bluuebunny/crossref_metadata_2025 huggingface dataset:
Ethnic Proximity and Ethnic Voting in Peru
Authors: MADRID RAÚL L.
Published: July 2011

Peru, in contrast to neighbouring Bolivia and Ecuador, has neither an important indigenous party nor a strong indigenous movement. Nevertheless, in recent years a growing gap has emerged in the voting patterns of indigenous and non-indigenous areas. This article maintains that this gap has developed because some Peruvian politicians, including Alberto Fujimori, Alejandro Toledo and Ollanta Humala, successfully wooed indigenous voters with a combination of ethnic and populist appeals. Like traditional populist leaders, they denounced the political elites, focused their campaigns on the poor and presented themselves as the saviours of Peru, but also forged ties to indigenous leaders, invoked indigenous symbols and embraced some ethnic demands. Although neither Fujimori, nor Toledo, nor Humala self-identified as indigenous, they successfully presented themselves as more ethnically proximate to the indigenous population than their main competitors, who represented the white Lima elite.
COLLECTIVE MEMORY IN THE FRONTIERS: A CASE STUDY FROM THE ANCIENT MAYA CENTER OF MINANHA, BELIZE
Authors: Iannone Gyles
Published: July 2011

The examination of the internal frontiers between ancient Maya polities is a topic that has received little focused attention. This article explores various topics associated with frontiers and frontier communities, including: (1) how they might be located archaeologically; (2) what their material correlates might be; and, (3) what we might expect in terms of their sociopolitical characteristics. Special attention is paid to the role that collective memory plays in both the definition and reaffirmation of territorial limits and in the production and reproduction of frontier identities. The ancient Maya center of Minanha is used as a case study.
THE MYSTERIOUS AND THE INVISIBLE: WRITING HISTORY IN AND OF COLONIAL YUCATAN
Authors: Restall Matthew
Published: July 2011

This brief essay argues that studying the non-Spanish inhabitants of Yucatan's past requires bridging the social distance generated by differences of time and culture and that the specific nature of that distance must first be understood. With respect to the Mayas, their mystique in the modern popular and academic imaginations is as much the creation of Maya elites in ancient and colonial times as it is the product of archaeologists and historians. To demystify the Maya, we must engage mundane as well as exotic sources and be aware of the obfuscating influence of those who interpreted Maya culture before us. A complete picture of colonial Yucatan and of the colonial Mayas must include Afro-Yucatecans, or Africans and their descendents in the peninsula. Rendered invisible by historical processes and lack of scholarly attention, Afro-Yucatecans must be fully examined if we are to fully grasp the Yucatec experience, including the Yucatec Maya experience.
A MAZAPA PHASE COPPER FIGURINE FROM ATETELCO, TEOTIHUACAN: DATA AND SPECULATIONS
Authors: Hosler Dorothy , Cabrera Ruben
Published: July 2011

Excavations of the Mazapan phase at the Atetelco complex at Teotihuacan by Ruben Cabrera and colleagues produced a small human copper figurine. Studies of Mesoamerican metallurgy have indicated that metallurgy was introduced to western Mexico arounda.d.700 from Colombia, and Ecuador. This particular figurine is a remarkable find. The dates (a.d.800–900) precede those of copper artifacts in the central Highlands. The manufacturing method (an open-backed casting) is rare in Mesoamerica but common in Colombia and Panama. Open-backed cast figurines that do appear in Mesoamerica come from the Cenote of Sacrifice, are made from copper-gold alloys, and were imported from lower Central America or Colombia. The Atetelco figurine is made from a copper-arsenic alloy. Copper-arsenic alloy objects are common in western Mexico (aftera.d.1150) and Ecuador and northern Peru (after abouta.d.300). The major question concerning the Atetelco figurine is whether it was fashioned in Ecuador or Colombia and imported, or whether it was produced in western Mexico. Our study of artifact chemistry, fabrication methods, and provenience indicate that the weight of the evidence points to western Mexico. We suggest that this fabrication technique (lost-wax castings of open-backed human figurines), while introduced from Colombia and lower Central America, never became a major component of the Mesoamerican metallurgical tradition.
Design and rationale of a genetic cohort study on congenital cardiac disease: experiences from a multi-institutional platform in Quebec
Authors: Dubé Marie-Pierre , Bigras Jean-Luc , Thibeault Maryse , Bureau Nathalie , Chetaille Philippe , Richter Andrea , Mercier Jocelyne , Bellavance Marc , Rohlicek Charles , Rozen Rima , Nemer Mona , Khairy Paul , Gendron Roxanne , Andelfinger Gregor
Published: July 2011

Background

Congenital cardiac disease is the most common malformation, and a substantial source of mortality and morbidity in children and young adults. A role for genetic factors is recognised for these malformations, but overall few predisposing loci have been identified. Here we report the rationale, design, and first results of a multi-institutional congenital cardiac disease cohort, assembled mainly from the French-Canadian population of the province of Quebec and centred on families with multiple affected members afflicted by cardiac malformations.

Methods

Families were recruited into the study, phenotyped and sampled for DNA in cardiology clinics over the first 3 years of enrolment. We performed segregation analysis and linkage simulations in the subgroup of families with left ventricular outflow tract obstruction (LVOTO).

Results

A total of 1603 participants from 300 families were recruited, with 169 out of 300 (56.3%) families having more than one affected member. For the LVOTO group, we estimate heritability to be 0.46–0.52 in our cohort. Simulation analysis demonstrated sufficient power to carry out linkage analyses, with an expected mean log-of-odds (LOD) score of 3.8 in 67 pedigrees with LVOTO.

Conclusion

We show feasibility and usefulness of a population-based biobank for genetic investigations into the causes of congenital cardiac disease. Heritability of LVOTO is high and could be accounted for by multiple loci. This platform is ideally suited for multiple analysis approaches, including linkage analysis and novel gene sequencing approaches, and will allow to establish segregation of risk alleles at family and population levels.
New York Heart Association class assessment by cardiologists and outpatients with congenital cardiac disease: a head-to-head comparison of three patient-based versions
Authors: Schoormans Dounya , Mager Yuma L. , Oort Frans J. , Sprangers Mirjam A.G. , Mulder Barbara J.M.
Published: July 2011

Background

The objective of this study was to compare three patient-based New York Heart Association assessments with cardiologist assessments in outpatients with congenital cardiac disease.

Methods

Consecutive adult outpatients completed three questionnaires in a random order: a patient-based translation of the New York Heart Association classes, a self-constructed questionnaire based on the New York Heart Association classes, and the Specific Activity Scale. The treating cardiologist assessed the New York Heart Association class on the same day. Patient–cardiologist agreement was assessed by calculating percent agreement and weighted kappa. We also explored the level of agreement for patients without co-morbidity.

Results

In all, 86 adults – with a median age of 35.8 years – including 46 women participated. An agreement of 75.6% (weighted kappa is 0.43; probability is smaller than 0.01), 70.6% (weighted kappa is 0.44; probability is smaller than 0.01), and 74.4% (weighted kappa is 0.28; probability is smaller than 0.01) was found between the cardiologist assessment and the patient-based translation, self-constructed questionnaire, and the Specific Activity Scale, respectively. The patient-based translation equally over- and underestimated the New York Heart Association class, whereas the self-constructed questionnaire overestimated and the Specific Activity Scale underestimated the New York Heart Association class. Agreement levels for patients without co-morbidity were higher than agreement levels for the total group.

Conclusion

The patient-based translation yielded adequate agreement with cardiologist-assessed New York Heart Association class, showed equal over- and underestimation, and was easy to complete. The patient-based translation with the instruction to only consider functional impairments caused by the congenital cardiac defect is recommended in future studies of outpatients with congenital cardiac disease.
Hospital Emergency Readiness Overview Study
Authors: Kollek Daniel , Cwinn A. Adam
Published: July 2011

In 2001, a survey of Canadian emergency departments indicated significant deficiencies in disaster preparedness. Since then, there have been efforts on the part of Provincial governments to remedy this situation. This survey repeats the original study with minor modifications to determine if there has been improvement. The Hospital Emergency Readiness Overview study demonstrates that despite improvements, there remain gaps in Canadian healthcare facility readiness for disaster, specifically one involving contaminated patients. It also highlights the lack of any standardized assessment of healthcare facilities' chemical, biological, radiological, or nuclear readiness.
Pre-Meeting Sunday Short Courses
Authors: Marko Mike
Published: July 2011

These full-day courses run from 8:30 AM to 5:00 PM on Sunday, August 7, 2011 (additional fees are required).
Pre-Meeting Congress on Opportunities, Artifacts and Interpretation of Aberration-Corrected Electron Microscopy Data
Authors: Batson Philip , Muller David , Allard Lawrence , Voyles Paul , Chi Miofang , O'Keefe Michael
Published: July 2011

This one-day pre-meeting congress, organized by the MSA Aberration-Corrected Electron Microscopy (ACEM) FIG, will be a forum for the discussion of the latest advances and solutions to problems associated with application of aberration correction technology. There will be platform presentations by both invited and contributed speakers, with poster presentations during a working lunch. Invited speakers will introduce innovations and issues, while contributors will highlight practical experiences and solutions to problems encountered during the application of ACEM to on-going experimental studies. This workshop includes: image collection/interpretation, new spectroscopies or other signals, artifacts and practical experiences in applications of ACEM to difficult situations such as hard/soft materials and in-situ experiments. All platform presentations will be intentionally kept short (∼15–20 minutes) to allow the maximum amount of interaction and information flow among attendees. Please send one page abstracts, including figures, to batson@rutgers.edu with the subject line: M&M PMC Abstract.
Dissoziative Störungen mit Beginn im Kindes- und Jugendalter
Authors: Jans Thomas , Warnke Andreas
Published: July 2011

Zu den dissoziativen Störungen vom Bewusstseinstyp werden psychogene Syndrome mit Amnesie, Stupor, Fugue, Trance- und Besessenheit, Identitätsdiffusion sowie Depersonalisation und Derealisation gerechnet. Dissoziative Symptome treten nicht nur im Rahmen dieser dissoziativen Störungen auf, sondern auch bei anderen, vor allem posttraumatischen Störungen. Dass behandlungsbedürftige dissoziative Syndrome bereits im Kindesalter vorkommen, ist unbestritten. Allerdings fehlen adaptierte diagnostische Kriterien für diese Altersgruppe. Stark ergänzungsbedürftig sind auch die Erkenntnisse zu Auftretenshäufigkeit, Komorbidität und Verlauf des Störungsbildes. Behandlungsstrategien sind nicht hinreichend evidenzbasiert. Vor diesem Hintergrund wird ein kurzer Überblick zu Symptomatik, Diagnostik, Komorbidität, Verlauf und Behandlung psychoformer dissoziativer Störungen im Kindes- und Jugendalter gegeben.
Frühe Emotionsdysregulation: Ein Indikator für psychische Störungen im Kindesalter?
Authors: Petermann Franz , Kullik Angelika
Published: July 2011

Die Rolle der Emotionsregulation im Säuglings- und Kleinkindalter für die Entwicklung psychischer Störungen findet bisher wenig Beachtung und soll diskutiert werden. Zunächst ist zu klären, was Emotionsregulation eigentlich bedeutet und welche konkreten Emotionsregulationsstrategien im frühen Kindesalter Anwendung finden. Auch der Emotionsdysregulation mangelt es an einer einheitlichen Definition. Es wird die Frage aufgeworfen, ob die für das DSM-V vorgeschlagene „Temper Dysregulation Disorder with Dysphoria“ Emotionsdysregulation diagnostiziert. Ferner muss Emotionsdysregulation in ihrem Zusammenhang mit psychischen Störungen betrachtet werden, wobei hier wenige Befunde für das frühe Kindesalter vorliegen. Dies macht die Klärung der Rolle der Emotionsregulation in der frühen Kindheit zum großen Problem.
Ceramide synthase 4 and de novo production of ceramides with specific N-acyl chain lengths are involved in glucolipotoxicity-induced apoptosis of INS-1 β-cells
Authors: Véret Julien , Coant Nicolas , Berdyshev Evgeny V. , Skobeleva Anastasia , Therville Nicole , Bailbé Danielle , Gorshkova Irina , Natarajan Viswanathan , Portha Bernard , Le Stunff Hervé
Published: July 2011

Pancreatic β-cell apoptosis induced by palmitate requires high glucose concentrations. Ceramides have been suggested to be important mediators of glucolipotoxicity-induced β-cell apoptosis. In INS-1 β-cells, 0.4 mM palmitate with 5 mM glucose increased the levels of dihydrosphingosine and dihydroceramides, two lipid intermediates in the de novo biosynthesis of ceramides, without inducing apoptosis. Increasing glucose concentrations to 30 mM amplified palmitate-induced accumulation of dihydrosphingosine and the formation of (dihydro)ceramides. Of note, glucolipotoxicity specifically induced the formation of C18:0, C22:0 and C24:1 (dihydro)ceramide molecular species, which was associated with the up-regulation of CerS4 (ceramide synthase 4) levels. Fumonisin-B1, a ceramide synthase inhibitor, partially blocked apoptosis induced by glucolipotoxicity. In contrast, apoptosis was potentiated in the presence of D,L-threo-1-phenyl-2-palmitoylamino-3-morpholinopropan-1-ol, an inhibitor of glucosylceramide synthase. Moreover, overexpression of CerS4 amplified ceramide production and apoptosis induced by palmitate with 30 mM glucose, whereas down-regulation of CerS4 by siRNA (short interfering RNA) reduced apoptosis. CerS4 also potentiates ceramide accumulation and apoptosis induced by another saturated fatty acid: stearate. Collectively, our results suggest that glucolipotoxicity induces β-cell apoptosis through a dual mechanism involving de novo ceramide biosynthesis and the formation of ceramides with specific N-acyl chain lengths rather than an overall increase in ceramide content.
Suppression of TG-interacting factor sensitizes arsenic trioxide-induced apoptosis in human hepatocellular carcinoma cells
Authors: Liu Zi-Miao , Tseng Joseph T. , Hong Duang-Yang , Huang Huei-Sheng
Published: July 2011

HCC (hepatocellular carcinoma) is among the most common and lethal cancers worldwide with a poor prognosis mainly due to a high recurrence rate and chemotherapy resistance. ATO (arsenic trioxide) is a multi-target drug that has been effectively used as an anticancer drug in acute promyelocytic leukaemia. However, a Phase II trial involving patients with HCC indicates that the use of arsenic as a single agent is not effective against HCC. TGIF (TG-interacting factor) is a transcriptional co-repressor that interferes with TGF-β (transforming growth factor-β) signalling which plays a growth-inhibitory role in HCC. In the present study, we demonstrated that ATO induced hepatocellular apoptosis via TGF-β/Smad signalling and led to downstream induction of p21WAF1/CIP1 (p21). However, ATO could also induce TGIF expression via a post-transcriptional regulation mechanism to antagonize this effect. Using a biotin-labelled RNA probe pull-down assay and in vivo RNA immunoprecipitation analysis, we identified that HuR (human antigen R) bound to the TGIF mRNA 3′-UTR (3′-untranslated region) and prevented it from degradation. ATO treatment increased the interaction between HuR and TGIF mRNA, and reduction of HuR expression inhibited ATO-induced TGIF expression. Moreover, the EGFR (epidermal growth factor receptor)/PI3K (phosphoinositide 3-kinase)/Akt pathway was shown to mediate the post-transcriptional regulation of TGIF in response to ATO. Finally, we also demonstrated that the down-regulation of TGIF could sensitize ATO-induced HepG2 cell apoptosis. Collectively, we propose that the EGFR/PI3K/Akt pathway may regulate the post-transcriptional regulation of TGIF expression to antagonize ATO-induced apoptosis in HCC. Blockage of the PI3K/Akt pathway or TGIF expression combined with ATO treatment may be a promising strategy for HCC therapy.
Ser756 of β2 integrin controls Rap1 activity during inside-out activation of αMβ2
Authors: Lim Jenson , Hotchin Neil A. , Caron Emmanuelle
Published: July 2011

During αMβ2-mediated phagocytosis, the small GTPase Rap1 activates the β2 integrin by binding to a region between residues 732 and 761. Using COS-7 cells transfected with αMβ2, we show that αMβ2 activation by the phorbol ester PMA involves Ser756 of β2. This residue is critical for the local positioning of talin and biochemically interacts with Rap1. Using the CaM (calmodulin) antagonist W7, we found Rap1 recruitment and the inside-out activation of αMβ2 to be affected. We also report a role for CaMKII (calcium/CaM-dependent kinase II) in the activation of Rap1 during integrin activation. These results demonstrate a distinct physiological role for Ser756 of β2 integrin, in conjunction with the actions of talin and Rap1, during αMβ2 activation in macrophages.
Evidence that ACN1 (acetate non-utilizing 1) prevents carbon leakage from peroxisomes during lipid mobilization in Arabidopsis seedlings
Authors: Allen Elizabeth , Moing Annick , Wattis Jonathan A. D. , Larson Tony , Maucourt Mickaël , Graham Ian A. , Rolin Dominique , Hooks Mark A.
Published: July 2011

ACN1 (acetate non-utilizing 1) is a short-chain acyl-CoA synthetase which recycles free acetate to acetyl-CoA in peroxisomes of Arabidopsis. Pulse-chase [2-13C]acetate feeding of the mutant acn1–2 revealed that acetate accumulation and assimilation were no different to that of wild-type, Col-7. However, the lack of acn1–2 led to a decrease of nearly 50% in 13C-labelling of glutamine, a major carbon sink in seedlings, and large decreases in primary metabolite levels. In contrast, acetyl-CoA levels were higher in acn1–2 compared with Col-7. The disappearance of eicosenoic acid was slightly delayed in acn1–2 indicating only a small effect on the rate of lipid breakdown. A comparison of transcript levels in acn1–2 and Col-7 showed that induced genes included a number of metabolic genes and also a large number of signalling-related genes. Genes repressed in the mutant were represented primarily by embryogenesis-related genes. Transcript levels of glyoxylate cycle genes also were lower in acn1–2 than in Col-7. We conclude that deficiency in peroxisomal acetate assimilation comprises only a small proportion of total acetate use, but this affects both primary metabolism and gene expression. We discuss the possibility that ACN1 safeguards against the loss of carbon as acetate from peroxisomes during lipid mobilization.
Variation in the organization and subunit composition of the mammalian pyruvate dehydrogenase complex E2/E3BP core assembly
Authors: Vijayakrishnan Swetha , Callow Philip , Nutley Margaret A. , McGow Donna P. , Gilbert David , Kropholler Peter , Cooper Alan , Byron Olwyn , Lindsay J. Gordon
Published: July 2011

Crucial to glucose homoeostasis in humans, the hPDC (human pyruvate dehydrogenase complex) is a massive molecular machine comprising multiple copies of three distinct enzymes (E1–E3) and an accessory subunit, E3BP (E3-binding protein). Its icosahedral E2/E3BP 60-meric ‘core’ provides the central structural and mechanistic framework ensuring favourable E1 and E3 positioning and enzyme co-operativity. Current core models indicate either a 48E2+12E3BP or a 40E2+20E3BP subunit composition. In the present study, we demonstrate clear differences in subunit content and organization between the recombinant hPDC core (rhPDC; 40E2+20E3BP), generated under defined conditions where E3BP is produced in excess, and its native bovine (48E2+12E3BP) counterpart. The results of the present study provide a rational basis for resolving apparent differences between previous models, both obtained using rhE2/E3BP core assemblies where no account was taken of relative E2 and E3BP expression levels. Mathematical modelling predicts that an ‘average’ 48E2+12E3BP core arrangement allows maximum flexibility in assembly, while providing the appropriate balance of bound E1 and E3 enzymes for optimal catalytic efficiency and regulatory fine-tuning. We also show that the rhE2/E3BP and bovine E2/E3BP cores bind E3s with a 2:1 stoichiometry, and propose that mammalian PDC comprises a heterogeneous population of assemblies incorporating a network of E3 (and possibly E1) cross-bridges above the core surface.
Interleukin enhancer-binding factor 3 functions as a liver receptor homologue-1 co-activator in synergy with the nuclear receptor co-activators PRMT1 and PGC-1α
Authors: Ohno Masae , Komakine Jun , Suzuki Eiko , Nishizuka Makoto , Osada Shigehiro , Imagawa Masayoshi
Published: July 2011

LRH-1 (liver receptor homologue-1), a transcription factor and member of the nuclear receptor superfamily, regulates the expression of its target genes, which are involved in bile acid and cholesterol homoeostasis. However, the molecular mechanisms of transcriptional control by LRH-1 are not completely understood. Previously, we identified Ku80 and Ku70 as LRH-1-binding proteins and reported that they function as co-repressors. In the present study, we identified an additional LRH-1-binding protein, ILF3 (interleukin enhancer-binding factor 3). ILF3 formed a complex with LRH-1 and the other two nuclear receptor co-activators PRMT1 (protein arginine methyltransferase 1) and PGC-1α (peroxisome proliferator-activated receptor γ co-activator-1α). We demonstrated that ILF3, PRMT1 and PGC-1α were recruited to the promoter region of the LRH-1-regulated SHP (small heterodimer partner) gene, encoding one of the nuclear receptors. ILF3 enhanced SHP gene expression in co-operation with PRMT1 and PGC-1α through the C-terminal region of ILF3. In addition, we found that the small interfering RNA-mediated down-regulation of ILF3 expression led to a reduction in the occupancy of PGC-1α at the SHP promoter and SHP expression. Taken together, our results suggest that ILF3 functions as a novel LRH-1 co-activator by acting synergistically with PRMT1 and PGC-1α, thereby promoting LRH-1-dependent gene expression.
The Arabidopsis phi class glutathione transferase AtGSTF2: binding and regulation by biologically active heterocyclic ligands
Authors: Dixon David P. , Sellars Jonathan D. , Edwards Robert
Published: July 2011

The plant-specific phi class of glutathione transferases (GSTFs) are often highly stress-inducible and expressed in a tissue-specific manner, suggestive of them having important protective roles. To date, these functions remain largely unknown, although activities associated with the binding and transport of reactive metabolites have been proposed. Using a sensitive and selective binding screen, we have probed the Arabidopsis thaliana GSTFs for natural product ligands from bacteria and plants. Uniquely, when overexpressed in bacteria, family members GSTF2 and GSTF3 bound a series of heterocyclic compounds, including lumichrome, harmane, norharmane and indole-3-aldehyde. When screened against total metabolite extracts from A. thaliana, GSTF2 also selectively bound the indole-derived phytoalexin camalexin, as well as the flavonol quercetin-3-O-rhamnoside. In each case, isothermal titration calorimetry revealed high-affinity binding (typically Kd<1 μM), which was enhanced in the presence of glutathione and by the other heterocyclic ligands. With GSTF2, these secondary ligand associations resulted in an allosteric enhancement in glutathione-conjugating activity. Together with the known stress responsiveness of GSTF2 and its association with membrane vesicles, these results are suggestive of roles in regulating the binding and transport of defence-related compounds in planta.
Palmitoylation of human proteinase-activated receptor-2 differentially regulates receptor-triggered ERK1/2 activation, calcium signalling and endocytosis
Authors: Botham Andrew , Guo Xiaodan , Xiao Yu Pei , Morice Alyn H. , Compton Steven J. , Sadofsky Laura R.
Published: July 2011

hPAR2 (human proteinase-activated receptor-2) is a member of the novel family of proteolytically activated GPCRs (G-protein-coupled receptors) termed PARs (proteinase-activated receptors). Previous pharmacological studies have found that activation of hPAR2 by mast cell tryptase can be regulated by receptor N-terminal glycosylation. In order to elucidate other post-translational modifications of hPAR2 that can regulate function, we have explored the functional role of the intracellular cysteine residue Cys361. We have demonstrated, using autoradiography, that Cys361 is the primary palmitoylation site of hPAR2. The hPAR2C361A mutant cell line displayed greater cell-surface expression compared with the wt (wild-type)-hPAR2-expressing cell line. hPAR2C361A also showed a decreased sensitivity and efficacy (intracellular calcium signalling) towards both trypsin and SLIGKV. In stark contrast, hPAR2C361A triggered greater and more prolonged ERK (extracellular-signal-regulated kinase) phosphorylation compared with that of wt-hPAR2 possibly through Gi, since pertussis toxin inhibited the ability of this receptor to activate ERK. Finally, flow cytometry was utilized to assess the rate and extent of receptor internalization following agonist challenge. hPAR2C361A displayed faster internalization kinetics following trypsin activation compared with wt-hPAR2, whereas SLIGKV had a negligible effect on internalization for either receptor. In conclusion, palmitoylation plays an important role in the regulation of PAR2 expression, agonist sensitivity, desensitization and internalization.
Modulation of doxorubicin resistance by the glucose-6-phosphate dehydrogenase activity
Authors: Polimeni Manuela , Voena Claudia , Kopecka Joanna , Riganti Chiara , Pescarmona Gianpiero , Bosia Amalia , Ghigo Dario
Published: July 2011

How anti-neoplastic agents induce MDR (multidrug resistance) in cancer cells and the role of GSH (glutathione) in the activation of pumps such as the MRPs (MDR-associated proteins) are still open questions. In the present paper we illustrate that a doxorubicin-resistant human colon cancer cell line (HT29-DX), exhibiting decreased doxorubicin accumulation, increased intracellular GSH content, and increased MRP1 and MRP2 expression in comparison with doxorubicin-sensitive HT29 cells, shows increased activity of the PPP (pentose phosphate pathway) and of G6PD (glucose-6-phosphate dehydrogenase). We observed the onset of MDR in HT29 cells overexpressing G6PD which was accompanied by an increase in GSH. The G6PD inhibitors DHEA (dehydroepiandrosterone) and 6-AN (6-aminonicotinamide) reversed the increase of G6PD and GSH and inhibited MDR both in HT29-DX cells and in HT29 cells overexpressing G6PD. In our opinion, these results suggest that the activation of the PPP and an increased activity of G6PD are necessary to some MDR cells to keep the GSH content high, which is in turn necessary to extrude anticancer drugs out of the cell. We think that our data provide a new further mechanism for GSH increase and its effects on MDR acquisition.
Laforin, a dual-specificity phosphatase involved in Lafora disease, is phosphorylated at Ser25 by AMP-activated protein kinase
Authors: Romá-Mateo Carlos , Solaz-Fuster Maria del Carmen , Gimeno-Alcañiz José Vicente , Dukhande Vikas V. , Donderis Jordi , Worby Carolyn A. , Marina Alberto , Criado Olga , Koller Antonius , Rodriguez De Cordoba Santiago , Gentry Matthew S. , Sanz Pascual
Published: July 2011

Lafora progressive myoclonus epilepsy [LD (Lafora disease)] is a fatal autosomal recessive neurodegenerative disorder caused by loss-of-function mutations in either the EPM2A gene, encoding the dual-specificity phosphatase laforin, or the EPM2B gene, encoding the E3-ubiquitin ligase malin. Previously, we and others showed that laforin and malin form a functional complex that regulates multiple aspects of glycogen metabolism, and that the interaction between laforin and malin is enhanced by conditions activating AMPK (AMP-activated protein kinase). In the present study, we demonstrate that laforin is a phosphoprotein, as indicated by two-dimensional electrophoresis, and we identify Ser25 as the residue involved in this modification. We also show that Ser25 is phosphorylated both in vitro and in vivo by AMPK. Lastly, we demonstrate that this residue plays a critical role for both the phosphatase activity and the ability of laforin to interact with itself and with previously established binding partners. The results of the present study suggest that phosphorylation of laforin-Ser25 by AMPK provides a mechanism to modulate the interaction between laforin and malin. Regulation of this complex is necessary to maintain normal glycogen metabolism. Importantly, Ser25 is mutated in some LD patients (S25P), and our results begin to elucidate the mechanism of disease in these patients.
Aberrant interaction of calmodulin with the ryanodine receptor develops hypertrophy in the neonatal cardiomyocyte
Authors: Gangopadhyay Jaya P. , Ikemoto Noriaki
Published: July 2011

We have shown previously that the inter-domain interaction between the two domains of RyR (ryanodine receptor), CaMBD [CaM (calmodulin)-binding domain] and CaMLD (CaM-like domain), activates the Ca2+ channel, and this process is called activation-link formation [Gangopadhyay and Ikemoto (2008) Biochem. J. 411, 415–423]. Thus CaM that is bound to CaMBD is expected to interfere the activation-link formation, thereby stabilizing the closed state of the channel under normal conditions. In the present paper, we report that, upon stimulation of neonatal cardiomyocytes with the pro-hypertrophy agonist ET-1 (endothelin-1), CaM dissociates from the RyR, which induces a series of intracellular events: increased frequency of Ca2+ transients, translocation of the signalling molecules CaM, CaMKII (CaM kinase II) and the transcription factor NFAT (nuclear factor of activated T-cells) to the nucleus. These events then lead to the development of hypertrophy. Importantly, an anti-CaMBD antibody that interferes with activation-link formation prevented all of these intracellular events triggered by ET-1 and prevented the development of hypertrophy. These results indicate that the aberrant formation of the activation link between CaMBD and CaMLD of RyR is a key step in the development of hypertrophy in cultured cardiomyocytes.
Human and mouse granzyme M display divergent and species-specific substrate specificities
Authors: de Poot Stefanie A.H. , Westgeest Marijn , Hostetter Daniel R. , van Damme Petra , Plasman Kim , Demeyer Kimberly , Broekhuizen Roel , Gevaert Kris , Craik Charles S. , Bovenschen Niels
Published: July 2011

Cytotoxic lymphocyte protease GrM (granzyme M) is a potent inducer of tumour cell death and a key regulator of inflammation. Although hGrM (human GrM) and mGrM (mouse GrM) display extensive sequence homology, the substrate specificity of mGrM remains unknown. In the present study, we show that hGrM and mGrM have diverged during evolution. Positional scanning libraries of tetrapeptide substrates revealed that mGrM is preferred to cleave after a methionine residue, whereas hGrM clearly favours a leucine residue at the P1 position. The kinetic optimal non-prime subsites of both granzymes were also distinct. Gel-based and complementary positional proteomics showed that hGrM and mGrM have a partially overlapping set of natural substrates and a diverged prime and non-prime consensus cleavage motif with leucine and methionine residues being major P1 determinants. Consistent with positional scanning libraries of tetrapeptide substrates, P1 methionine was more frequently used by mGrM as compared with hGrM. Both hGrM and mGrM cleaved α-tubulin with similar kinetics. Strikingly, neither hGrM nor mGrM hydrolysed mouse NPM (nucleophosmin), whereas human NPM was hydrolysed efficiently by GrM from both species. Replacement of the putative P1′–P2′ residues in mouse NPM with the corresponding residues of human NPM restored cleavage of mouse NPM by both granzymes. This further demonstrates the importance of prime sites as structural determinants for GrM substrate specificity. GrM from both species efficiently triggered apoptosis in human but not in mouse tumour cells. These results indicate that hGrM and mGrM not only exhibit divergent specificities but also trigger species-specific functions.
ATF4 deficiency protects mice from high-carbohydrate-diet-induced liver steatosis
Authors: Li Houkai , Meng Qingshu , Xiao Fei , Chen Shanghai , Du Ying , Yu Junjie , Wang Chunxia , Guo Feifan
Published: July 2011

Chronic feeding of HCD (high-carbohydrate diet) is one of the major contributors to the prevailing of metabolic diseases. ATF4 (activating transcription factor 4) has been shown to play an important role in the regulation of glucose metabolism and obesity development; however, it is unclear how ATF4−/− mice respond to HCD. In the present study, we show that 8 weeks of HCD results in significant higher accumulation of TAGs (triacylglycerols) in livers and impairment in glucose tolerance in ATF4+/+ mice, but not in ATF4−/− mice, compared with those on a normal diet. Meanwhile, energy expenditure is further enhanced by HCD in ATF4−/− mice. Moreover, we show that ATF4 deficiency suppresses HCD-induced SCD1 (stearoyl-CoA desaturase 1) expression, furthermore, oral supplementation of the main product of SCD1 oleate (18:1) increases TAG accumulation in livers of ATF4−/− mice. Taken together, these results suggest that ATF4 deficiency is protective for HCD-induced hepatic steatosis and impairment of glucose tolerance and insulin sensitivity. Furthermore, the resistance to hepatic steatosis is at least in part due to suppression of SCD1 expression under HCD.
Interaction between bacterial outer membrane proteins and periplasmic quality control factors: a kinetic partitioning mechanism
Authors: Wu Si , Ge Xi , Lv Zhixin , Zhi Zeyong , Chang Zengyi , Zhao Xin Sheng
Published: July 2011

The OMPs (outer membrane proteins) of Gram-negative bacteria have to be translocated through the periplasmic space before reaching their final destination. The aqueous environment of the periplasmic space and high permeability of the outer membrane engender such a translocation process inevitably challenging. In Escherichia coli, although SurA, Skp and DegP have been identified to function in translocating OMPs across the periplasm, their precise roles and their relationship remain to be elucidated. In the present paper, by using fluorescence resonance energy transfer and single-molecule detection, we have studied the interaction between the OMP OmpC and these periplasmic quality control factors. The results of the present study reveal that the binding rate of OmpC to SurA or Skp is much faster than that to DegP, which may lead to sequential interaction between OMPs and different quality control factors. Such a kinetic partitioning mechanism for the chaperone–substrate interaction may be essential for the quality control of the biogenesis of OMPs
Reconstitution in liposomes of the functionally active human OCTN1 (SLC22A4) transporter overexpressed in Escherichia coli
Authors: Pochini Lorena , Scalise Mariafrancesca , Galluccio Michele , Amelio Linda , Indiveri Cesare
Published: July 2011

The hOCTN1 (human organic cation transporter 1) overexpressed in Escherichia coli and purified by Ni-chelating chromatography has been reconstituted in liposomes by detergent removal with a batch-wise procedure. The reconstitution was optimized with respect to the protein concentration, the detergent/phospholipid ratio and the time of incubation with Amberlite XAD-4 resin. Time-dependent [14C]tetraethylammonium, [3H]carnitine or [3H]ergothioneine uptake was measured in proteoliposomes with activities ratios of 8:1.3:1 respectively. Optimal activity was found at pH 8.0. The transport depended on intraliposomal ATP. [14C]tetraethylammonium transport was inhibited by several compounds. The most effective were acetyl-choline and γ-butyrobetaine, followed by acetylcarnitine and tetramethylammonium. Reagents such as pyridoxal 5-phosphate, MTSES [sodium (2-sulfonatoethyl) methanethiosulfonate] and mercurials strongly inhibited the transport. From kinetic analysis of tetraethylammonium transport a Km of 0.77 mM was calculated. Acetylcholine and γ-butyrobetaine behaved as competitive inhibitors of TEA (tetraethylammonium) transport with Ki values of 0.44 and 0.63 mM respectively.
The phosphatidylinositol transfer protein RdgBβ binds 14-3-3 via its unstructured C-terminus, whereas its lipid-binding domain interacts with the integral membrane protein ATRAP (angiotensin II type I receptor-associated protein)
Authors: Garner Kathryn , Li Michelle , Ugwuanya Natalie , Cockcroft Shamshad
Published: July 2011

PITPs [PI (phosphatidylinositol) transfer proteins] bind and transfer PI between intracellular membranes and participate in many cellular processes including signalling, lipid metabolism and membrane traffic. The largely uncharacterized PITP RdgBβ (PITPNC1; retinal degeneration type B β), contains a long C-terminal disordered region following its defining N-terminal PITP domain. In the present study we report that the C-terminus contains two tandem phosphorylated binding sites (Ser274 and Ser299) for 14-3-3. The C-terminus also contains PEST sequences which are shielded by 14-3-3 binding. Like many proteins containing PEST sequences, the levels of RdgBβ are regulated by proteolysis. RdgBβ is degraded with a half-life of 4 h following ubiquitination via the proteasome. A mutant RdgBβ which is unable to bind 14-3-3 is degraded even faster with a half-life of 2 h. In vitro, RdgBβ is 100-fold less active than PITPα for PI transfer, and RdgBβ proteins (wild-type and a mutant that cannot bind 14-3-3) expressed in COS-7 cells or endogenous proteins from heart cytosol do not exhibit transfer activity. When cells are treated with PMA, the PITP domain of RdgBβ interacts with the integral membrane protein ATRAP (angiotensin II type I receptor-associated protein; also known as AGTRAP) causing membrane recruitment. We suggest that RdgBβ executes its function following recruitment to membranes via its PITP domain and the C-terminal end of the protein could regulate entry to the hydrophobic cavity.
A role for heparan sulfate proteoglycans in Plasmodium falciparum sporozoite invasion of anopheline mosquito salivary glands
Authors: Armistead Jennifer S. , Wilson Iain B.H. , van Kuppevelt Toin H. , Dinglasan Rhoel R.
Published: July 2011

HS (heparan sulfate) has been shown to be an important mediator of Plasmodium sporozoite homing and invasion of the liver, but the role of this glycosaminoglycan in mosquito vector host–sporozoite interactions is unknown. We have biochemically characterized the function of AgOXT1 (Anopheles gambiae peptide-O-xylosyltransferase 1) and confirmed that AgOXT1 can modify peptides representing model HS and chondroitin sulfate proteoglycans in vitro. Moreover, we also demonstrated that the mosquito salivary gland basal lamina proteoglycans are modified by HS. We used RNA interference-mediated knockdown of HS biosynthesis in A. gambiae salivary glands to determine whether Plasmodium falciparum sporozoites that are released from mosquito midgut oocysts use salivary gland HS as a receptor for tissue invasion. Our results suggest that salivary gland basal lamina HS glycosaminoglycans only partially mediate midgut sporozoite invasion of this tissue, and that in the absence of HS, the presence of other surface co-receptors is sufficient to facilitate parasite entry.
Aβ42 oligomers, but not fibrils, simultaneously bind to and cause damage to ganglioside-containing lipid membranes
Authors: Williams Thomas L. , Johnson Benjamin R. G. , Urbanc Brigita , Jenkins A. Toby A. , Connell Simon D. A. , Serpell Louise C.
Published: July 2011

Aβ (amyloid-β peptide) assembles to form amyloid fibres that accumulate in senile plaques associated with AD (Alzheimer's disease). The major constituent, a 42-residue Aβ, has the propensity to assemble and form soluble and potentially cytotoxic oligomers, as well as ordered stable amyloid fibres. It is widely believed that the cytotoxicity is a result of the formation of transient soluble oligomers. This observed toxicity may be associated with the ability of oligomers to associate with and cause permeation of lipid membranes. In the present study, we have investigated the ability of oligomeric and fibrillar Aβ42 to simultaneously associate with and affect the integrity of biomimetic membranes in vitro. Surface plasmon field-enhanced fluorescence spectroscopy reveals that the binding of the freshly dissolved oligomeric 42-residue peptide binds with a two-step association with the lipid bilayer, and causes disruption of the membrane resulting in leakage from vesicles. In contrast, fibrils bind with a 2-fold reduced avidity, and their addition results in approximately 2-fold less fluorophore leakage compared with oligomeric Aβ. Binding of the oligomers may be, in part, mediated by the GM1 ganglioside receptors as there is a 1.8-fold increase in oligomeric Aβ binding and a 2-fold increase in permeation compared with when GM1 is not present. Atomic force microscopy reveals the formation of defects and holes in response to oligomeric Aβ, but not preformed fibrillar Aβ. The results of the present study indicate that significant membrane disruption arises from association of low-molecular-mass Aβ and this may be mediated by mechanical damage to the membranes by Aβ aggregation. This membrane disruption may play a key role in the mechanism of Aβ-related cell toxicity in AD.
Crystal structure of the glutamate receptor GluA1 N-terminal domain
Authors: Yao Guorui , Zong Yinong , Gu Shenyan , Zhou Jie , Xu Huaxi , Mathews Irimpan I. , Jin Rongsheng
Published: July 2011

The AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) subfamily of iGluRs (ionotropic glutamate receptors) is essential for fast excitatory neurotransmission in the central nervous system. The malfunction of AMPARs (AMPA receptors) has been implicated in many neurological diseases, including Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. The active channels of AMPARs and other iGluR subfamilies are tetramers formed exclusively by assembly of subunits within the same subfamily. It has been proposed that the assembly process is controlled mainly by the extracellular ATD (N-terminal domain) of iGluR. In addition, ATD has also been implicated in synaptogenesis, iGluR trafficking and trans-synaptic signalling, through unknown mechanisms. We report in the present study a 2.5 Å (1 Å=0.1 nm) resolution crystal structure of the ATD of GluA1. Comparative analyses of the structure of GluA1-ATD and other subunits sheds light on our understanding of how ATD drives subfamily-specific assembly of AMPARs. In addition, analysis of the crystal lattice of GluA1-ATD suggests a novel mechanism by which the ATD might participate in inter-tetramer AMPAR clustering, as well as in trans-synaptic protein–protein interactions.
Human lysosomal α-D-mannosidase regulation in promyelocytic leukaemia cells
Authors: Urbanelli Lorena , Magini Alessandro , Ercolani Luisa , Trivelli Francesco , Polchi Alice , Tancini Brunella , Emiliani Carla
Published: July 2011

Lysosomal α-D-mannosidase is an exoglycosidase involved in the ordered degradation of N-linked oligosaccharides. It is ubiquitously expressed, although the main transcript is more abundant in peripheral blood leucocytes. Here we report that α-D-mannosidase enzyme activity is very high in the promyelocytic leukaemia cell lines HL60 and NB4, as compared with other leukaemic cell lines or cells from different human sources. The MAN2B1 transcript level correlates with enzyme activity, indicating a transcriptional up-regulation of the α-D-mannosidase gene. The promoter was then characterized in HEK-293 cells (human embryonic kidney 293 cells) and HL60 cells; regulatory sequences crucial for its activity were determined by reporter gene assay in HEK-293 cells and located in the region −101/−71 with respect to the first ATG codon. Supershift assay demonstrated that Sp1 (specificity protein 1) bound to this sequence both in HEK-293 and HL60 cells. However, 5′-RACE (5′-rapid amplification of cDNA ends) indicated the use of multiple upstream TSSs (transcription start sites) in HL60 with respect to HEK-293 cells and gel shift analysis of the sequence −373/−269 demonstrated a specific binding by NF-κB (nuclear factor κB) transcription factor in HL60 but not in HEK-293 cells. We concluded that despite the α-D-mannosidase promoter showing typical features of housekeeping gene promoters, α-D-mannosidase transcription is specifically regulated in HL60 by NF-κB transcription factor.
Characterization of bone‐marrow‐derived rat mesenchymal stem cells depending on donor age
Authors: Gala Kamila , Burdzińska Anna , Idziak Marta , Makula Jolanta , Pa̧czek Leszek
Published: July 2011

It is generally accepted that autologous transfers, as non‐immunogenic, constitute the safest approach in cellular transplantations. However, this attitude is often associated with the need for isolation and extracorporeal propagation of cells derived from aged patients. Thus the knowledge about relationship between aging and the properties of MSCs (mesenchymal stem cells) is crucial in developing new clinical strategies. The aim of this study was to perform complex comparison of MSC derived from young and aged individuals, which included phenotype, proliferating rate, osteogenic and adipogenic potential and secretory activity. Evaluated populations were isolated from bone marrow of 3‐month‐old and 24‐month‐old rats. There was no significant difference in membrane antigen expression and PDT (population doubling time). Additionally, the adipogenic and osteogenic potential did not vary between studied populations. The reaction of MSCs to either mitogen [bFGF (basic fibroblast growth factor)] or oxidative stress (H2O2)in vitrodisplayed a very similar pattern in both analysed populations. There was no difference in TGFβ1 (transforming growth factor β1) and VEGF (vascular endothelial growth factor) secretion measured by ELISA test and gene expression evaluated by real‐time PCR. However, the expression of the gene for IL‐1α (interleukin‐1α) was 8‐fold lower in oMSC (MSC isolated from old rats). These results indicate that aging individuals can be considered as candidates for autologous transplantation of bone‐marrow‐derived MSCs.