker expression and HPV status were correlated with outcomes.

Results

We identified 25 patients with sufficient tumor samples. Median age at diagnosis was 30 years (range, 20–39 years). p16INK4a overexpression was observed in 11 of 25 patients, whereas HPV‐16 positivity was observed in none of the tumor samples by ISH and 2 of the tumor samples by PCR. p16INK4a positivity was correlated with improved relapse‐free survival (hazard ratio [HR] = 0.23, p = .01) and overall survival (HR = 0.28, p = .05). Neither EGFR, pEGFR, p53, nor excision repair cross‐complementing rodent repair deficiency, complementation group 1 (ERCC1) expression correlated with outcome on univariate analysis.

Conclusions

p16INK4a overexpression was common and was a marker of favorable prognosis. p16INK4a overexpression was not a reliable predictor of HPV positivity in our cohort. © 2010 Wiley Periodicals, Inc. Head Neck, 2011
Precipitation measurement and derivation of precipitation inclination in a windy mountainous area in northern Costa Rica
Authors: Frumau K. F. Arnoud , Bruijnzeel L. A. (Sampurno) , Tobón Conrado
Published: December 2010

Over small‐scale topography in windy areas, precipitation tends to be redistributed by wind through the modification of precipitation inclination. The latter is often derived from wind speed and conventional rain gauge records by application of relations—derived mainly for convective rainfall conditions—between (1) precipitation intensity and drop diameter, and (2) drop diameter and terminal fall velocity. However, it remains to be seen whether such relationships give valid results for the typically low precipitation intensities prevailing in tropical montane cloud forests. On the basis of the assumption that the total amount of near‐surface atmospheric liquid water, defined hereafter as the ‘potential precipitation’, is most likely to be identical over small distances, this paper introduces a device to measure amounts of potential precipitation. The gauge captures both the vertical and horizontal component of precipitation, and this allows derivation of precipitation inclination using simple trigonometry. Results on precipitation inclinations obtained with the ‘potential precipitation gauge’ on a wet and windy site in northern Costa Rica suggest the droplets to be smaller than those predicted by the commonly used relationships referred to above. The current gauge is also shown to be more effective in catching inclined precipitation than two different types of spherical gauges. In addition, ‘effective’ fall velocities were determined for each precipitation event using derivated precipitation inclination and wind speed. The assumption of spatially similar potential precipitation amounts and effective fall velocities throughout a small catchment allows the catchment‐wide determination of precipitation inclination and therefore of hydrologically effective precipitation from a single‐point measurement of potential precipitation and wind speed in combination with modelled spatial wind speed pattern. This approach is believed to yield appreciably better estimates of spatial precipitation inputs compared to reliance on conventional rain gauges and derivation of terminal fall velocities from precipitation intensity records. Copyright © 2010 John Wiley & Sons, Ltd.
Large‐scale modelling of channel flow and floodplain inundation dynamics and its application to the Pantanal (Brazil)
Authors: Paz Adriano Rolim da , Collischonn Walter , Tucci Carlos E. M. , Padovani Carlos R.
Published: December 2010

For large‐scale sites, difficulties for applying coupled one‐dimensional (1D)/2D models for simulating floodplain inundation may be encountered related to data scarcity, complexity for establishing channel–floodplain connections, computational cost, long duration of floods and the need to represent precipitation and evapotranspiration processes. This paper presents a hydrologic simulation system, named SIRIPLAN, developed to accomplish this aim. This system is composed by a 1D hydrodynamic model coupled to a 2D raster‐based model, and by two modules to compute the vertical water balance over floodplain and the water exchanges between channel and floodplain. Results are presented for the Upper Paraguay River Basin (UPRB), including the Pantanal, one of the world's largest wetlands. A total of 3965 km of river channels and 140 000 km2 of floodplains are simulated for a period of 11 years. Comparison of observed and calculated hydrographs at 15 gauging stations showed that the model was capable to simulate distinct, complex flow regimes along main channels, including channel‐floodplain interactions. The proposed system was also able to reproduce the Pantanal seasonal flood pulse, with estimated inundated areas ranging from 35 000 km2 (dry period) to more than 120 000 km2 (wet period). Floodplain inundation maps obtained with SIRIPLAN were consistent with previous knowledge of Pantanal dynamics, but comparison with inundation extent provided by a previous satellite‐based study indicates that permanently flooded areas may have been underestimated. The results obtained are promising, and further work will focus on improving vertical processes representation over floodplains and analysing model sensitivity to floodplain parameters, time step and precipitation estimates uncertainty. Copyright © 2010 John Wiley & Sons, Ltd.
HFE polymorphisms influence the response to chemotherapeutic agents via induction of p16INK4A
Authors: Lee Sang Y. , Liu Siying , Mitchell Ryan M. , Slagle‐Webb Becky , Hong Young‐Soo , Sheehan Jonas M. , Connor James R.
Published: December 2010

HFE is a protein that impacts cellular iron uptake. HFE gene variants are identified as risk factors or modifiers for multiple diseases. Using HFE stably transfected human neuroblastoma cells, we found that cells carrying the C282Y HFE variant do not differentiate when exposed to retinoic acid. Therefore, we hypothesized HFE variants would impact response to therapeutic agents. Both the human neuroblastoma and glioma cells that express the C282Y HFE variant are resistant to Temodar, geldanamycin and γ‐radiation. A gene array analysis revealed that p16INK4A (p16) expression was increased in association with C282Y expression. Decreasing p16 protein by siRNA resulted in increased vulnerability to all of the therapeutic agents suggesting that p16 is responsible for the resistance. Decreasing HFE expression by siRNA resulted in a 85% decrease in p16 expression in the neuroblastoma cells but not the astrocytoma cells. These data suggest a potential direct relationship between HFE and p16 that may be cell specific or mediated by different pathways in the different cell types. In conclusion, the C282Y HFE variant impacts the vulnerability of cancer cells to current treatment strategies apparently by increasing expression of p16. Although best known as a tumor suppressor, there are multiple reports that p16 is elevated in some forms of cancer. Given the frequency of the HFE gene variants, as high as 10% of the Caucasian population, these data provide compelling evidence that the C282Y HFE variant should be part of a pharmacogenetic strategy for evaluating treatment efficacy in cancer cells.
HPV involvement in tonsillar cancer: Prognostic significance and clinically relevant markers
Authors: Rotnáglová Eliška , Tachezy Ruth , Saláková Martina , Procházka Bohumír , Košl'abová Eva , Veselá Eva , Ludvíková Viera , Hamšíková Eva , Klozar Jan
Published: December 2010

The association of high‐risk human papillomaviruses (HR HPVs) with tonsillar cancer (TC) has been documented. Because patients with HPV‐associated tumors show better survival rates, modification of their treatment regimen is being considered. It is therefore crucial to find markers for the identification of patients whose tumors are linked to viral infection. A cohort of 109 patients with primary TC was screened for HPV DNA presence in the tumor tissues and HPV‐specific antibodies in sera. Data regarding risk factors and clinical parameters were collected. Forty‐five specimens were analyzed for the expression of viral E6 and E2‐region mRNA, and the p16 and p53 protein expression status was assessed by immunohistochemistry. The overall prevalence of HPV DNA in TC tissues was 65.1%. Ninety‐three percent of HR HPV DNA‐positive samples expressed E6*I mRNA. E2‐region mRNA expression was detected in 36% of positive samples, which implies that the virus is integrated in 64% of HPV DNA/RNA‐positive tumors. p16 overexpression and the presence of antibodies specific to HPV16 E6/E7 oncoproteins correlated well with HPV DNA and RNA presence. The disease‐specific survival rate of patients with HPV DNA‐positive tumors was significantly higher than that of HPV DNA‐negative patients. In addition to providing further evidence of the involvement of HPV infection in the etiopathogenesis of a proportion of TC cases, our study demonstrates that p16 immunostaining and anti‐E6/E7 antibodies as surrogate markers of HPV involvement represent specific, sensitive and clinically accessible assays for the identification of TC patients who have a considerably better prognosis.
Combined CADM1 and MAL promoter methylation analysis to detect (pre‐)malignant cervical lesions in high‐risk HPV‐positive women
Authors: Overmeer Renée M. , Louwers Jacqueline A. , Meijer Chris J.L.M. , van Kemenade Folkert J. , Hesselink Albertus T. , Daalmeijer Nathalie Fransen , Wilting Saskia M. , Heideman Daniëlle A.M. , Verheijen René H.M. , Zaal Afra , Marchien van Baal W. , Berkhof Johannes , Snijders Peter J.F. , Steenbergen Renske D.M.
Published: December 2010

Given the lower specificity for high‐grade cervical lesions of high‐risk human papillomavirus (hrHPV) testing compared to cytology, additional triage testing for hrHPV test‐positive women is needed to detect high‐grade cervical lesions. Here, we tested whether combined methylation analysis for cell adhesion molecule 1 (CADM1) and T‐lymphocyte maturation associated protein (MAL), both functionally involved in cervical carcinogenesis, could serve as such a triage marker. Four quantitative methylation‐specific PCRs (qMSP), two for CADM1 (regions M12 and M18) and MAL (regions M1 and M2) each, were applied to 261 cervical tissue specimens ranging from no neoplasia to carcinoma. When qMSPs were combined and positivity for at least one of the qMSPs in the combination was taken into account, the highest positivity rates for cervical intraepithelial neoplasia grade 3 (CIN3) lesions (97%) and squamous cell‐ and adeno‐carcinomas (99%) were obtained by combining a single CADM1 marker with a single MAL marker. Subsequent qMSP analysis of 70 GP5+/6+‐PCR hrHPV‐positive scrapings revealed that a two‐marker panel consisting of CADM1‐M18 and MAL‐M1 was most discriminative, detecting 90% of women with CIN3 (n = 30), whereas it showed a positive result in only 13.5% of women without cervical disease (n = 40). Finally, we applied hrHPV GP5+/6+‐PCR testing followed by CADM1‐M18/MAL‐M1 methylation analysis to a cohort of 79 women visiting the outpatient colposcopy clinic. hrHPV testing revealed a sensitivity of 97% and a specificity of 33% for CIN3+. Additional CADM1‐M18/MAL‐M1 methylation analysis on the hrHPV‐positive women increased the specificity to 78% with a sensitivity of 70%. In conclusion, the methylation marker panel CADM1‐M18 and MAL‐M1 may serve as an alternative molecular triage tool for hrHPV‐positive women.
Polymorphonuclear granulocytes in human head and neck cancer: Enhanced inflammatory activity, modulation by cancer cells and expansion in advanced disease
Authors: Trellakis Sokratis , Bruderek Kirsten , Dumitru Claudia A. , Gholaman Hossein , Gu Xiang , Bankfalvi Agnes , Scherag André , Hütte Jan , Dominas Nina , Lehnerdt Götz F. , Hoffmann Thomas K. , Lang Stephan , Brandau Sven
Published: December 2010

The progression of epithelial cancer is associated with an intense immunological interaction between the tumor cells and immune cells of the host. However, little is known about the interaction between tumor cells and polymorphonuclear granulocytes (PMNs) in patients with head and neck squamous cell carcinoma (HNSCC). In our study, we investigated systemic PMN‐related alterations in HNSCC, the role of tumor‐infiltrating PMNs and their modulation by the tumor microenvironment. We assessed the infiltration of HNSCC tissue by PMNs (retrospectively) and systemic PMN‐related alterations in blood values (prospectively) in HNSCC patients (n = 99 and 114, respectively) and control subjects (n = 41). PMN recruitment, apoptosis and inflammatory activity were investigated in an in vitro system of peripheral blood PMNs and a human HNSCC cell line (FaDu). HNSCC tissue exhibited considerable infiltration by PMNs, and strong infiltration was associated with poorer survival in advanced disease. PMN count, neutrophil‐to‐lymphocyte ratio and serum concentrations of CXCL8 (interleukin‐8), CCL4 (MIP‐1β) and CCL5 (RANTES) were significantly higher in the peripheral blood of HNSCC patients than in that of controls. In vitro, HNSCC‐conditioned medium inhibited apoptosis of PMNs, increased chemokinesis and chemotaxis of PMNs, induced release of lactoferrin and matrix metalloproteinase 9 by PMNs and enhanced the secretion of CCL4 by PMN. Our findings demonstrate alterations in PMN biology in HNSCC patients. In vitro, tumor‐derived factors modulate cellular functions of PMNs and increase their inflammatory activity. Thus, the interaction between HNSCC and PMNs may contribute to host‐mediated changes in the tumor microenvironment.
Functional characterization of a BRAF insertion mutant associated with pilocytic astrocytoma
Authors: Eisenhardt Anja E. , Olbrich Heike , Röring Michael , Janzarik Wibke , Anh Ton Nu Van , Cin Huriye , Remke Marc , Witt Hendrik , Korshunov Andrey , Pfister Stefan M. , Omran Heymut , Brummer Tilman
Published: December 2010

Pilocytic astrocytoma (PA) is emerging as a tumor entity with dysregulated Ras/Raf/MEK/ERK signaling. Common genetic lesions observed in PA, which are linked to aberrant ERK pathway activity, include either NF1 inactivation, KRAS or BRAF gain‐of‐function mutations. To investigate the mutation spectrum within the proto‐oncogene encoding the Ser/Thr‐kinase B‐Raf in more detail, we analyzed 64 primary tumor samples from children with PA including two patients with neurofibromatosis type 1 (NF1). The well‐known BRAFV600E mutation was found in 6/64 (9.38%) of our samples. For the first time, we report concomitant presence of a somatic BRAFV600E mutation in an NF1 patient indicating that more than one Ras/ERK pathway component can be affected in PA. Furthermore, 2/64 (3.13%) of our samples carried a 3‐bp insertion in BRAF resulting in the duplication of threonine 599. This conserved residue is located within the activation segment and, if phosphorylated in a Ras‐dependent manner, plays a key role in Raf activation. Here, we demonstrate that this mutant (B‐RafinsT) and another B‐Raf mutant, which carries two additional threonine residues at this position, display an in vitro kinase activity and cellular MEK/ERK activation potential comparable to those of B‐RafV600E. Notably, replacement of threonines by valine residues had similar effects on B‐Raf activity, suggesting that the distortion of the peptide backbone by additional amino acids rather than the insertion of additional, potential phosphorylation sites destabilizes the inactive conformation of the kinase domain. We also demonstrate that B‐RafinsT and B‐RafV600E, but not B‐Rafwt, provoke drastic morphological alterations in human astrocytes.
KLF1 directly coordinates almost all aspects of terminal erythroid differentiation
Authors: Tallack Michael R. , Perkins Andrew C.
Published: December 2010

The molecular events and transcriptional mechanisms that underlie erythropoiesis are of great interest to biologists and hematologists since disorders of erythrocytes are common and remain relatively poorly understood. Kruppel‐like factor 1 (KLF1) is a critical transcription factor for erythropoiesis in mice and man. Recently the use of chromatin immunoprecipitation (ChIP) coupled to next‐generation DNA sequencing (ChIP‐seq) has led to an updated understanding of how KLF1 functions in vivo. The full extent of KLF1 target genes have provided new insights into erythropoiesis, and have established that KLF1 controls almost all aspects of erythroid cell development and maturation. © 2010 IUBMB IUBMB Life, 62(12): 886–890
Naturally occurring organic osmolytes: From cell physiology to disease prevention
Authors: Khan Shagufta H. , Ahmad Nihal , Ahmad Faizan , Kumar Raj
Published: December 2010

Osmolytes are naturally occurring organic compounds, which represent different chemical classes including amino acids, methylamines, and polyols. By accumulating high concentrations of osmolytes, organisms adapt to perturbations that can cause structural changes in their cellular proteins. Osmolytes shift equilibrium toward natively‐folded conformations by raising the free energy of the unfolded state. As osmolytes predominantly affect the protein backbone, the balance between osmolyte–backbone interactions and amino acid side chain–solvent interactions determines protein folding. Abnormal cell volume regulation significantly contributes to the pathophysiology of several disorders, and cells respond to these changes by importing, exporting, or synthesizing osmolytes to maintain volume homeostasis. In recent years, it has become quite evident that cells regulate many biological processes such as protein folding, protein disaggregation, and protein–protein interactions via accumulation of specific osmolytes. Many genetic diseases are attributed to the problems associated with protein misfolding/aggregation, and it has been shown that certain osmolytes can protect these proteins from misfolding. Thus, osmolytes can be utilized as therapeutic targets for such diseases. In this review article, we discuss the role of naturally occurring osmolytes in protein stability, underlying mechanisms, and their potential use as therapeutic molecules. © 2010 IUBMB IUBMB Life, 62(12): 891–895
Dynamic interaction between breast cancer cells and osteoblastic tissue: Comparison of Two‐ and Three‐dimensional cultures
Authors: Krishnan Venkatesh , Shuman Laurie A. , Sosnoski Donna M. , Dhurjati Ravi , Vogler Erwin A. , Mastro Andrea M.
Published: December 2010

Breast cancer cell colonization of osteoblast monolayers grown in standard tissue culture (2D) is compared to colonization of a multi‐cell‐layer osteoblastic tissue (3D) grown in a specialized bioreactor. Colonization of 3D tissue recapitulates events observed in clinical samples including cancer penetration of tissue, growth of microcolonies, and formation of “Single cell file” commonly observed in end‐stage pathological bone tissue. By contrast, adherent cancer cell colonies did not penetrate 2D tissue and did not form cell files. Thus, it appears that 3D tissue is a more biologically (clinically) relevant model than 2D monolayers in which to study cancer cell interactions with osteoblastic tissue. This direct comparison of 2D and 3D formats is implemented using MC3T3‐E1 murine osteoblasts and MDA‐MB‐231 human metastatic breast cancer cells, or the metastasis‐suppressed line, MDA‐MB‐231BRMS1, for comparison. When osteoblasts were co‐cultured with metastatic cells, production of osteocalcin (a mineralization marker) decreased and secretion of the pro‐inflammatory cytokine IL‐6 increased in both 2D and 3D formats. Cancer cell penetration of the 3D tissue coincided with a changed osteoblast morphology from cuboidal to spindle‐shaped, and with osteoblasts alignment parallel to the cancer cells. Metastasis‐suppressed cells did not penetrate 3D tissue, did not cause a change in osteoblast morphology or align in rows. Moreover, they proliferated much less in the 3D culture than in the 2D culture in a manner similar to their growth in bone. In both systems, the cancer cells proliferated to a greater extent with immature osteoblasts compared to more mature osteoblasts. J. Cell. Physiol. 226: 2150–2158, 2011. © 2010 Wiley‐Liss, Inc.
PKCε promotes cardiac mitochondrial and metabolic adaptation to chronic hypobaric hypoxia by GSK3β inhibition
Authors: McCarthy Joy , Lochner Amanda , Opie Lionel H. , Sack Michael N. , Essop M. Faadiel
Published: December 2010

PKCε is central to cardioprotection. Sub‐proteome analysis demonstrated co‐localization of activated cardiac PKCε (aPKCε) with metabolic, mitochondrial, and cardioprotective modulators like hypoxia‐inducible factor 1α (HIF‐1α). aPKCε relocates to the mitochondrion, inactivating glycogen synthase kinase 3β (GSK3β) to modulate glycogen metabolism, hypertrophy and HIF‐1α. However, there is no established mechanistic link between PKCε, p‐GSK3β and HIF1‐α. Here we hypothesized that cardiac‐restricted aPKCε improves mitochondrial response to hypobaric hypoxia by altered substrate fuel selection via a GSK3β/HIF‐1α‐dependent mechanism. aPKCε and wild‐type (WT) mice were exposed to 14 days of hypobaric hypoxia (45 kPa, 11% O2) and cardiac metabolism, functional parameters, p‐GSK3β/HIF‐1α expression, mitochondrial function and ultrastructure analyzed versus normoxic controls. Mitochondrial ADP‐dependent respiration, ATP production and membrane potential were attenuated in hypoxic WT but maintained in hypoxic aPKCε mitochondria (P < 0.005, n = 8). Electron microscopy revealed a hypoxia‐associated increase in mitochondrial number with ultrastructural disarray in WT versus aPKCε hearts. Concordantly, left ventricular work was diminished in hypoxic WT but not aPKCε mice (glucose only perfusions). However, addition of palmitate abrogated this (P < 0.05 vs. WT). aPKCε hearts displayed increased glucose utilization at baseline and with hypoxia. In parallel, p‐GSK3β and HIF1‐α peptide levels were increased in hypoxic aPKCε hearts versus WT. Our study demonstrates that modest, sustained PKCε activation blunts cardiac pathophysiologic responses usually observed in response to chronic hypoxia. Moreover, we propose that preferential glucose utilization by PKCε hearts is orchestrated by a p‐GSK3β/HIF‐1α‐mediated mechanism, playing a crucial role to sustain contractile function in response to chronic hypobaric hypoxia. J. Cell. Physiol. 226: 2457–2468, 2011. © 2010 Wiley‐Liss, Inc.
Functional role of rho‐kinase in ameloblast differentiation
Authors: Otsu Keishi , Kishigami Ryota , Fujiwara Naoki , Ishizeki Kiyoto , Harada Hidemitsu
Published: December 2010

During tooth development, inner enamel epithelial (IEE) cells differentiate into enamel‐secreting ameloblasts, a polarized and elongated cellular population. The molecular underpinnings of this morphogenesis and cytodifferentiation, however, are not well understood. Here, we show that Rho‐associated coiled‐coil‐containing protein kinase (ROCK) regulates ameloblast differentiation and enamel formation. In mouse incisor organ cultures, inhibition of ROCK, hindered IEE cell elongation and disrupted polarization of differentiated ameloblasts. Expression of enamel matrix proteins, such as amelogenin and ameloblastin, and formation of the terminal band structure of actin and E‐cadherin were also perturbed. Cultures of dental epithelial cells revealed that ROCK regulates cell morphology and cell adhesion through localization of actin bundles, E‐cadherin, and β‐catenin to cell membranes. Moreover, inhibition of ROCK promoted cell proliferation. Small interfering RNA specific for ROCK1 and ROCK2 demonstrated that the ROCK isoforms performed complementary functions in the regulation of actin organization and E‐cadherin‐mediated cell–cell adhesion. Thus, our results have uncovered a novel role for ROCK in amelogenesis. J. Cell. Physiol. 226: 2527–2534, 2011. © 2010 Wiley‐Liss, Inc.
Overexpression of gastrokine 1 in gastric cancer cells induces Fas‐mediated apoptosis
Authors: Rippa Emilia , La Monica Giovanna , Allocca Rosa , Romano Maria Fiammetta , De Palma Maurizio , Arcari Paolo
Published: December 2010

Gastrokine 1 (GKN1) is involved in the replenishment of the surface lumen epithelial cell layer, in maintaining the mucosal integrity, and could play a role in cell proliferation and differentiation. In fact, after injury of the gastric mucosa, restoration may occur very rapidly in the presence of GKN1. In contrast, if the protein is downregulated, the repair process may be hampered; however, application of GKN1 to gastrointestinal cells promoted epithelial restoration. Because GKN1 possesses some mitogenic effects on intestinal epithelial cells (IEC‐6) whereas this protein was also capable of inhibiting proliferation in gastric cancer cells (MKN28), we decided to study its involvement in apoptosis to understand the role of GKN1 in the modulation of inflammatory damage or tumorigenesis in gastric mucosa. We found by cytofluorimetry, Western blot and RT‐PCR that the overexpression of GKN1 in gastric cancer cell lines (AGS and MKN28) stimulated the expression of Fas receptor. Moreover, compared to control cells, a significant increase of apoptosis, evaluated by TUNEL, was observed when GKN1 transfected cells were treated with a monoclonal antibody (IgM) anti‐Fas. The activation of Fas expression was also observed by the overexpression of GKN1 in other cancer cell lines. Moreover, in GKN1‐overexpressing gastric cancer cells exposed to FasL, the activation of caspase‐3 was also observed by Western blot and fluorescence assays. Our data represent the first report for GKN1 as modulator of apoptotic signals and suggest that GKN1 might play an important role for tissue repair during the early stages of neoplastic transformation. J. Cell. Physiol. 226: 2571–2578, 2011. © 2010 Wiley‐Liss, Inc.
The effect of endothelin‐1 on Src‐family tyrosine kinases and Na,K‐ATPase activity in porcine lens epithelium
Authors: Mandal A. , Shahidullah M. , Beimgraben C. , Delamere N.A.
Published: December 2010

Previous studies show Src family kinase (SFK) activation is involved in a response that stimulates Na,K‐ATPase. Here, we tested whether SFK activation is involved in the Na,K‐ATPase response to endothelin‐1 (ET‐1). Intact porcine lenses were exposed to 100 nM ET‐1 for 5–30 min. Then, the epithelium was removed and used for Na,K‐ATPase activity measurement and Western blot analysis of SFK activation. Na,K‐ATPase activity was reduced by ∼30% in lenses exposed to ET‐1 for 15 min. The response was abolished by the SFK inhibitor PP2 or the ET receptor antagonist, PD145065. Activation of a ∼61 kDa SFK was evident from an increase in Y416 phosphorylation, which reached a maximum at 15 min ET‐1 treatment, and a decrease in Y527 phosphorylation. PP2 prevented SFK activation. Since Fyn, Src, Hck, and Yes may contribute to the observed 61 kDa band, these SFKs were isolated by immunoprecipitation and analyzed. Based on Y416 phosphorylation, ET‐1 appeared to activate Fyn, while Src and Hck were inhibited and Yes was unaltered. ET‐1 requires SFK activation to cause Na,K‐ATPase inhibition. ET‐1 elicits a different pattern of SFK activation from that reported earlier for purinergic agonists that stimulate Na,K‐ATPase activity and activate Src. In the ET‐1 response Src is inhibited and Fyn is activated. The findings suggest SFK phosphorylation is involved in a regulatory mechanism for Na,K‐ATPase. Knowing this may help us understand drug actions on Na,K‐ATPase. Faulty regulation of Na,K‐ATPase in the lens could contribute to cataract formation since an abnormal sodium content is associated with lens opacification. J. Cell. Physiol. 226: 2555–2561, 2011. © 2010 Wiley‐Liss, Inc.
The requirement for proteomics to unravel stem cell regulatory mechanisms
Authors: Williamson Andrew J.K. , Whetton Anthony D.
Published: December 2010

Stem cells are defined by their ability to self‐renew and to differentiate, the processes whereby these events are achieved is the subject of much investigation. These studies include cancer stem cell populations, where eradication of this specific population is the ultimate goal of treatment. Whilst cellular signalling events and transcription factor complex‐mediated changes in gene expression have been analysed in some detail within stem cells, full systematic understanding of the events promoting self‐renewal or the commitment process leading to formation of a specific cell type require a systems biology approach. This in turn demands a need for proteomic analysis of post‐translational regulation of protein levels, protein interactions, protein post‐translational modification (e.g. ubiquitination, methylation, acetylation, phosphorylation) to identify networks for stem cell regulation. Furthermore, the phenomenon of induced pluripotency via cellular reprogramming also can be understood optimally using combined molecular biology and proteomics approaches; here we describe current research employing proteomics and mass spectrometry to dissect stem cell regulatory mechanisms. J. Cell. Physiol. 226: 2478–2483, 2011. © 2010 Wiley‐Liss, Inc.
Neutral sphingomyelinase, NADPH oxidase and reactive oxygen species. Role in acute hypoxic pulmonary vasoconstriction
Authors: Frazziano Giovanna , Moreno Laura , Moral‐Sanz Javier , Menendez Carmen , Escolano Lucía , Gonzalez Constancio , Villamor Eduardo , Alvarez‐Sala Jose Luis , Cogolludo Angel L. , Perez‐Vizcaino Francisco
Published: December 2010

The molecular mechanisms underlying hypoxic pulmonary vasoconstriction (HPV) are not yet properly understood. Mitochondrial electron transport chain (ETC) and NADPH oxidase have been proposed as possible oxygen sensors, with derived reactive oxygen species (ROS) playing key roles in coupling the sensor(s) to the contractile machinery. We have recently reported that activation of neutral sphingomyelinase (nSMase) and protein kinase C ζ (PKCζ) participate in the signalling cascade of HPV. Herein, we studied the significance of nSMase in controlling ROS production rate in rat pulmonary artery (PA) smooth muscle cells and thereby HPV in rat PA. ROS production (analyzed by dichlorofluorescein and dihydroethidium fluorescence) was increased by hypoxia in endothelium‐denuded PA segments and their inhibition prevented hypoxia‐induced voltage‐gated potassium channel (KV) inhibition and pulmonary vasoconstriction. Consistently, H2O2, or its analogue t‐BHP, decreased KV currents and induced a contractile response, mimicking the effects of hypoxia. Inhibitors of mitochondrial ETC (rotenone) and NADPH oxidase (apocynin) prevented hypoxia‐induced ROS production, KV channel inhibition and vasoconstriction. Hypoxia induced p47phox phosphorylation and its interaction with caveolin‐1. Inhibition of nSMase (GW4869) or PKCζ prevented p47phox phosphorylation and ROS production. The increase in ceramide induced by hypoxia (analyzed by immunocytochemistry) was inhibited by rotenone. Exogenous ceramide increased ROS production in a PKCζ sensitive manner. We propose an integrated signalling pathway for HPV which includes nSMase‐PKCζ‐NADPH oxidase as a necessary step required for ROS production and vasoconstriction. J. Cell. Physiol. 226: 2633–2640, 2011. © 2010 Wiley‐Liss, Inc.
Proteomic and functional analysis of human sperm detergent resistant membranes
Authors: Nixon Brett , Mitchell Lisa A. , Anderson Amanda L. , Mclaughlin Eileen A. , O'bryan Moira K. , Aitken R. John
Published: December 2010

Mammalian spermatozoa attain the ability to fertilize an oocyte as they negotiate the female reproductive tract. This acquisition of functional competence is preceded by an intricate cascade of biochemical and functional changes collectively known as “capacitation.” Among the universal correlates of the capacitation process is a remarkable remodeling of the lipid and protein architecture of the sperm plasma membrane. While the mechanisms that underpin this dynamic reorganization remain enigmatic, emerging evidence has raised the prospect that it may be coordinated, in part, by specialized membrane microdomains, or rafts. In the present study we have demonstrated that human spermatozoa express recognized markers of membrane rafts. Further, upon depletion of membrane cholesterol through either physiological (capacitation) or pharmacological (methyl‐β‐cyclodextrin) intervention, these membrane rafts appear to undergo a polarized redistribution to the peri‐acrosomal region of the sperm head. This finding encourages speculation that membrane rafts represent platforms for the organization of proteins involved in sperm–oocyte interactions. Support for this notion rests with the demonstration that membrane rafts isolated on the basis of their biochemical composition in the form of detergent resistant membranes (DRMs), possess the ability to adhere to homologous zona pellucidae. Furthermore a comprehensive proteomic analysis of the DRMs identified a number of proteins known for their affinity for the zona pellucida in addition to other candidates putatively involved in the mediation of downstream binding and/or fusion with the oolemma. Collectively these data afford novel insights into the subcellular localization and potential functions of membrane rafts in human spermatozoa. J. Cell. Physiol. 226: 2651–2665, 2011. © 2010 Wiley‐Liss, Inc.
Role of Doppler sonography in the diagnosis of pulmonary sequestration in an elderly patient: A case report
Authors: Huang Li‐Kuo , Tsai Ming‐Ji , Chao Heng‐Sheng , Chen Chun‐Ku , Chang Shi‐Chuan
Published: December 2010

An 83‐year‐old man presented with an asymptomatic mass‐like lesion in left lower lobe on chest radiograph and CT, with no change over the past 7 years. Because of discrepancy between clinical and radiological manifestations, chest color Doppler sonography was done and identified a large tortuous pulsating vessel with systemic arterial waveform flowing toward the probe and entering the lesion at its apex. Subsequent contrast‐enhanced reconstructed CT scans of the chest with angiography confirmed the diagnosis of intralobar pulmonary sequestration. © 2010 Wiley Periodicals, Inc. J Clin Ultrasound, 2011