bluuebunny/crossref_metadata_2025 huggingface dataset:
The convergent evolution of aluminium resistance in plants exploits a convenient currency
Authors: Ryan Peter R. , Delhaize Emmanuel
Published: March 2010

Suspicions that soluble aluminium (Al) is detrimental to plant growth were reported more than 100 years ago. The rhizotoxicity of Al3+ is now accepted as the major limitation to plant production on acidic soils. Plants differ in their susceptibility to Al3+ toxicity and significant variation can occur within species, even in some major crops. The physiology of Al3+ resistance in some species has been understood for 15 years but the molecular biology has been elucidated only recently. The first gene controlling Al3+ resistance was cloned from wheat (Triticum aestivum L.) in 2004 but others have now been identified in Arabidopsis, barley (Hordeum vulgare L.), rye (Secale cereale L.), sorghum (Sorghum bicolour (L.) Moench) and rice (Oryza sativa L.) with strong additional candidates in wheat and oilseed rape (Brassica napus L.). These genes confer resistance in different ways, but one mechanism occurs in nearly all species examined so far. This mechanism relies on the release of organic anions from roots which bind with the harmful Al3+ cations in the apoplast and detoxify them. The genes controlling this response come from at least two distinct families, suggesting that convergent evolution has occurred. We discuss the processes driving this convergence of protein function and offer opinions for why organic anions are central to the mechanisms of resistance in disparate species. We propose that mutations which modify protein expression or their activation by Al3+ have played important roles in co-opting different transport proteins from other functions.
Isolation and characterisation of two MATE genes in rye
Authors: Yokosho Kengo , Yamaji Naoki , Ma Jian Feng
Published: March 2010

Multidrug and toxic compound extrusion (MATE) proteins are widely present in bacteria, fungi, plants and mammals. Recent studies have showed that a group of plant MATE genes encodes citrate transporter, which are involved in the detoxification of aluminium or translocation of iron from the roots to the shoots. In this study, we isolated two homologous genes (ScFRDL1 and ScFRDL2) from this family in rye (Secale cereale L.). ScFRDL1 shared 94.2% identity with HvAACT1, an Al-activated citrate transporter in barley (Hordeum vulgare L.) and ScFRDL2 shared 80.6% identity with OsFRDL2, a putative Al-responsive protein in rice (Oryza sativa L.). Both genes were mainly expressed in the roots, however, they showed different expression patterns. Expression of ScFRDL1 was unaffected by Al treatment, but up-regulated by Fe-deficiency treatment. In contrast, expression of ScFRDL2 was greatly induced by Al but not by Fe deficiency. The Al-induced up-regulation of ScFRDL2 was found in both the root tips and basal roots. Furthermore, the expression pattern of ScFRDL2 was consistent with citrate secretion pattern. Immunostaining showed that ScFRDL1 was localised at all cells in the root tips and central cylinder and endodermis in the basal root. Taken together, our results suggest that ScFRDL1 was involved in efflux of citrate into the xylem for Fe translocation from the roots to the shoots, while ScFRDL2 was involved in Al-activated citrate secretion in rye.
The agroecological matrix as alternative to the land-sparing/agriculture intensification model
Authors: Perfecto Ivette , Vandermeer John
Published: March 2010

Among the myriad complications involved in the current food crisis, the relationship between agriculture and the rest of nature is one of the most important yet remains only incompletely analyzed. Particularly in tropical areas, agriculture is frequently seen as the antithesis of the natural world, where the problem is framed as one of minimizing land devoted to agriculture so as to devote more to conservation of biodiversity and other ecosystem services. In particular, the “forest transition model” projects an overly optimistic vision of a future where increased agricultural intensification (to produce more per hectare) and/or increased rural-to-urban migration (to reduce the rural population that cuts forest for agriculture) suggests a near future of much tropical aforestation and higher agricultural production. Reviewing recent developments in ecological theory (showing the importance of migration between fragments and local extinction rates) coupled with empirical evidence, we argue that there is little to suggest that the forest transition model is useful for tropical areas, at least under current sociopolitical structures. A model that incorporates the agricultural matrix as an integral component of conservation programs is proposed. Furthermore, we suggest that this model will be most successful within a framework of small-scale agroecological production.
Notch-activated signaling cascade interacts with mitochondrial remodeling proteins to regulate cell survival
Authors: Perumalsamy Lakshmi R. , Nagala Manjula , Sarin Apurva
Published: March 2010

Survival of differentiated cells is one of several processes regulated by Notch activity, although the general principles underlying this function remain to be characterized. Here, we probe the mechanism underlying Notch-mediated survival, building on emerging evidence that apoptotic responses coordinated by specialized intermediates converge on mitochondria, identifying a core event in death pathways. The Bcl-2 family protein Bax is one such intermediate, which in a unifying response to diverse apoptotic stimuli nucleates multiprotein assemblies on mitochondria, committing cells to irrevocable damage. Using Bax as the prototype stimulus, we analyze Notch signaling for potential interactions with mitochondria, probe intrinsic properties of the Notch receptor, and describe key intermediates in the Notch-activated signaling cascade. Ligand-dependent processing was necessary to generate the Notch intracellular domain (NIC) although signaling was independent of canonical interactions with nuclear factors. Notably, antiapoptotic activity was recapitulated by NIC recombinants, localized outside the nucleus, and compromised by enforced nuclear sequestration. NIC signaled via the kinase Akt to prevent the loss of mitochondrial function, contiguity, and consequent nuclear damage, outcomes critically depend on mitochondrial remodeling proteins Mitofusins-(Mfn)-1 and 2. Thus, the NIC-Akt-Mfn signaling cascade identifies a pathway regulating cell-survival, independent of canonical functions associated with NIC activity.
Exploring the within- and between-class correlation distributions for tumor classification
Authors: Wei Xuelian , Li Ker-Chau
Published: March 2010

To many biomedical researchers, effective tumor classification methods such as the support vector machine often appear like a black box not only because the procedures are complex but also because the required specifications, such as the choice of a kernel function, suffer from a clear guidance either mathematically or biologically. As commonly observed, samples within the same tumor class tend to be more similar in gene expression than samples from different tumor classes. But can this well-received observation lead to a useful procedure of classification and prediction? To address this issue, we first conceived a statistical framework and derived general conditions to serve as the theoretical foundation that supported the aforementioned empirical observation. Then we constructed a classification procedure that fully utilized the information obtained by comparing the distributions of within-class correlations with between-class correlations via Kullback–Leibler divergence. We compared our approach with many machine-learning techniques by applying to 22 binary- and multiclass gene-expression datasets involving human cancers. The results showed that our method performed as efficiently as support vector machine and Naïve Bayesian and outperformed other learning methods (decision trees, linear discriminate analysis, and
k
-nearest neighbor). In addition, we conducted a simulation study and showed that our method would be more effective if the arriving new samples are subject to the often-encountered baseline shift or increased noise level problems. Our method can be extended for general classification problems when only the similarity scores between samples are available.
Explanatory Depth
Authors: Weslake Brad
Published: March 2010

I defend an account of explanatory depth according to which explanations in the nonfundamental sciences can be deeper than explanations in fundamental physics.
An Objective Justification of Bayesianism I: Measuring Inaccuracy
Authors: Leitgeb Hannes , Pettigrew Richard
Published: March 2010

In this article and its sequel, we derive Bayesianism from the following norm: Accuracy—an agent ought to minimize the inaccuracy of her partial beliefs. In this article, we make this norm mathematically precise. We describe epistemic dilemmas an agent might face if she attempts to follow Accuracy and show that the only measures of inaccuracy that do not create these dilemmas are the quadratic inaccuracy measures. In the sequel, we derive Bayesianism from Accuracy and show that Jeffrey Conditionalization violates Accuracy unless Rigidity is assumed. We describe the alternative updating rule that Accuracy mandates in the absence of Rigidity.
The Structure and Dynamics of Scientific Theories: A Hierarchical Bayesian Perspective
Authors: Henderson Leah , Goodman Noah D. , Tenenbaum Joshua B. , Woodward James F.
Published: March 2010

Hierarchical Bayesian models (HBMs) provide an account of Bayesian inference in a hierarchically structured hypothesis space. Scientific theories are plausibly regarded as organized into hierarchies in many cases, with higher levels sometimes called ‘paradigms’ and lower levels encoding more specific or concrete hypotheses. Therefore, HBMs provide a useful model for scientific theory change, showing how higher-level theory change may be driven by the impact of evidence on lower levels. HBMs capture features described in the Kuhnian tradition, particularly the idea that higher-level theories guide learning at lower levels. In addition, they help resolve certain issues for Bayesians, such as scientific preference for simplicity and the problem of new theories.
Knowing with Images: Medium and Message
Authors: Kulvicki John
Published: March 2010

Problems concerning scientists’ uses of representations have received quite a bit of attention recently. The focus has been on how such representations get their contents and on just what those contents are. Less attention has been paid to what makes certain kinds of scientific representations different from one another and thus well suited to this or that epistemic end. This article considers the latter question with particular focus on the distinction between images and graphs on the one hand and descriptions and related representations on the other.
Investigating agreement between different data sources using Bayesian state-space models: an application to estimating NE Atlantic mackerel catch and stock abundance
Authors: Simmonds E. John , Portilla Enrique , Skagen Dankert , Beare Doug , Reid Dave G.
Published: March 2010

Simmonds, E. J., Portilla, E., Skagen, D., Beare, D., and Reid, D. G. 2010. Investigating agreement between different data sources using Bayesian state-space models: an application to estimating NE Atlantic mackerel catch and stock abundance. – ICES Journal of Marine Science, 67: 1138–1153. Bayesian Markov chain Monte Carlo methods are ideally suited to analyses of situations where there are a variety of data sources, particularly where the uncertainties differ markedly among the data and the estimated parameters can be correlated. The example of Northeast Atlantic (NEA) mackerel is used to evaluate the agreement between available data from egg surveys, tagging, and catch-at-age using multiple models within the Bayesian framework WINBUGS. The errors in each source of information are dealt with independently, and there is extensive exploration of potential sources of uncertainty in both the data and the model. Model options include variation by age and over time of both selectivity in the fishery and natural mortality, varying the precision and calculation method for spawning-stock biomass derived from an egg survey, and the extent of missing catches varying over time. The models are compared through deviance information criterion and Bayesian posterior predictive p-values. To reconcile mortality estimated from the different datasets the landings and discards reported would have to have been between 1.7 and 3.6 times higher than the recorded catches.
Emergence of CTX-M-3, TEM-1 and a new plasmid-mediated MOX-4 AmpC in a multiresistant Aeromonas caviae isolate from a patient with pneumonia
Authors: Ye Ying , Xu Xi-Hai , Li Jia-Bin
Published: March 2010

Aeromonas species rarely cause pulmonary infection. We report, for what is believed to be the first time, a case of severe pneumonia in a cancer patient caused by Aeromonas caviae. Detailed microbiological investigation revealed that this isolate carried three β-lactamase-encoding genes (encoding MOX-4, CTX-M-3 and TEM-1) conferring resistance to all β-lactams but imipenem. The β-lactamase with a pI of 9.0 was transferred by conjugation and associated with a 7.3 kb plasmid, as demonstrated by Southern blot hybridization. Analysis of the nucleotide and amino acid sequences showed a new ampC gene that was closely related to those encoding the MOX-1, MOX-2 and MOX-3 β-lactamases. This new plasmid-mediated AmpC β-lactamase from China was named MOX-4. This is believed to be the first report of MOX-4, CTX-M-3 and TEM-1 β-lactamases in a multiresistant A. caviae.
Is exposure to mercury a driving force for the carriage of antibiotic resistance genes?
Authors: Skurnik David , Ruimy Raymond , Ready Derren , Ruppe Etienne , Bernède-Bauduin Claire , Djossou Felix , Guillemot Didier , Pier Gerald B. , Andremont Antoine
Published: March 2010

The mercury resistance genemerAhas often been found together with antibiotic resistance genes in human commensalEscherichia coli. To study this further, we analysed mercury resistance in collections of strains from various populations with different levels of mercury exposure and various levels of antibiotic resistance. The first population lived in France and had no known mercury exposure. The second lived in French Guyana and included a group of Wayampi Amerindians with a known high exposure to mercury. Carriage rates of mercury resistance were assessed by measuring the MIC and by detecting themerAgene. Mercury-resistantE. coliwas found significantly more frequently in the populations that had the highest carriage rates of antibiotic-resistantE. coliand in parallel antibiotic resistance was higher in the population living in an environment with a high exposure to mercury, suggesting a possible co-selection. Exposure to mercury might be a specific driving force for the acquisition and maintenance of mobile antibiotic resistance gene carriage in the absence of antibiotic selective pressure.
Genetic characterization and diversity of Streptococcus agalactiae isolates with macrolide resistance
Authors: Brzychczy-Włoch Monika , Gosiewski Tomasz , Bodaszewska Małgorzata , Pabian Wojciech , Bulanda Małgorzata , Kochan Piotr , Strus Magdalena , Heczko Piotr B.
Published: March 2010

Macrolide resistance in 169Streptococcus agalactiae[group B streptococcus (GBS)] isolates originating from pregnant carriers was investigated. Using multiplex PCR the presence of genes encoding erythromycin resistance and capsular polysaccharides, as well as surface proteins, was determined. Random amplification of polymorphic DNA (RAPD) and PFGE were used to characterize specific clones among the isolates. In the examined population of women, erythromycin-resistant strains were found in 4.5 % of patients, whereas clindamycin-resistant strains were found in 3 % of patients, which was 16 % of strains resistant to erythromycin and 10 % of strains resistant to clindamycin among GBS isolates, respectively. Among the isolates, the largest percentage was represented by the constitutive macrolide–lincosamide–streptogramin B (cMLSB) phenotype (63 %), then the inductive macrolide–lincosamide–streptogramin B (iMLSB) phenotype (26 %) and the macrolide resistance (M) phenotype (11 %). TheermBgene was indicated in all isolates with the cMLSBphenotype and V serotype, whereasmefA/mefEgenes were found in isolates with the M phenotype and Ia serotype. Among resistance isolates, serotype V was predominant (67 %), followed by serotypes II (15 %), Ia (11 %) and III (7 %). The most common surface protein encoding genes werealp3(70 %), thenrib(11 %),epsilon(7.5 %),bca(7.5 %) andalp2(4 %). A statistically significant relationship between macrolide resistance, serotype V and thealp3gene was demonstrated. PFGE, in comparison to the RAPD method, gave better genetic discrimination of GBS isolates. A relatively high genetic diversity among investigated strains was shown. In addition, the largest genetic homogeneity was found in serotype V.
TheArabidopsisStem Cell Factor POLTERGEIST Is Membrane Localized and Phospholipid Stimulated
Authors: Gagne Jennifer M. , Clark Steven E.
Published: March 2010

Stem cell maintenance and differentiation are tightly regulated in multicellular organisms. In plants, proper control of the stem cell populations is critical for extensive postembryonic organogenesis. The Arabidopsis thaliana protein phosphatase type 2C proteins POLTERGEIST (POL) and PLL1 are essential for maintenance of both the root and shoot stem cells. Specifically, POL and PLL1 are required for proper specification of key asymmetric cell divisions during stem cell initiation and maintenance. POL and PLL1 are known to be integral components of the CLE/WOX signaling pathways, but the location and mechanisms by which POL and PLL1 are regulated within these pathways are unclear. Here, we show that POL and PLL1 are dual-acylated plasma membrane proteins whose membrane localization is required for proper function. Furthermore, this localization places POL and PLL1 in proximity of the upstream plasma membrane receptors that regulate their activity. Additionally, we find that POL and PLL1 directly bind to multiple lipids and that POL is catalytically activated by phosphatidylinositol (4) phosphate [PI(4)P] in vitro. Based on these results, we propose that the upstream receptors in the CLE/WOX signaling pathways may function to either limit PI(4)P availability or antagonize PI(4)P stimulation of POL/PLL1. Significantly, the findings presented here suggest that phospholipids play an important role in promoting stem cell specification.
Epidermal growth factor’s activation of Ras is inhibited by four cardiac hormones
Authors: Sun Ying , Eichelbaum Ehrentraud J. , Lenz Anne , Wang Hai , Vesely David L.
Published: March 2010

Eur J Clin Invest 2010; 40 (5): 408–413

Background  Four cardiac hormones synthesized by the same gene, i.e. atrial natriuretic peptide, vessel dilator, kaliuretic peptide and long‐acting natriuretic peptide, have anticancer effects in vitro and in vivo. Epidermal growth factor’s mechanism of cancer formation involves the activation of Ras.

Materials and methods  These four cardiac hormones were evaluated for their ability to inhibit mitogen (epidermal growth factor) activation of Ras.

Results  Epidermal growth factor increased the activation of Ras by 68%, 85% and 90% at its 1, 2 and 5 ng mL−1 concentrations. Vessel dilator, long‐acting natriuretic peptide, atrial natriuretic peptide and kaliuretic peptide inhibited 5 ng mL−1 epidermal growth factor’s stimulation of Ras by 73%, 79%, 33% and 45%, respectively, at their 1 μM concentrations. Their effects on epidermal growth factor’s activation of Ras were specific with addition of the cardiac hormones’ respective antibodies (5 μM) blocking 95%, 93%, 100% and 100% (P < 0·001 for each) of their ability to inhibit epidermal growth factor’s stimulation of Ras.

Conclusions  Four cardiac hormones specifically inhibit epidermal growth factor’s activation of Ras. This investigation would suggest that these cardiac hormones’ anticancer effects involve the inhibition of mitogens such as epidermal growth factor’s ability to activate Ras as well as inhibiting unstimulated basal activity of Ras.
Closure strategies as a tool for fisheries management in metapopulations subjected to catastrophic events
Authors: LITTLE L. R. , GRAFTON R. Q. , KOMPAS T. , SMITH A. D. M.
Published: March 2010

Abstract  Spatial management measures in the form of no‐take areas used in fisheries management can provide a buffer against catastrophic events. Dynamic area closures, like rotational closures, have also been used as a management tool particularly for sessile organisms. In this study, bioeconomic models are developed to investigate dynamic closure strategies for use as a management tool in the harvest of a metapopulation consisting of two local sub‐populations. The models provide an optimal strategy that maximises the sum of discounted net returns with a fixed harvest level [i.e. total allowable catch (TAC)] by opening and closing the sub‐populations of a metapopulation, subject to random negative catastrophic effects. Results showed the optimal policy for opening and closing a single exploited population depends on the degree and pattern of migration between it and other sub‐populations. When the harvest or TAC can be applied to either sub‐population, the optimal closure strategy depends on the abundance of both populations, crucially, even if they are biologically independent. The results provide insights into the management of stochastically fluctuating populations including more mobile species that are frequently not subject to no‐take controls.
Seasonal and long‐term changes in fishing depth of Lake Constance whitefish
Authors: THOMAS G. , RÖSCH R. , ECKMANN R.
Published: March 2010

Abstract  The ecosystem of Lake Constance in central Europe has undergone profound modifications over the last six decades. Seasonal and inter‐annual changes in the vertical distribution patterns of whitefish were examined and related to changes in biotic and abiotic gradients. Between 1958 and 2007, the average fishing depth in late summer and autumn was related to two factors influencing food supply of whitefish – lake productivity and standing stock biomass. In years with low food supply, whitefish were harvested from greater depths, where temperatures were up to 4 °C lower. The whitefish’s distribution towards colder water might be a bioenergetic optimisation behaviour whereby fish reduce metabolic losses at lower temperatures, or it may result from a reassessment of habitat preference under conditions of limited food supply, according to the ideal free distribution theory.
The effect of desalivation on the malignant transformation of the tongue epithelium and associated stromal myofibroblasts in a rat 4‐nitroquinoline 1‐oxide‐induced carcinogenesis model
Authors: Vered Marilena , Grinstein‐Koren Osnat , Reiter Shoshana , Allon Irit , Dayan Dan
Published: March 2010

The aim of our study was to analyse desalivated rat tongue epithelium for histopathological changes, proliferating cell nuclear antigen (PCNA), and epithelium‐associated stromal myofibroblasts [SMF; α‐smooth muscle actin (αSMA)] following 0.001% 4‐nitroquinoline 1‐oxide (4NQO) administration in drinking water. Results were compared with those of identically treated but salivated specimens. 4NQO was administered for 7, 14, 22 and 28 weeks. Tongue length was divided into anterior, middle and posterior ‘thirds’. The histopathological changes per ‘third’ were scored as normal epithelium, hyperplasia, dysplasia, carcinoma‐in‐situ, and superficial and invasive carcinoma. The PCNA and αSMA stains were assessed by a point‐counting method. At all time points, the histopathological changes in the anterior and middle thirds were higher in the desalivated than in the salivated group (P < 0.05) but almost identical in the posterior third (P > 0.05). PCNA scores were significantly lower in the desalivated vs. the salivated group at almost all time points and tongue thirds (P < 0.05). SMF were usually scarce in both groups, but there was a significant surge in the posterior third at 28 weeks: the score in the desalivated group was only about one‐half that of the salivated group (P < 0.05). The absence of saliva seems to promote malignant transformation of the tongue epithelium in the early stages. PCNA cannot be regarded as a marker of proliferation and probably contributes to this process by other mechanisms. Emergence of SMF seems to be highly dependent on growth factors from saliva in addition to factors from cancerous cells.
Tissue changes in senescent gerbil prostate after hormone deprivation leads to acquisition of androgen insensitivity
Authors: Campos Silvana G. P. , Gonçalves Bianca F. , Scarano Wellerson R. , Corradi Lara S. , Santos Fernanda C. A. , Custodio Ana M. G. , Vilamaior Patricia S. L. , Góes Rejane M. , Taboga Sebastião R.
Published: March 2010

The present study examined the response of the prostate epithelium of senescent gerbils submitted to orchiectomy and with or without steroidal blockade. Animals were divided into five groups, all surgically castrated except the control group composed of intact animals. In the experimental groups, doses of flutamide and/or tamoxifen were applied for 1, 3, 7 and 30 days postcastration. The structural methods applied reveal that castration, whether associated or not with anti‐steroidal drugs, promoted short‐ and long‐term decrease in wet and relative weights of the prostate. The quantitative decline of epithelial compartment proportion observed at the end of treatment was due to the sum of slight changes in the epithelium and lumen. The apoptotic index had risen significantly at 1 day and declined at 7 days postcastration. Androgen receptor (AR) expression decreased after 3 days of hormonal ablation, coinciding with the highest levels of apoptosis and cell proliferation observed in all treated groups. The majority of cells remained differentiated in all groups due to CK 8/18 expression. Some animals remained with injuries such as carcinomas and adenocarcinomas after hormonal ablation. In the latter a mixture of AR‐positive and AR‐negative cells was identified. Microinvasive carcinomas found in the group treated for 30 days consisted of PCNA‐positive, inflammatory and non‐proliferating cells. Low apoptosis incidence and bcl‐2 positive cells were observed in these lesions. The treatments promoted a reduction of lesions in older gerbils, but treatment‐resistant tumours will improve understanding of the events that lead to hormone resistance.
Soluble gp130 promotes intestinal epithelial hyperplasia during reovirus infection
Authors: Montufar‐Solis Dina , Garza Tomas , Vigneswaran Nadarajah , Klein John R.
Published: March 2010

Soluble gp130 (sgp130) has been shown to suppress the inflammatory response of autoimmune pathologies; however, its effects on virus infection are not known. Here, we report that intraperitoneal treatment of mice with sgp130‐Fc fusion protein at the time of oral reovirus serotype 3 infection resulted in altered morphopathological changes that were evident by less shortening of intestinal villi length and crypt depth after infection. That the effect mediated by sgp130 treatment was due to an increase in intestinal crypt cell proliferation was demonstrated by an increase in the number of crypt mitotic figures. This was further confirmed by increased immunoreactivity to the Cdc47 proliferation‐associated antigen in crypts of sgp130‐treated virus‐infected mice compared to infected non‐treated mice. These findings suggest that sgp130 may have a beneficial effect during intestinal virus infection by disrupting interleukin‐6 trans‐signalling, thereby reducing the local inflammatory response.
Proteomic analysis of osteogenic sarcoma: association of tumour necrosis factor with poor prognosis
Authors: Cates Justin M. M. , Friedman David B. , Seeley Erin H. , Dupont William D. , Schwartz Herbert S. , Holt Ginger E. , Caprioli Richard M. , Young Pampee P.
Published: March 2010

A significant proportion of patients with osteogenic sarcoma die from lung metastasis within 5 years of diagnosis. Molecular signatures that predict pulmonary metastasis from primary osteogenic sarcoma and identify those patients at risk would be clinically useful as prognostic markers. Protein expression profiles of two clonally related murine osteogenic sarcoma cell lines with low (K12) and high (K7M2) metastatic potential were compared using two different proteomic technologies, two‐dimensional difference gel electrophoresis and cell profiling by matrix‐assisted laser desorption/ionization mass spectrometry. Interrogation of a molecular pathways network database suggested several additional candidate molecules that potentially predict metastatic potential of primary osteogenic sarcoma. Two such proteins, macrophage migration inhibitory factor and tumour necrosis factor were selected for further validation studies. Western blots confirmed increased expression of both cytokines in K7M2 cells compared to K12 cells. Levels of migration inhibitory factor and tumour necrosis factor were semi‐quantitatively measured in human osteogenic sarcoma samples by immunohistochemistry and were correlated with clinicopathologic parameters and patient outcomes. Multivariate survival analysis demonstrated that tumour necrosis factor expression in chemotherapy naïve osteogenic sarcoma is an independent prognostic factor for overall and metastasis‐free survival. No significant differences in adverse outcomes were observed based on macrophage migration inhibitory factor expression.
An early commitment to expression of a particular TCRVβ chain on CD8+ T cells responding to attenuated Plasmodium berghei sporozoites is maintained following challenge with infectious sporozoites
Authors: LUMSDEN J. M. , CRANMER M. A. , KRZYCH U.
Published: March 2010

Protection induced by irradiated Plasmodium berghei sporozoites (Pbγ‐spz) in mice is linked to CD8+ T cells specific for exo‐erythrocytic‐stage Ags, and intrahepatic memory CD8+ T cells are associated with protracted protection. However, the Ag specificity of the protective CD8+ T cells remains largely unknown. In this study, we characterized the TCR Vβ usage by intrahepatic CD8+ T cells during γ‐spz immunization and after the challenge with infectious Pb sporozoites. The repertoire of naïve (TN) and central memory (TCM) CD8+ T cells was diverse and conserved between individual mice, and did not change with immunization. In contrast, preferential usage of one or more TCR Vβ subset was observed in effector memory (TEM) CD8+ T cells after immunization. The expanded TCR Vβ varied between individual mice but Vβ4, 6, 7, 8.3, 9 and 11 were the most frequently expressed. In addition, there was a correlation in the TCR Vβ usage by γ‐spz‐induced CD8+ TEM in the liver and blood of individual mice. The expansion pattern of blood CD8+ TEM did not change with challenge and remained the same for 8 weeks thereafter. These results demonstrate that immunization with γ‐spz skews the TCR Vβ repertoire of CD8+ TEM, and commitment to a particular TCR Vβ expression is maintained long‐term.
Thymic stromal lymphopoietin is present in human breast milk
Authors: Macfarlane Trisha V. , Seager Anna L. , Moller Maria , Morgan Gareth , Thornton Catherine A.
Published: March 2010

Macfarlane TV, Seager AL, Moller M, Morgan G, Thornton CA. Thymic stromal lymphopoietin (TSLP) is present in human breast milk. Pediatr Allergy Immunol 2010: 21: e454–e456. © 2010 The Authors Journal compilation © 2010 Blackwell Munksgaard

Thymic stromal lymphopoietin (TSLP) has emerged as a potential key mediator of both the initiation and maintenance of Th2 responsiveness and thereby the allergic inflammatory cascade. Given the possible role of TSLP in initiating Th2‐mediated allergic disease more detailed investigation of TSLP expression and responsiveness in early life is warranted. Therefore, the presence of TSLP in breast milk was evaluated. Defatted human breast milk collected at 3–5 days and 11–26 days postpartum (n = 22/group) and commercially available ready‐made baby formula (n = 3) were analysed for TSLP with a specific ELISA. TSLP was detectable in breast milk but not in commercial ready‐made formulas. Levels in breast milk were significantly higher (p < 0.001) at 3–5 days than at 11–26 days postpartum. The potential role of breast milk TSLP in the development of immune function within the neonatal gastrointestinal tract and the development of allergy in childhood remain to be determined.
Structural Studies of the Equine Infectious Anemia Virus trans ‐Activator Protein
Authors: Willbold Dieter , Volkmann Andrea , Metzger Armin U. , Sticht Heinrich , Rosin‐Arbesfeld Rina , Gazit Arnona , Yaniv Abraham , Frank Rainer W. , Rösch Paul
Published: March 2010

Trans‐activator(tat) proteins are necessary components for the completion of the T replication cycle of lentiviruses. The three‐dimensional structure of the equine infectious anemia virus (EIAV) tat protein (e‐tat) was studied with CD spectroscopy, NMR spectroscopy, and restrained molecular‐dynamics calculations. No stable elements of regular secondary structure were detected, but the sequence regions responsible for nucleic acid binding showed helix‐forming tendency, e‐tat exhibits a flexible tertiary structure, and only the amino acids comprising the core sequence region form a well‐defined tertiary fold. The three‐dimensional structure allows discussion of biochemical data as well as data from molecular biological investigations of lentiviral tat proteins.
New Ligands Binding to the Human Leukocyte Antigen Class II Molecule DRB1*0101 Based on the Activity Pattern of an Undecapeptide Library
Authors: Fleckenstein Burkhard , Kalbacher Hubert , Muller Claude P. , Stoll Dieter , Halder Thomas , Jung Günther , Wiesmüller Karl‐Heinz
Published: March 2010

Major histocompatibility complex (MHC) class II molecules present peptide antigens to CD4+‐T cells. These heterogeneous peptides are derived from internalized exogenous proteins or from endogenous membrane proteins that are processed by the antigen‐presenting cell. Peptides are bound to the MHC class II molecules in an extended conformation and extend out of the binding groove. The aim of this study was to estimate the influence of every amino acid in all the possible undecapeptide amides (2.048×1014 individuals) on the binding to human MHC‐DRB1*0101 molecules (HLA‐DR1) and to identify new peptide ligands. 220 undecapeptide sublibraries, O/X10, each composed of ten degenerate positions and one defined position, were screened for binding to isolated HLA‐DR1. Competition of the sublibraries with a fluorescence‐labeled peptide ligand allowed definition of the amino acids favourable or unfavourable for DR1‐binding at every sequence position. From the activity pattern of the undecapeptide library, 54 individual peptides were deduced (27 potential hits and 27 potential falls) and prepared by chemical synthesis. As anticipated, 27 positive and 27 negative results were obtained from the competition experiments. The 27 peptides that bind obey the rules for the HLA‐DR1‐binding motif. The synthetic peptide library approach proved to be valuable for the design of synthetic MHC class II ligands and thus can be considered as a basis for drug design in immunotherapy.
Nucleotide Binding to the Heat‐Shock Protein DnaK as Studied by ESR Spectroscopy
Authors: Neuhofen Sonja , Theyssen Holger , Reinstein Jochen , Trommer Wolfgang E. , Vogel Pia D.
Published: March 2010

We employed ESR spectroscopy using spin‐labeled adenine nucleotides to investigate nucleotide binding to the 70‐kDa heat shock protein, DnaK, from Escherichia coli. Binding stoichiometries of 1 mol/mol for both ATP and ADP to previously nucleotide‐depleted protein in the presence of Mg2+ were determined directly and under equilibrium binding conditions. Of the spin‐labeled adenine nucleotides available to us, only the derivatives with the spin label attached to the C8 position of the adenine moiety, 8‐SL‐AdoP3 and 8‐SL‐AdoP2 [8‐(2,2,6,6‐tetramethyl‐piperidin‐4‐yl‐1‐oxyl‐)amino‐adenosine‐5′‐triphosphate or diphosphate], were bound sufficiently tightly by the heat‐shock protein, resulting in ESR spectra typical for immobilized radicals. In the absence of Mg2+, only approximately 0.5 mol were bound. Subsequent addition of Mg2+, however, led to the previously observed maximum binding of 1 mol/mol. Both 8‐SL‐AdoP3, and 8‐SL‐AdoP2 were fully exchangeable upon addition of excess ATP or ADP suggesting that the analogs bound directly to the nucleotide binding sites within the protein. 8‐SL‐AdoP2 release was also observed in the presence of the co‐chaperone GrpE, indicating that the spin‐labeled analogs of adenine nucleotides function like the natural nucleotidesubstrates of the heat‐shock protein. Small differences in the ESR spectra of 8‐SL‐AdoP3 and 8‐SL‐AdoP2 in complex with DnaK were observed.
Roles of Tyr103 and Tyr264 in the Regulation of RecA‐DNA Interactions by Nucleotide Cofactors
Authors: Morimatsu Katsumi , Maraboeuf Fabrice , Hagmar Per , Nordén Bengt , Horii Toshihiro , Takahashi Masayuki
Published: March 2010

The DNA‐binding mode of the RecA protein, in particular its dependence on nucleotide cofactor, has been investigated by monitoring the fluorescence and linear‐dichroism signals of a tryptophan residue inserted in the RecA to replace tyrosine at position 103 or 264. These residues are important for cofactor and DNA binding, as evidenced from their fluorescence changes upon binding of cofactor and DNA [Morimatsu, K., Horii, T. & Takahashi, M. (1995) Eur. J. Biochem. 228, 779–785]. The substitution of these residues with tryptophan does not affect the structure or biological function of the complex and can therefore be exploited to gain structural information in terms of the orientation and environment of the inserted reporter chromophore. The fluorescence change upon formation of the ternary cofactor RecA DNA complex was much smaller than the sum of the changes induced by cofactor or DNA alone. This difference indicates that the cofactor and DNA interact with RecA via common components. The fluorescence change caused by DNA in the presence of cofactor was almost independent of the base composition of DNA, in contrast to the interaction in the absence of cofactor. Hence, the contact mode between the selected residues and DNA in the complex may depend significantly on the cofactor. Linear‐dichroism measurements indicate that the cofactor does not markedly alter the organization of RecA filament. Linear dichroism shows that neither the aromatic moiety of residue 103 nor that of residue 264 is intercalated between the DNA bases. The textural changes reported for the helical pitch and contour length of RecA fiber upon interaction with cofactor and DNA may derive from a subtle change in orientation of the RecA subunits in the filament.
Properties of a Cl−‐Conductive Pathway(s) in Microsomes from Rat Kidney Inner Medulla
Authors: Benharouoa Mohamed , Lipecka Joanna , Fanen Pascale , Baudoin‐Legros Maryvonne , Banting George , Fritsch Janine , Edelman Aleksander
Published: March 2010

The properties of a protein‐kinase‐A(PKA)‐activated Cl−‐conductive pathway(s) in alkaline phosphatase‐enriched microsomes from the rat inner medulla (IMV) were investigated. Transcripts of cystic fibrosis transmembrane regulator (CFTR) were detected by reverse transcription/polymerase analysis of total RNA from the inner medulla, while immunoblot analysis using anti‐CFTR antibodies detected a 170‐kDa protein in the IMV. The PKA Cl− conductive pathway(s) was studied by measuring the rate of valinomycin‐induced microsomal swelling by light scattering. PKA increased the rate of valinomycin‐induced swelling of vesicles consistent with the presence of Cl−‐conductive pathway(s). The pharmacological properties and anion selectivity of the PKA‐activated Cl− ‐conductive pathway(s) were similar to those of the CFTR Cl− channel. Our results show that a CFTR Cl− channel and possibly another cAMP‐activated pathway(s) may participate in Cl− secretion in the rat inner medulla.
Tetrameric and Octameric Lactate Dehydrogenase from the Hyperthermophilic Bacterium Thermotoga maritima
Authors: Dams Thomas , Ostendorp Ralf , Ott Markus , Rutkat Kerstin , Jaenicke Rainer
Published: March 2010

Lactate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima has been functionally expressed in Escherichia coli. As shown by gel‐permeation chromatography, dynamic light scattering, and ultracentrifugation, the recombinant protein forms homotetrameric and homooctameric assemblies with identical spectral properties and a common subunit molecular mass (35 kDa). Dynamic light scattering and sedimentation equilibrium experiments proved that both species are monodisperse, thus excluding their interconversion in the given ranges of concentration (0.02–50 mg/ml) and temperature (20–80°C). Rechromatography confirms this finding: the octamer does not dissociate at low enzyme concentrations, nor do tetramers dimerize at the given upper limit of concentration. Renaturation of pure tetramers or octamers after preceding guanidine denaturation leads to redistribution of the two species; increased temperature favors octamer formation.

Thermal analysis and denaturation by chaotropic agents do not allow the free energies of stabilization of the two forms to be quantified, because heat coagulation and kinetic partitioning between reconstitution and aggregation cause irreversible side reactions. Guanidine denaturation of the octamer leads to a highly cooperative dissociation to tetramers which subsequently dissociate and unfold to yield metastable dimers and, finally, fully unfolded monomers. Evidently, there is no tight coupling of the two tetramers within the stable octameric quaternary structure. Electron microscopy clearly corroborates this conclusion: image processing shows that the dumb‐bell‐shaped octamer is made up of two tetramers connected via surface contacts without significant changes in the dimensions of the constituent parts.
Inherited Mutations within the Calcium‐Binding Sites of the Integrin αIIb Subunit (Platelet Glycoprotein IIb)
Authors: Jackson Denise E. , Poncz Mortimer , Holyst M. Trudy , Newman Peter J.
Published: March 2010

To examine the effects of naturally occurring inherited mutations on the ability of the integrin α‐subunit, also termed glycoprotein IIb (GPIIb), to bind metal ions, we prepared small synthetic peptides that encompassed individual cation‐binding domains, and recombinant GPIIb polypeptides that encompassed all four Ca2+‐binding domains, and examined their interactions with divalent cations by means of Tb3+‐luminescence spectroscopy. Replacement of the highly conserved Gly418 residue, located within the flanking region of the fourth Ca2+‐binding domain of GPIIb, with a negatively charged Asp residue resulted in marked reduction in the ability to bind divalent cations. A variant form of GPIIb with a deletion of two amino acids at the −1 and X positions of the fourth Ca2+‐binding domain of GPIIb also failed to bind metal ions in a normal manner. In contrast, a Glanzmann mutation at the −1 position of the first Ca2+‐binding domain of GPIIb had no effect on divalent‐cation‐binding ability with either synthetic peptides or recombinant GPIIb polypeptides. These data support the hypothesis that the highly conserved Gly normally found 7–8 residues N‐terminal to integrin metal‐binding domains plays a critical role in the maintenance of the conformation or orientation of surrounding EF‐hand structures so that they can effectively interact with and bind divalent cations. Furthermore, inherited mutations at or near the divalent‐cation‐binding domains of GPIIb do not necessarily exert their biochemical effects by disruption of cation binding, but can interfere with integrin biogenesis in a Ca2+‐independent manner.
Functional Expression of a Fragment of Human Dihydroorotate Dehydrogenase by Means of the Baculovirus Expression Vector System, and Kinetic Investigation of the Purified Recombinant Enzyme
Authors: Knecht Wolfgang , Bergjohann Ute , Gonski Sigrid , Kirschbaum Bernhard , Lóffler Monika
Published: March 2010

Human mitochondrial dihydroorotate dehydrogenase (the fourth enzyme of pyrimidine de novo synthesis) has been overproduced by means of a recombinant baculovirus that contained the human cDNA fragment for this protein. After virus infection and protein expression in Trichoplusia ni cells (BTI‐Tn‐5B1‐4), the subcellular distribution of the recombinant dihydroorotate dehydrogenase was determined by two distinct enzyme‐activity assays and by Western blot analysis with anti‐(dihydroorotate dehydrogenase) Ig. The targeting of the recombinant protein to the mitochondria of the insect cells was verified. The activity of the recombinant enzyme in the mitochondria of infected cells was about 740‐fold above the level of dihydroorotate dehydrogenase in human liver mitochondria. In a three‐step procedure, dihydroorotate dehydrogenase was purified to a specific activity of greater than 50 U/mg. Size‐exclusion chromatography showed a molecular mass of 42 kDa and confirmed the existence of the fully active enzyme as a monomeric species. Fluorimetric cofactor analysis revealed the presence of FMN in recombinant dihydroorotate dehydrogenase. By kinetics analysis, Km values for dihydroorotate and ubiquinone‐50 were found to be 4 μM and 9.9 μM, respectively, while Km values for dihydroorotate and decylubiquinone were 9.4 μM and 13.7 μM, respectively. The applied expression system will allow preparation of large quantities of the enzyme for structure and function studies. Purified recombinant human dihydroorotate dehydrogenase was tested for its sensitivity to a reported inhibitor A77 1726 (2‐hydroxyethylidene‐cyanoacetic acid 4‐trifluoromethyl anilide), which is the active metabolite of the isoxazol derivative leflunomide [5‐methyl‐N‐(4‐trifluoromethyl‐phenyl)‐4‐isoxazole carboximide]. An IC50 value of 1 μM was determined for A77 1726. Detailed kinetics experiments revealed uncompetitive inhibition with respect to dihydroorotate (Kiu= 0.94 μM) and non‐competitive inhibition with respect to decylubiquinone (Kic= 1.09 μM, Kiu= 1.05 μM). These results suggest that the immunomodulating agent A77 1726 (currently in clinical phase III studies for the treatment of rheumatoid arthritis) is a very good inhibitor of human dihydroorotate dehydrogenase.
Time use, parenting practice and conduct problems in four‐ to five‐year‐old Australian children
Authors: Yu Mong‐Lin , Ziviani Jenny , Baxter Janeen , Haynes Michele
Published: March 2010

Background:  Conduct problems (CoP) represent one of the most common mental health issues manifesting in childhood, with the potential to influence the developmental trajectory of children negatively. Early identification of children at risk of developing CoP is a key strategy to their effective management. Evidence suggests that parenting practices are important contributors to CoP; however, these practices can also interact with the activities in which children engage and these have not yet been addressed in combination.

Method:  A cross‐sectional study of a nationally representative sample of 4936 four‐ to five‐year‐old children from Wave 1 of the Longitudinal Study of Australian Children was undertaken to examine the relationship between CoP, parenting practices and time use.

Results:  All children were at a lower risk of CoP if they were exposed to less hostile and consistent parenting practices and if they did not have sleep problems as reported by their parents. However, boys were more vulnerable if they had fathers who had not undertaken tertiary education, and spent more time in risk‐oriented physical activities.

Conclusion:  Parenting practices are affirmed as a significant independent predictor of risk for developing CoP. The nature of activities in which children engaged, particularly boys, also has the potential to influence the manifestation of CoP. Occupational therapy services for children with CoP are best provided in the context of family‐centred practice and should incorporate an examination of daily time use.