bluuebunny/crossref_metadata_2025 huggingface dataset:
Relative dominance of hydrologic versus biogeochemical factors on solute export across impact gradients
Authors: Thompson S. E. , Basu N. B. , Lascurain J. , Aubeneau A. , Rao P. S. C.
Published: June 2011

Many processes lead to variability of catchment concentration‐discharge relationships, but exports of geogenic (weathering derived) solutes and nutrients (nitrogen and phosphorus species) from agricultural basins display relatively constant concentrations despite large variations in streamflow. These “chemostatic” responses are hypothesized to arise when a large mass store, the parent material for geogenics or chemically recalcitrant legacies of fertilization in agricultural catchments, buffers concentration variability. This hypothesis implies that (1) chemostatic behavior should be a general response to elevated external inputs to a catchment and (2) chemostatic behavior should be predictable from theory. Data‐ and model‐based analyses were used to explore these hypotheses. We evaluated concentration variability relative to discharge (expressed as the ratio of the coefficients of variation of concentration and flow, or CVC/CVQ) across a gradient of increasing exported load, as a proxy for an external impact gradient. The CVC/CVQ of multiple solutes declined with increasing exported load. Exceptions included the geogenic solutes, which showed chemostatic responses for all sites, phosphorus, and some nitrogen species. Nitrate showed a suggestive pattern in CVC/CVQ with export, but further data are needed to confirm its generality. A simple model of runoff generation and solute export suggested that the decline in CVC/CVQ arises if the internal mass store is distributed homogeneously in space and there is sufficient time for mass transfer to reach steady state between runoff events. Export from catchments may become more predictable in impacted watersheds, simplifying water quality prediction but inducing strong hysteresis in recovery and making restoration efforts challenging.
Convergent tracer tests in multilayered aquifers: The importance of vertical flow in the injection borehole
Authors: Riley Michael S. , Tellam John H. , Greswell Richard B. , Durand Véronique , Aller Maria F.
Published: June 2011

A mathematical model describing the steady state flows in a forced gradient tracer test between an injection and pumping borehole in a multilayered sandstone aquifer has been developed that includes the effect of vertically variable background heads. A second model describing the recovery of tracer from a layer in which there are discharges due to vertical flow in the injection borehole is also presented. Application of the models to field tracer test data indicates that the observed recoveries, which are not proportional to the abstraction rate in each layer, are consistent with the hydraulic behavior of the aquifer when natural vertical head gradients are taken into account. Investigation with the models illustrates that the vertical distribution of tracer recovery depends strongly upon the background heads and that tracer tests conducted in the same aquifer, but at different times, may interrogate different aquifer layers. It is also shown generally that for a given abstraction rate the vertical distribution of tracer recovery in small‐scale tracer tests is controlled largely by the transmissivity distribution but that as the spatial scale of the test increases, the distribution of recovery becomes proportional to the discharges from the injection borehole because of vertical flows within it, which may be natural or induced by pumping in the monitoring borehole. Uncertainties inherent in the design of forced gradient tracer tests in multilayered aquifers and the problems of applying the results of such tests to natural gradient contaminant migration are discussed.
Metatranscriptomic analysis of autonomously collected and preserved marine bacterioplankton
Authors: Ottesen Elizabeth A , Marin Roman , Preston Christina M , Young Curtis R , Ryan John P , Scholin Christopher A , DeLong Edward F
Published: June 2011

Planktonic microbial activity and community structure is dynamic, and can change dramatically on time scales of hours to days. Yet for logistical reasons, this temporal scale is typically under-sampled in the marine environment. In order to facilitate higher-resolution, long-term observation of microbial diversity and activity, we developed a protocol for automated collection and fixation of marine microbes using the Environmental Sample Processor (ESP) platform. The protocol applies a preservative (RNALater) to cells collected on filters, for long-term storage and preservation of total cellular RNA. Microbial samples preserved using this protocol yielded high-quality RNA after 30 days of storage at room temperature, or onboard the ESP at in situ temperatures. Pyrosequencing of complementary DNA libraries generated from ESP-collected and preserved samples yielded transcript abundance profiles nearly indistinguishable from those derived from conventionally treated replicate samples. To demonstrate the utility of the method, we used a moored ESP to remotely and autonomously collect Monterey Bay seawater for metatranscriptomic analysis. Community RNA was extracted and pyrosequenced from samples collected at four time points over the course of a single day. In all four samples, the oxygenic photoautotrophs were predominantly eukaryotic, while the bacterial community was dominated by Polaribacter-like Flavobacteria and a Rhodobacterales bacterium sharing high similarity with Rhodobacterales sp. HTCC2255. However, each time point was associated with distinct species abundance and gene transcript profiles. These laboratory and field tests confirmed that autonomous collection and preservation is a feasible and useful approach for characterizing the expressed genes and environmental responses of marine microbial communities.
Saliva microbiomes distinguish caries-active from healthy human populations
Authors: Yang Fang , Zeng Xiaowei , Ning Kang , Liu Kuan-Liang , Lo Chien-Chi , Wang Wei , Chen Jie , Wang Dongmei , Huang Ranran , Chang Xingzhi , Chain Patrick S , Xie Gary , Ling Junqi , Xu Jian
Published: June 2011

The etiology of dental caries remains elusive because of our limited understanding of the complex oral microbiomes. The current methodologies have been limited by insufficient depth and breadth of microbial sampling, paucity of data for diseased hosts particularly at the population level, inconsistency of sampled sites and the inability to distinguish the underlying microbial factors. By cross-validating 16S rRNA gene amplicon-based and whole-genome-based deep-sequencing technologies, we report the most in-depth, comprehensive and collaborated view to date of the adult saliva microbiomes in pilot populations of 19 caries-active and 26 healthy human hosts. We found that: first, saliva microbiomes in human population were featured by a vast phylogenetic diversity yet a minimal organismal core; second, caries microbiomes were significantly more variable in community structure whereas the healthy ones were relatively conserved; third, abundance changes of certain taxa such as overabundance of Prevotella Genus distinguished caries microbiota from healthy ones, and furthermore, caries-active and normal individuals carried different arrays of Prevotella species; and finally, no ‘caries-specific’ operational taxonomic units (OTUs) were detected, yet 147 OTUs were ‘caries associated’, that is, differentially distributed yet present in both healthy and caries-active populations. These findings underscored the necessity of species- and strain-level resolution for caries prognosis, and were consistent with the ecological hypothesis where the shifts in community structure, instead of the presence or absence of particular groups of microbes, underlie the cariogenesis.
Evolution and diversification of Pseudomonas aeruginosa in the paranasal sinuses of cystic fibrosis children have implications for chronic lung infection
Authors: Hansen Susse Kirkelund , Rau Martin Holm , Johansen Helle Krogh , Ciofu Oana , Jelsbak Lars , Yang Lei , Folkesson Anders , Jarmer Hanne Østergaard , Aanæs Kasper , von Buchwald Christian , Høiby Niels , Molin Søren
Published: June 2011

The opportunistic pathogen Pseudomonas aeruginosa is a frequent colonizer of the airways of patients suffering from cystic fibrosis (CF). Depending on early treatment regimens, the colonization will, with high probability, develop into chronic infections sooner or later, and it is important to establish under which conditions the switch to chronic infection takes place. In association with a recently established sinus surgery treatment program for CF patients at the Copenhagen CF Center, colonization of the paranasal sinuses with P. aeruginosa has been investigated, paralleled by sampling of sputum from the same patients. On the basis of genotyping and phenotypic characterization including transcription profiling, the diversity of the P. aeruginosa populations in the sinuses and the lower airways was investigated and compared. The observations made from several children show that the paranasal sinuses constitute an important niche for the colonizing bacteria in many patients. The paranasal sinuses often harbor distinct bacterial subpopulations, and in the early colonization phases there seems to be a migration from the sinuses to the lower airways, suggesting that independent adaptation and evolution take place in the sinuses. Importantly, before the onset of chronic lung infection, lineages with mutations conferring a large fitness benefit in CF airways such as mucA and lasR as well as small colony variants and antibiotic-resistant clones are part of the sinus populations. Thus, the paranasal sinuses potentially constitute a protected niche of adapted clones of P. aeruginosa, which can intermittently seed the lungs and pave the way for subsequent chronic lung infections.
A Nutritional Comparison of Foods and Beverages Marketed to Children in Two Advertising Policy Environments
Authors: Potvin Monique Potvin , Dubois Lise , Wanless Alissa
Published: June 2011

Childhood obesity is associated with children's exposure to food/beverage marketing. Policy options in this area are being sought in order to reduce childhood obesity rates on a population‐level. We examined the nutritional quality of foods advertised to children during their preferred television viewing in Ontario (Canada), where advertising is self‐regulated by industry, and in Quebec (Canada), where a child‐directed advertising ban exists. A total of 428 children aged 10–12 years completed television viewing diaries for 7 days. Thirty‐two television stations were recorded simultaneously between 6 am and midnight. A content analysis of 90 h of English Ontario, French Quebec, and English Quebec children's preferred viewing was then undertaken. A total of 429 food and beverage advertisements were analyzed and their nutritional quality was assessed. Food advertisements in the Quebec French sample were statistically significantly higher in total fat, saturated fat and protein, and lower in carbohydrates and sugar per 100 g, and as a percentage of energy than food ads in the two English samples. A statistically significantly lower percentage of the Quebec French food advertisements were classified as either high fat, sugar or sodium and a smaller proportion of food ads were classified as “less healthy” compared to the Ontario and Quebec English samples. These results suggest that the Quebec advertising ban is influencing the macronutrient profile of advertised foods viewed by French Quebec children during their preferred viewing and that their promotions are marginally healthier than that viewed by the English samples.
Six‐Minute Walk Test Improved Forearm Skin Blood Flow in Tunisian Obese Women
Authors: Miâdi‐Messaoud Hanène , Chouchane Afef , Ben Helmi Ben , Debbabi Haytham , Ben‐Jebria Abdelaziz , Tabka Zouhair
Published: June 2011

The purpose of this study was to investigate whether 6‐min walk test (6MWT) would improve the forearm skin blood flow (FSBF) response to acetylcholine (ACh), an endothelium‐dependent vasodilator, in Tunisian women over a wide range of BMI. The FSBF was measured noninvasively using a laser Doppler flowmeter in response to local infusion of a cumulative dose of ACh, before and after the 6MWT for 102 healthy women; the results were expressed as percentage of baseline. The 6MWT was monitored and recorded. The mean response of FSBF to ACh was significantly greater before as well as after the 6MWT in lean (1,235 ± 123% vs. 1,644 ± 140%) than in overweight (630 ± 62% vs. 1,080 ± 66%) and obese subjects (402 ± 38% vs. 795 ± 40%) (P < 0.0001). Our regression analysis also revealed that the maximal FSBF response to ACh (i.e., its efficacy) was inversely correlated with BMI both before as well as after the 6MWT (r = −0.828, P < 0.0001; r = −0.859, P < 0.0001, respectively), and the efficacies of ACh in the three groups were all significantly elevated following the 6MWT (P < 0.0001). As indicated by ANOVA test, the 6MWT improved the FSBF responses of the lean, overweight, and obese subjects, by 33, 71, and 98%, respectively. We confirm that obesity induced a reduction of skin vasodilatory reserve and altered both endothelial‐dependent relaxation and wall compliance. However, our new data clearly demonstrated that the 6MWT not only improved significantly the FSBF responses in the three groups of women, but the obese patients appeared to benefit more from the 6MWT than the overweight and the lean subjects.
Television Viewing and Obesity in 300 Women: Evaluation of the Pathways of Energy Intake and Physical Activity
Authors: Tucker Larry A. , Tucker Jared M.
Published: June 2011

We assessed the roles of energy intake and physical activity in the relationships among television (TV) viewing, body composition, and obesity using high‐quality measurement methods. Adult women (n = 300) reported TV viewing behavior, which was categorized into infrequent (≤1 h/day), moderate (2 h/day), and frequent (≥3 h/day) viewing. Body fat percentage (BF%) was assessed using plethysmography (Bod Pod) and BMI was calculated from height and body weight. Energy intake and physical activity, including time spent in sedentary, moderate, and vigorous physical activity (PA), were objectively measured using 7‐day weighed food records and 7‐day accelerometry, respectively. The mean BF% of frequent TV viewers (34.6 ± 6.9%) was significantly greater (F = 3.9, P = 0.0218) than those of moderate (31.5 ± 6.7%) and infrequent viewers (30.8 ± 7.0%); however, BMI did not differ across the TV viewing groups (F = 0.8, P = 0.4172). Controlling statistically for differences in age, education, time in sedentary activity, time in moderate activity, and energy intake, considered individually, had no influence on the relationships between TV viewing and BF%, nor TV and BMI. Moreover, the relationship between TV and BF% remained significant after adjusting for differences in BMI (F = 3.6, P = 0.0276). However, adjusting for total PA reduced the relationship between TV and BF% to nonsignificance (F = 2.5, P = 0.0810), as did time spent in vigorous PA (F = 2.2, P = 0.1307). These data suggest a strong relationship between TV viewing and BF%. This association appears to be due, in part, to differences in total PA, particularly vigorous PA, but not time spent in sedentary activity, moderate activity, or energy intake.
Intraperitoneal Fat Is Associated With Thickening of the Thoracic Aorta in Individuals at High Risk for Cardiovascular Events
Authors: Chughtai Haroon L. , Morgan Timothy M. , Hamilton Craig A. , Charoenpanichkit Charaslak , Ding Jingzhong , Brinkley Tina E. , Hundley W. Gregory
Published: June 2011

Increased intraperitoneal (IP) fat is associated with increased cardiovascular (CV) risk, but mechanisms for this increase in risk are not completely established. We performed this study to assess whether IP fat is associated with ascending aortic wall thickness (AOWT), a risk factor for CV events. Four hundred and forty‐one consecutive participants, aged 55–85 years, with risk factors for CV events underwent magnetic resonance measures of AOWT and abdominal fat (subcutaneous (SC) fat + IP fat). For the ascending aorta, mean wall thickness of the 4th quartile of the IP fat was higher relative to the 1st quartile (P ≤ 0.001). This difference persisted after accounting for SC fat (P ≤ 0.001), as well as age, gender, height, weight, smoking, diabetes, hypertension, low‐density lipoprotein‐cholesterol (LDL‐C), high‐density lipoprotein‐cholesterol (HDL‐C), and C‐reactive protein (CRP) (P < 0.03). Elevated IP fat volume is associated with an increase in ascending AOWT, a condition that promotes CV events in middle aged and elderly adults.
Altering the stability of the Cdc8 overlap region modulates the ability of this tropomyosin to bind co-operatively to actin and regulate myosin
Authors: East Daniel A. , Sousa Duncan , Martin Stephen R. , Edwards Thomas A. , Lehman William , Mulvihill Daniel P.
Published: July 2011

Tm (tropomyosin) is an evolutionarily conserved α-helical coiled-coil protein, dimers of which form end-to-end polymers capable of associating with and stabilizing actin filaments, and regulating myosin function. The fission yeast Schizosaccharomyces pombe possesses a single essential Tm, Cdc8, which can be acetylated on its N-terminal methionine residue to increase its affinity for actin and enhance its ability to regulate myosin function. We have designed and generated a number of novel Cdc8 mutant proteins with N-terminal substitutions to explore how stability of the Cdc8 overlap region affects the regulatory function of this Tm. By correlating the stability of each protein, its propensity to form stable polymers, its ability to associate with actin and to regulate myosin, we have shown that the stability of the N-terminal of the Cdc8 α-helix is crucial for Tm function. In addition we have identified a novel Cdc8 mutant with increased N-terminal stability, dimers of which are capable of forming Tm polymers significantly longer than the wild-type protein. This protein had a reduced affinity for actin with respect to wild-type, and was unable to regulate actomyosin interactions. The results of the present paper are consistent with acetylation providing a mechanism for modulating the formation and stability of Cdc8 polymers within the fission yeast cell. The data also provide evidence for a mechanism in which Tm dimers form end-to-end polymers on the actin filament, consistent with a co-operative model for Tm binding to actin.
CREBL2, interacting with CREB, induces adipogenesis in 3T3-L1 adipocytes
Authors: Ma Xi , Zhang Heyu , Yuan Lan , Jing Hao , Thacker Phil , Li Defa
Published: July 2011

The factors that influence preadipocyte determination remain poorly understood. In the present paper, we report that CREBL2 [CREB (cAMP-response-element-binding protein)-like 2], a novel bZIP_1 protein, is up-regulated during MDI-induced preadipocyte differentiation. During both overexpression and under physiological conditions, CREBL2 interacted and was entirely co-localized with CREB. Overexpression of CREBL2 was sufficient to promote adipogenesis via up-regulating the expression of PPARγ (peroxisome-proliferator-activated receptor γ) and C/EBPα (CCAAT/enhancer-binding protein α) and accelerate lipogenesis accompanied with increased GLUT (glucose transporter) 1 and GLUT4. CREBL2 knockdown restrained adipogenic conversion and lipogenesis. Additionally, depletion of CREB could completely block the effects of overexpressed CREBL2, whereas an increase in CREB could not drive adipogenesis in the absence of CREBL2, indicating that the roles for CREBL2 on adipogenesis were CREB-dependent. Furthermore, siCREBL2 [siRNA (short interfering RNA) against CREBL2] could down-regulate CREB transcriptional activity and suppress CREB phosphorylation. CREB knockdown decreased the CREBL2 protein levels and vice versa. Collectively, the results of the present study indicate that CREBL2 plays a critical role in adipogenesis and lipogenesis via interaction with CREB.
Transforming growth factor-β2 promotes Snail-mediated endothelial–mesenchymal transition through convergence of Smad-dependent and Smad-independent signalling
Authors: Medici Damian , Potenta Scott , Kalluri Raghu
Published: July 2011

EndMT (endothelial–mesenchymal transition) is a critical process of cardiac development and disease progression. However, little is know about the signalling mechanisms that cause endothelial cells to transform into mesenchymal cells. In the present paper we show that TGF-β2 (transforming growth factor-β2) stimulates EndMT through the Smad, MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase], PI3K (phosphinositide 3-kinase) and p38 MAPK signalling pathways. Inhibitors of these pathways prevent TGF-β2-induced EndMT. Furthermore, we show that all of these pathways are essential for increasing expression of the cell-adhesion-suppressing transcription factor Snail. Inhibition of Snail with siRNA (small interfering RNA) prevents TGF-β2-induced EndMT. However, overexpression of Snail is not sufficient to cause EndMT. Chemical inhibition of GSK-3β (glycogen synthase kinase-3β) allows EndMT to be induced by Snail overexpression. Expression of a mutant Snail protein that is resistant to GSK-3β-dependent inactivation also promotes EndMT. These results provide the foundation for understanding the roles of specific signalling pathways in mediating EndMT.
Divergent effect of mammalian PLCζ in generating Ca2+ oscillations in somatic cells compared with eggs
Authors: Phillips Sally V. , Yu Yuansong , Rossbach Andreas , Nomikos Michail , Vassilakopoulou Vyronia , Livaniou Evangelia , Cumbes Bevan , Lai F. Anthony , George Christopher H. , Swann Karl
Published: July 2011

Sperm PLCζ (phospholipase Cζ) is a distinct phosphoinositide-specific PLC isoform that is proposed to be the physiological trigger of egg activation and embryo development at mammalian fertilization. Recombinant PLCζ has the ability to trigger Ca2+ oscillations when expressed in eggs, but it is not known how PLCζ activity is regulated in sperm or eggs. In the present study, we have transfected CHO (Chinese-hamster ovary) cells with PLCζ fused with either YFP (yellow fluorescent protein) or luciferase and found that PLCζ-transfected cells did not display cytoplasmic Ca2+ oscillations any differently from control cells. PLCζ expression was not associated with changes in CHO cell resting Ca2+ levels, nor with a significantly changed Ca2+ response to extracellular ATP compared with control cells transfected with either YFP alone, a catalytically inactive PLCζ or luciferase alone. Sperm extracts containing PLCζ also failed to cause Ca2+ oscillations in CHO cells. Despite these findings, PLCζ-transfected CHO cell extracts exhibited high recombinant protein expression and PLC activity. Furthermore, either PLCζ-transfected CHO cells or derived cell extracts could specifically cause cytoplasmic Ca2+ oscillations when microinjected into mouse eggs. These data suggest that PLCζ-mediated Ca2+ oscillations may require specific factors that are only present within the egg cytoplasm or be inhibited by factors present only in somatic cell lines.
Protein network study of human AF4 reveals its central role in RNA Pol II-mediated transcription and in phosphorylation-dependent regulatory mechanisms
Authors: Esposito Gabriella , Cevenini Armando , Cuomo Alessandro , de Falco Francesca , Sabbatino Dario , Pane Fabrizio , Ruoppolo Margherita , Salvatore Francesco
Published: July 2011

AF4 belongs to a family of proteins implicated in childhood lymphoblastic leukaemia, FRAXE (Fragile X E site) mental retardation and ataxia. AF4 is a transcriptional activator that is involved in transcriptional elongation. Although AF4 has been implicated in MLL (mixed-lineage leukaemia)-related leukaemogenesis, AF4-dependent physiological mechanisms have not been clearly defined. Proteins that interact with AF4 may also play important roles in mediating oncogenesis, and are potential targets for novel therapies. Using a functional proteomic approach involving tandem MS and bioinformatics, we identified 51 AF4-interacting proteins of various Gene Ontology categories. Approximately 60% participate in transcription regulatory mechanisms, including the Mediator complex in eukaryotic cells. In the present paper we report one of the first extensive proteomic studies aimed at elucidating AF4 protein cross-talk. Moreover, we found that the AF4 residues Thr220 and Ser212 are phosphorylated, which suggests that AF4 function depends on phosphorylation mechanisms. We also mapped the AF4-interaction site with CDK9 (cyclin-dependent kinase 9), which is a direct interactor crucial for the function and regulation of the protein. The findings of the present study significantly expand the number of putative members of the multiprotein complex formed by AF4, which is instrumental in promoting the transcription/elongation of specific genes in human cells.
Optimized native gel systems for separation of thylakoid protein complexes: novel super- and mega-complexes
Authors: Järvi Sari , Suorsa Marjaana , Paakkarinen Virpi , Aro Eva-Mari
Published: July 2011

Gel-based analysis of thylakoid membrane protein complexes represents a valuable tool to monitor the dynamics of the photosynthetic machinery. Native-PAGE preserves the components and often also the conformation of the protein complexes, thus enabling the analysis of their subunit composition. Nevertheless, the literature and practical experimentation in the field sometimes raise confusion owing to a great variety of native-PAGE and thylakoid-solubilization systems. In the present paper, we describe optimized methods for separation of higher plant thylakoid membrane protein complexes by native-PAGE addressing particularly: (i) the use of detergent; (ii) the use of solubilization buffer; and (iii) the gel electrophoresis method. Special attention is paid to separation of high-molecular-mass thylakoid membrane super- and mega-complexes from Arabidopsis thaliana leaves. Several novel super- and mega-complexes including PS (photosystem) I, PSII and LHCs (light-harvesting complexes) in various combinations are reported.
NMR spectroscopy of 14-3-3ζ reveals a flexible C-terminal extension: differentiation of the chaperone and phosphoserine-binding activities of 14-3-3ζ
Authors: Williams Danielle M. , Ecroyd Heath , Goodwin Katy L. , Dai Huanqin , Fu Haian , Woodcock Joanna M. , Zhang Lixin , Carver John A.
Published: July 2011

Intracellular 14-3-3 proteins bind to many proteins, via a specific phosphoserine motif, regulating diverse cellular tasks including cell signalling and disease progression. The 14-3-3ζ isoform is a molecular chaperone, preventing the stress-induced aggregation of target proteins in a manner comparable with that of the unrelated sHsps (small heat-shock proteins). 1H-NMR spectroscopy revealed the presence of a flexible and unstructured C-terminal extension, 12 amino acids in length, which protrudes from the domain core of 14-3-3ζ and is similar in structure and length to the C-terminal extension of mammalian sHsps. The extension stabilizes 14-3-3ζ, but has no direct role in chaperone action. Lys49 is an important functional residue within the ligand-binding groove of 14-3-3ζ with K49E 14-3-3ζ exhibiting markedly reduced binding to phosphorylated and non-phosphorylated ligands. The R18 peptide binds to the binding groove of 14-3-3ζ with high affinity and also reduces the interaction of 14-3-3ζ ligands. However, neither the K49E mutation nor the presence of the R18 peptide affected the chaperone activity of 14-3-3ζ, implying that the C-terminal extension and binding groove of 14-3-3ζ do not mediate interaction with target proteins during chaperone action. Other region(s) in 14-3-3ζ are most likely to be involved, i.e. the protein's chaperone and phosphoserine-binding activities are functionally and structurally separated.
Adenosine 5′-phosphosulfate reductase (APR2) mutation inArabidopsisimplicates glutathione deficiency in selenate toxicity
Authors: Grant Kevron , Carey Nicole M. , Mendoza Miguel , Schulze John , Pilon Marinus , Pilon-Smits Elizabeth A. H. , van Hoewyk Doug
Published: July 2011

APR2 is the dominant APR (adenosine 5′-phosphosulfate reductase) in the model plant Arabidopsis thaliana, and converts activated sulfate to sulfite, a key reaction in the sulfate reduction pathway. To determine whether APR2 has a role in selenium tolerance and metabolism, a mutant Arabidopsis line (apr2-1) was studied. apr2-1 plants had decreased selenate tolerance and photosynthetic efficiency. Sulfur metabolism was perturbed in apr2-1 plants grown on selenate, as observed by an increase in total sulfur and sulfate, and a 2-fold decrease in glutathione concentration. The altered sulfur metabolism in apr2-1 grown on selenate did not reflect typical sulfate starvation, as cysteine and methionine levels were increased. Knockout of APR2 also increased the accumulation of total selenium and selenate. However, the accumulation of selenite and selenium incorporation in protein was lower in apr2-1 mutants. Decreased incorporation of selenium in protein is typically associated with increased selenium tolerance in plants. However, because the apr2-1 mutant exhibited decreased tolerance to selenate, we propose that selenium toxicity can also be caused by selenate's disruption of glutathione biosynthesis leading to enhanced levels of damaging ROS (reactive oxygen species).
Aldo-keto reductase family 1, member B10 is secreted through a lysosome-mediated non-classical pathway
Authors: Luo Di-xian , Huang Mei C. , Ma Jun , Gao Zachary , Liao Duan-fang , Cao Deliang
Published: July 2011

AKR1B10 (aldo-keto reductase family 1, member B10) protein is primarily expressed in normal human small intestine and colon, but overexpressed in several types of human cancers and considered as a tumour marker. In the present study, we found that AKR1B10 protein is secreted from normal intestinal epithelium and cultured cancer cells, as detected by a newly developed sandwich ELISA and Western blotting. The secretion of AKR1B10 was not affected by the protein-synthesis inhibitor cycloheximide and the classical protein-secretion pathway inhibitor brefeldin A, but was stimulated by temperature, ATP, Ca2+ and the Ca2+ carrier ionomycin, lysosomotropic NH4Cl, the G-protein activator GTPγS and the G-protein coupling receptor N-formylmethionyl-leucyl-phenylalanine. The ADP-ribosylation factor inhibitor 2-(4-fluorobenzoylamino)-benzoic acid methyl ester and the phospholipase C inhibitor U73122 inhibited the secretion of AKR1B10. In cultured cells, AKR1B10 was present in lysosomes and was secreted with cathepsin D, a lysosomal marker. In the intestine, AKR1B10 was specifically expressed in mature epithelial cells and secreted into the lumen at 188.6–535.7 ng/ml of ileal fluids (mean=298.1 ng/ml, n=11). Taken together, our results demonstrate that AKR1B10 is a new secretory protein belonging to a lysosome-mediated non-classical protein-secretion pathway and is a potential serum marker.
Exo70, a subunit of the exocyst complex, interacts with SNEVhPrp19/hPso4 and is involved in pre-mRNA splicing
Authors: Dellago Hanna , Löscher Marlies , Ajuh Paul , Ryder Ursula , Kaisermayer Christian , Grillari-Voglauer Regina , Fortschegger Klaus , Gross Stefan , Gstraunthaler Anna , Borth Nicole , Eisenhaber Frank , Lamond Angus I. , Grillari Johannes
Published: July 2011

The Cdc5L (cell division cycle 5-like) complex is a spliceosomal subcomplex that also plays a role in DNA repair. The complex contains the splicing factor hPrp19, also known as SNEV or hPso4, which is involved in cellular life-span regulation and proteasomal breakdown. In a recent large-scale proteomics analysis for proteins associated with this complex, proteins involved in transcription, cell-cycle regulation, DNA repair, the ubiquitin–proteasome system, chromatin remodelling, cellular aging, the cytoskeleton and trafficking, including four members of the exocyst complex, were identified. In the present paper we report that Exo70 interacts directly with SNEVhPrp19/hPso4 and shuttles to the nucleus, where it associates with the spliceosome. We mapped the interaction site to the N-terminal 100 amino acids of Exo70, which interfere with pre-mRNA splicing in vitro. Furthermore, Exo70 influences the splicing of a model substrate as well as of its own pre-mRNA in vivo. In addition, we found that Exo70 is alternatively spliced in a cell-type- and cell-age- dependent way. These results suggest a novel and unexpected role of Exo70 in nuclear mRNA splicing, where it might signal membrane events to the splicing apparatus.
Demonstration that endoplasmic reticulum-associated degradation of glycoproteins can occur downstream of processing by endomannosidase
Authors: Kukushkin Nikolay V. , Alonzi Dominic S. , Dwek Raymond A. , Butters Terry D.
Published: July 2011

During quality control in the ER (endoplasmic reticulum), nascent glycoproteins are deglucosylated by ER glucosidases I and II. In the post-ER compartments, glycoprotein endo-α-mannosidase provides an alternative route for deglucosylation. Previous evidence suggests that endomannosidase non-selectively deglucosylates glycoproteins that escape quality control in the ER, facilitating secretion of aberrantly folded as well as normal glycoproteins. In the present study, we employed FOS (free oligosaccharides) released from degrading glycoproteins as biomarkers of ERAD (ER-associated degradation), allowing us to gain a global rather than single protein-centred view of ERAD. Glucosidase inhibition was used to discriminate between glucosidase- and endomannosidase-mediated ERAD pathways. Endomannosidase expression was manipulated in CHO (Chinese-hamster ovary)-K1 cells, naturally lacking a functional version of the enzyme, and HEK (human embryonic kidney)-293T cells. Endomannosidase was shown to decrease the levels of total FOS, suggesting decreased rates of ERAD. However, following pharmacological inhibition of ER glucosidases I and II, endomannosidase expression resulted in a partial switch between glucosylated FOS, released from ER-confined glycoproteins, to deglucosylated FOS, released from endomannosidase-processed glycoproteins transported from the Golgi/ERGIC (ER/Golgi intermediate compartment) to the ER. Using this approach, we have identified a previously unknown pathway of glycoprotein flow, undetectable by the commonly employed methods, in which secretory cargo is targeted back to the ER after being processed by endomannosidase.
GATA-4 transcription factor regulates hepatic hepcidin expression
Authors: Island Marie-Laure , Fatih Nadia , Leroyer Patricia , Brissot Pierre , Loreal Olivier
Published: July 2011

Hepcidin, a hormone mainly synthesized by hepatocytes and secreted in plasma, controls iron bioavailability. Thus, by inducing the internalization of the iron exporter ferroportin, it regulates iron release from macrophages, enterocytes and hepatocytes towards plasma. Abnormal levels of hepcidin expression alter plasma iron parameters and lead to iron metabolism disorders. Understanding the mechanisms controlling hepcidin (HAMP encodes hepcidin) gene expression is therefore an important goal. We identified a potential GATA-binding site within the human hepcidin promoter. Indeed, in hepatic HepG2 cells, luciferase experiments demonstrated that mutation of this GATA-binding site impaired the hepcidin promoter transcriptional activity in basal conditions. Gel-retardation experiments showed that GATA-4 could bind to this site. Co-transfection of a GATA-4 expression vector with a hepcidin promoter reporter construct enhanced hepcidin promoter transcriptional activity. Furthermore, modulation of GATA4 mRNA expression using specific siRNAs (small interfering RNAs) down-regulated endogenous hepcidin gene expression. Finally, we found that mutation of the GATA-binding site impaired the interleukin-6 induction of hepcidin gene expression, but did not prevent the bone morphogenetic protein-6 response. In conclusion, the findings of the present study (i) indicate that GATA-4 may participate in the control of hepcidin expression, and (ii) suggest that alteration of its expression could contribute to the development of iron-related disorders.
Drosophila Sal and Salr are transcriptional repressors
Authors: Sánchez Jonatan , Talamillo Ana , González Monika , Sánchez-Pulido Luis , Jiménez Silvia , Pirone Lucia , Sutherland James D. , Barrio Rosa
Published: July 2011

The SALL (Spalt-like) family of zinc-finger transcription factors is conserved in metazoans. In Drosophila Sal (Spalt) and Salr (Spalt-related) control the expression of genes involved in wing and central nervous system development, including cell adhesion and cytoskeletal proteins. In humans, SALL mutations associate with congenital disorders such as the Townes–Brocks and Okihiro syndromes. Human and Drosophila SALL proteins are modified by SUMO (small ubiquitin-related modifier), which influences their subnuclear localization. In the present study, we have analysed the transcriptional activity of Drosophila Sall proteins in cultured cells. We show that both Sal and Salr act as transcriptional repressors in Drosophila cells where they repress transcription through an AT-rich sequence. Furthermore, using the UAS/Gal4 heterologous system, Drosophila Sal and Salr repress transcription in human cells. Under our experimental conditions, only in the case of Salr is the repression activity dependent on the HDAC (histone deacetylase) complex. This complex might interact with the C-terminal zinc fingers of Salr. We describe the differential subcellular localizations of Sal and Salr fragments and identify their repression domains. Surprisingly, both repressors also contain transcription activation domains. In addition, under our experimental conditions SUMOylation has differential effects on Sal and Salr repressor activity. Phylogenetic comparison between nematodes, insects and vertebrates identifies conserved peptide sequences that are presumably critical for SALL protein function.
PLU1 histone demethylase decreases the expression of KAT5 and enhances the invasive activity of the cells
Authors: Yoshida Masakazu , Ishimura Akihiko , Terashima Minoru , Enkhbaatar Zanabazar , Nozaki Naohito , Satou Kenji , Suzuki Takeshi
Published: July 2011

PLU1 is a candidate oncogene that encodes H3K4 (Lys4 of histone H3) demethylase. In the present study, we found that ectopic expression of PLU1 enhanced the invasive potential of the weakly invasive cells dependent on its demethylase activity. PLU1 was shown to repress the expression of the KAT5 gene through its H3K4 demethylation on the promoter. The regulation of KAT5 by PLU1 was suggested to be responsible for PLU1-induced cell invasion. First, knockdown of KAT5 similarly increased the invasive potential of the cells. Secondly, knockdown of PLU1 in the highly invasive cancer cells increased KAT5 expression and reduced the invasive activity. Thirdly, simultaneous knockdown of KAT5 partially relieved the suppression of cell invasion imposed by PLU1 knockdown. Finally, we found that CD82, which was transcriptionally regulated by KAT5, might be a candidate effector of cell invasion promoted by PLU1. The present study demonstrated a functional contribution of PLU1 overexpression with concomitant epigenetic dysregulation in cancer progression.
The structure of a Burkholderia pseudomallei immunophilin–inhibitor complex reveals new approaches to antimicrobial development
Authors: Norville Isobel H. , O'Shea Katherine , Sarkar-Tyson Mitali , Zheng Suxin , Titball Richard W. , Varani Gabriele , Harmer Nicholas J.
Published: July 2011

Mips (macrophage infectivity potentiators) are a subset of immunophilins associated with virulence in a range of micro-organisms. These proteins possess peptidylprolyl isomerase activity and are inhibited by drugs including rapamycin and tacrolimus. We determined the structure of the Mip homologue [BpML1 (Burkholderia pseudomallei Mip-like protein 1)] from the human pathogen and biowarfare threat B. pseudomallei by NMR and X-ray crystallography. The crystal structure suggests that key catalytic residues in the BpML1 active site have unexpected conformational flexibility consistent with a role in catalysis. The structure further revealed BpML1 binding to a helical peptide, in a manner resembling the physiological interaction of human TGFβRI (transforming growth factor β receptor I) with the human immunophilin FKBP12 (FK506-binding protein 12). Furthermore, the structure of BpML1 bound to the class inhibitor cycloheximide N-ethylethanoate showed that this inhibitor mimics such a helical peptide, in contrast with the extended prolyl-peptide mimicking shown by inhibitors such as tacrolimus. We suggest that Mips, and potentially other bacterial immunophilins, participate in protein–protein interactions in addition to their peptidylprolyl isomerase activity, and that some roles of Mip proteins in virulence are independent of their peptidylprolyl isomerase activity.
Active-pocket size differentiating insectile from bacterial chitinolytic β-N-acetyl-D-hexosaminidases
Authors: Liu Tian , Zhang Haitao , Liu Fengyi , Chen Lei , Shen Xu , Yang Qing
Published: July 2011

Chitinolytic β-N-acetyl-D-hexosaminidase is a branch of the GH20 (glycoside hydrolase family 20) β-N-acetyl-D-hexosaminidases that is only distributed in insects and micro-organisms, and is therefore a potential target for the action of insecticides. PUGNAc [O-(2-acetamido-2-deoxy-D-glucopyransylidene)-amino-N-phenylcarbamate] was initially identified as an inhibitor against GH20 β-N-acetyl-D-hexosaminidases. So far no crystal structure of PUGNAc in complex with any GH20 β-N-acetyl-D-hexosaminidase has been reported. We show in the present study that the sensitivities of chitinolytic β-N-acetyl-D-hexosaminidases towards PUGNAc can vary by 100-fold, with the order being OfHex1 (Ostrinia furnacalis β-N-acetyl-D-hexosaminidase)<SmCHB (Serratia marcescens chitobiase)<SpHex (Streptomyces plicatus β-N-acetyl-D-hexosaminidase). To explain this difference, the crystal structures of wild-type OfHex1 as well as mutant OfHex1(V327G) in complex with PUGNAc were determined at 2.0 Å (1 Å=0.1 nm) and 2.3 Å resolutions and aligned with the complex structures of SpHex and SmCHB. The results showed that the sensitivities of these enzymes to PUGNAc were determined by the active pocket size, with OfHex1 having the largest but narrowest entrance, whereas SpHex has the smallest entrance, suitable for holding the inhibitor, and SmCHB has the widest entrance. By widening the size of the active pocket entrance of OfHex1 through replacing the active site Val327 with a glycine residue, the sensitivity of OfHex1 to PUGNAc became similar to that of SmCHB. The structural differences among chitinolytic β-N-acetyl-D-hexosaminidases leading to different sensitivities to PUGNAc may be useful for developing species-specific pesticides and bactericides.
Calcium homoeostasis modulator 1 (CALHM1) reduces the calcium content of the endoplasmic reticulum (ER) and triggers ER stress
Authors: Gallego-Sandín Sonia , Alonso María Teresa , García-Sancho Javier
Published: July 2011

CALHM1 (calcium homoeostasis modulator 1), a membrane protein with similarity to NMDA (N-methyl-D-aspartate) receptor channels that localizes in the plasma membrane and the ER (endoplasmic reticulum) of neurons, has been shown to generate a plasma-membrane Ca2+ conductance and has been proposed to influence Alzheimer's disease risk. In the present study we have investigated the effects of CALHM1 on intracellular Ca2+ handling in HEK-293T [HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40)] cells by using targeted aequorins for selective monitorization of Ca2+ transport by organelles. We find that CALHM1 increases Ca2+ leak from the ER and, more importantly, reduces ER Ca2+ uptake by decreasing both the transport capacity and the Ca2+ affinity of SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase). As a result, the Ca2+ content of the ER is drastically decreased. This reduction in the Ca2+ content of the ER triggered the UPR (unfolded protein response) with induction of several ER stress markers, such as CHOP [C/EBP (CCAAT/enhancer-binding protein)-homologous protein], ERdj4, GRP78 (glucose-regulated protein of 78 kDa) and XBP1 (X-box-binding protein 1). Thus CALHM1 might provide a relevant link between Ca2+ homoeostasis disruption, ER stress and cell damage in the pathogenesis of neurodegenerative diseases
Arachidonic acid modulates Na+ currents by non-metabolic and metabolic pathways in rat cerebellar granule cells
Authors: Fang Yan-Jia , Zhou Meng-Hua , Gao Xiao-Fei , Gu Hua , Mei Yan-Ai
Published: July 2011

AA (arachidonic acid), which possesses both neurotoxic and neurotrophic activities, has been implicated as a messenger in both physiological and pathophysiological processes. In the present study, we investigated the effects of both extracellular and intracellular application of AA on the activity of NaV (voltage-gated Na+ channels) in rat cerebellar GCs (granule cells). The extracellular application of AA inhibited the resultant INa (NaV current), wherein the current–voltage curve shifted to a negative voltage direction. Because this effect could be reproduced by treating the GCs with ETYA (eicosa-5,8,11,14-tetraynoic acid) or a membrane-impermeable analogue of AA, AA-CoA (arachidonoyl coenzyme A), we inferred that AA itself exerted the observed modulatory effects on INa. In contrast, intracellular AA significantly augmented the elicited INa peak when the same protocol that was used for extracellular AA was followed. The observed INa increase that was induced by intracellular AA was mimicked by the AA cyclo-oxygenase metabolite PGE2 (prostaglandin E2), but not by ETYA. Furthermore, cyclo-oxygenase inhibitors decreased INa and quenched AA-induced channel activation, indicating that the effect of intracellular AA on NaV was possibly mediated through AA metabolites. In addition, the PGE2-induced activation of INa was mimicked by cAMP and quenched by a PKA (protein kinase A) inhibitor, a Gs inhibitor and EP (E-series of prostaglandin) receptor antagonists. The results of the present study suggest that extracellular AA modulates NaV channel activity in rat cerebellar GCs without metabolic conversion, whereas intracellular AA augments the INa by PGE2-mediated activation of cAMP/PKA pathways. These observations may explain the dual character of AA in neuronal pathogenesis.
Modulation of microtubule dynamics by a TIR domain protein from the intracellular pathogen Brucella melitensis
Authors: Radhakrishnan Girish K. , Harms Jerome S. , Splitter Gary A.
Published: July 2011

TIR (Toll/interleukin-1 receptor) domain-containing proteins play a crucial role in innate immunity in eukaryotes. Brucella is a highly infectious intracellular bacterium that encodes a TIR domain protein (TcpB) to subvert host innate immune responses to establish a beneficial niche for pathogenesis. TcpB inhibits NF-κB (nuclear factor κB) activation and pro-inflammatory cytokine secretions mediated by TLR (Toll-like receptor) 2 and TLR4. In the present study, we have demonstrated that TcpB modulates microtubule dynamics by acting as a stabilization factor. TcpB increased the rate of nucleation as well as the polymerization phases of microtubule formation in a similar manner to paclitaxel. TcpB could efficiently inhibit nocodazole- or cold-induced microtubule disassembly. Microtubule stabilization by TcpB is attributed to the BB-loop region of the TIR domain, and a point mutation affected the microtubule stabilization as well as the TLR-suppression properties of TcpB.