Role of Na+-K+-ATPase in insulin-induced lactate release by skeletal muscle.

Hyperinsulinemia increases lactate release by various organs and tissues. Whereas it has been shown that aerobic glycolysis is linked to Na+-K+-ATPase activity, we hypothesized that stimulation by insulin of skeletal muscle Na+-K+-ATPase is responsible for increased muscle lactate production. To test this hypothesis, we assessed muscle lactate release in healthy volunteers from the [13C]lactate concentration in the effluent dialysates of microdialysis probes inserted into the tibialis anterior muscles on both sides and infused with solutions containing 5 mmol/l [U-13C]glucose. On one side, the microdialysis probe was intermittently infused with the same solution additioned with 2.10(-5) M ouabain. In the basal state, [13C]lactate concentration in the dialysate was not affected by ouabain. During a euglycemic-hyperinsulinemic clamp, [13C]lactate concentration increased by 135% in the dialysate without ouabain, and this stimulation was nearly entirely reversed by ouabain (56% inhibition compared with values in the dialysate collected from the contralateral probe). These data indicate that insulin stimulates muscle lactate release by activating Na+-K+-ATPase in healthy humans.

The effect of nitrogen and sulphur fertilization on yield and quality of kohlrabi (Brassica oleracea, L.)

In a greenhouse pot experiment with kohlrabi, variety Luna, we explored the joint effect of N (0.6 g N per pot = 6 kg of soil) and S in the soil (25-35-45 mg kg-1 of S) on yields, on N, S and NO3- content in tubers and leaves, and on alterations in the amino acids concentration in the tubers. S fertilisation had no effect on tuber yields. The ranges of N content in tubers and leaves were narrow (between 1.42-1.48 % N and 1.21-1.35 % N, respectively) and the effect of S fertilisation was insignificant. S concentration in the tubers ranged between 0.59 and 0.64 % S. S fertilisation had a more pronounced effect on the S concentration in leaf tissues where it increased from 0.50 to 0.58 or to 0.76 % S under the applied dose. The NO3- content was higher in tubers than in leaves. Increasing the S level in the soil significantly reduced NO3- concentrations in the tubers by 42.2-53.6 % and in the leaves by 8.8-21.7 %. Increasing the S content in the soil reduced the concentration of cysteine + methionine by 16-28 %. The values of valine, tyrosine, aspartic acid and serine were constant. In the S0, S1, and S2 treatments the levels of threonine, isoleucine, leucine, arginine, the sum of essential amino acids and alanine decreased from 37 to 9 %. The histidine concentration increased with increasing S fertilisation. S fertilisation of kohlrabi can be recommended to stabilize the yield and reduce the undesirable NO3- contained in the parts used for consumption.

Beta cell mass and growth after syngeneic islet cell transplantation in normal and streptozocin diabetic C57BL/6 mice

In islet transplantation, nonimmunological factors such as limited growth capacity or increased death rate could reduce the beta cell mass in the graft and lead to failure of the transplant. We studied the evolution of beta cell replication and mass after transplantation of insufficient, minimally sufficient, or excessive islet tissue. Streptozocin diabetic C57BL/6 mice received 150 or 300 syngeneic islets under the kidney capsule and normal mice received 300 islets. In streptozocin diabetic mice 300 islets restored normoglycemia; beta cell replication in transplanted islets was similar to replication in normal pancreas and beta cell mass in the graft remained constant. In contrast, 150 islets were insufficient to achieve normoglycemia; beta cell replication was increased initially but not by 18 or 30 d despite persistent hyperglycemia, and beta cell mass fell progressively. When islets were transplanted into normal recipients, beta cell replication remained normal but beta cells underwent atrophy and mass in the graft was substantially reduced. Therefore, with a successful islet transplant, in diabetic mice beta cell replication and mass remain constant. In contrast, when insufficient islet tissue is transplanted an initial increase in beta cell replication can not compensate for a decline in beta cell mass. When excessive islet tissue is transplanted, beta cell mass is reduced despite normal beta cell replication.

Comparative modular analysis of gene expression in vertebrate organs.

ABSTRACT: BACKGROUND: The degree of conservation of gene expression between homologous organs largely remains an open question. Several recent studies reported some evidence in favor of such conservation. Most studies compute organs' similarity across all orthologous genes, whereas the expression level of many genes are not informative about organ specificity. RESULTS: Here, we use a modularization algorithm to overcome this limitation through the identification of inter-species co-modules of organs and genes. We identify such co-modules using mouse and human microarray expression data. They are functionally coherent both in terms of genes and of organs from both organisms. We show that a large proportion of genes belonging to the same co-module are orthologous between mouse and human. Moreover, their zebrafish orthologs also tend to be expressed in the corresponding homologous organs. Notable exceptions to the general pattern of conservation are the testis and the olfactory bulb. Interestingly, some co-modules consist of single organs, while others combine several functionally related organs. For instance, amygdala, cerebral cortex, hypothalamus and spinal cord form a clearly discernible unit of expression, both in mouse and human. CONCLUSIONS: Our study provides a new framework for comparative analysis which will be applicable also to other sets of large-scale phenotypic data collected across different species.

Quality of care and survival of haemodialysed patients in western Switzerland.

BACKGROUND: Many factors affect survival in haemodialysis (HD) patients. Our aim was to study whether quality of clinical care may affect survival in this population, when adjusted for demographic characteristics and co-morbidities. METHODS: We studied survival in 553 patients treated by chronic HD during March 2001 in 21 dialysis facilities in western Switzerland. Indicators of quality of care were established for anaemia control, calcium and phosphate product, serum albumin, pre-dialysis blood pressure (BP), type of vascular access and dialysis adequacy (spKt/V) and their baseline values were related to 3-year survival. The modified Charlson co-morbidity index (including age) and transplantation status were also considered as a predictor of survival. RESULTS: Three-year survival was obtained for 96% of the patients; 39% (211/541) of these patients had died. The 3-year survival was 50, 62 and 69%, respectively, in patients who had 0-2, 3 and >or=4 fulfilled indicators of quality of care (test for linear trend, P < 0.001). In a Cox multivariate analysis model, the absence of transplantation, a higher modified Charlson's score, decreased fulfilment of indicators of good clinical care and low pre-dialysis systolic BP were independent predictors of death. CONCLUSION: Good clinical care improves survival in HD patients, even after adjustment for availability of transplantation and co-morbidities.

Efficacy of enzyme replacement therapy in Fabry disease.

Enzyme replacement therapy has recently been introduced to treat Fabry disease, a rare X-linked lysosomal storage disorder. The disease occurs due to deficient activity of alpha-galactosidase A, leading to progressive accumulation of globotriaosylceramide in multiple organs and tissues. Renal, cardiac and cerebrovascular manifestations of the disease result in premature death in both hemizygous males and heterozygous females. This paper outlines the clinical signs, symptoms and diagnosis of Fabry disease, and the development of the two available enzyme replacement therapies -- agalsidase alfa and agalsidase beta. Agalsidase alfa and agalsidase beta are produced in a human cell line and in Chinese hamster ovary cells, respectively, resulting in products with the same amino acid sequence as the native human enzyme, but with different patterns of glycosylation. Correct post-translational glycosylation is important in terms of the pharmacokinetics, biodistribution, clinical efficacy and tolerability of genetically engineered protein therapeutics. Differences in glycosylation, which may affect immunogenicity and mannose-6-phosphate receptor-mediated cellular internalisation of administered enzyme, possibly account for the differences in dosing, clinical effects and safety profiles reported for agalsidase alfa and agalsidase beta.

Identification of different heart tissues from MRI C-SENC images using an unsupervised multi-stage fuzzy clustering technique.

PURPOSE: To objectively characterize different heart tissues from functional and viability images provided by composite-strain-encoding (C-SENC) MRI. MATERIALS AND METHODS: C-SENC is a new MRI technique for simultaneously acquiring cardiac functional and viability images. In this work, an unsupervised multi-stage fuzzy clustering method is proposed to identify different heart tissues in the C-SENC images. The method is based on sequential application of the fuzzy c-means (FCM) and iterative self-organizing data (ISODATA) clustering algorithms. The proposed method is tested on simulated heart images and on images from nine patients with and without myocardial infarction (MI). The resulting clustered images are compared with MRI delayed-enhancement (DE) viability images for determining MI. Also, Bland-Altman analysis is conducted between the two methods. RESULTS: Normal myocardium, infarcted myocardium, and blood are correctly identified using the proposed method. The clustered images correctly identified 90 +/- 4% of the pixels defined as infarct in the DE images. In addition, 89 +/- 5% of the pixels defined as infarct in the clustered images were also defined as infarct in DE images. The Bland-Altman results show no bias between the two methods in identifying MI. CONCLUSION: The proposed technique allows for objectively identifying divergent heart tissues, which would be potentially important for clinical decision-making in patients with MI.

Retention half times in the skeleton of plutonium and Sr-90 from above-ground nuclear tests: A retrospective study of the Swiss population

Plutonium and Sr-90 are considered to be among the most radiotoxic nuclides produced by the nuclear fission process. In spite of numerous studies on mammals and humans there is still no general agreement on the retention half time of both radionuclides in the skeleton in the general population. Here we determined plutonium and Sr-90 in human vertebrae in individuals deceased between 1960 and 2004 in Switzerland. Plutonium was measured by sensitive SF-ICP-MS techniques and Sr-90 by radiometric methods. We compared our results to the ones obtained for other environmental compartments to reveal the retention half time of NBT fallout Pu-239 and Sr-90 in trabecular bones of the Swiss population. Results show that plutonium has a retention half time of 40 +/- 14 years. In contrast Sr-90 has a shorter retention half time of 13.5 +/- 1.0 years. Moreover Sr-90 retention half time in vertebrae is shown to be linked to the retention half time in food and other environmental compartments. These findings demonstrate that the renewal of the vertebrae through calcium homeostatic control is faster for Sr-90 excretion than for plutonium excretion. The precise determination of the retention half time of plutonium in the skeleton will improve the biokinetic model of plutonium metabolism in humans. (C) 2010 Elsevier Ltd. All rights reserved.

Effect of Nitrogen and Potassium Rates on Early Development of Macaw Palm

ABSTRACT The economic exploitation of macaw palm [Acrocomia aculeate(Jacq.) Lodd. ex Mart.] is currently in transition, from extractivism to agricultural cultivation, thus requiring studies on the fertilization of the crop. This study evaluated the response of three genotypes of macaw palm to increasing rates of nitrogen and potassium, grown in the field until the 2nd year and to establish reference contents of mineral nutrients in the leaf. The experiment was a split-plot randomized block design with five main treatments (N and K rates) and three secondary treatments (genotypes), with three replications, each plot containing three plants. Plant height, leaf number, vigor, and nutrient contents in leaf tissues were evaluated at the end of 2nd year of cultivation. Differential responses were observed among genotypes, indicating that some genotypes are more efficient in the use of mineral inputs. There was a differentiated and positive response to increasing side-dressed N and K rates in the vegetative development of macaw genotypes until the 2nd year of field cultivation, indicating variability in the species in terms of nutrient use efficiency. The N and K fertilization rate corresponding to 360 g N + 480 g K2O per plant, in four split applications over the two years of cultivation, was insufficient to induce maximum vegetative development in the three macaw genotypes. There was no variation in macro- and micronutrient contents in leaf dry matter of the three macaw genotypes.

Differential role of naïve and memory CD4 T-cell subsets in primary alloresponses.

The T cell response to major histocompatibility complex (MHC) alloantigens occurs via two main pathways. The direct pathway involves the recognition of intact allogeneic MHC:peptide complexes on donor cells and provokes uniquely high frequencies of responsive T cells. The indirect response results from alloantigens being processed like any other protein antigen and presented as peptide by autologous antigen-presenting cells. The frequencies of T cells with indirect allospecificity are orders of magnitude lower and comparable to other peptide-specific responses. In this study, we explored the contributions of naïve and memory CD4(+) T cells to these two pathways. Using an adoptive transfer and skin transplantation model we found that naive and memory CD4(+) T cells, both naturally occurring and induced by sensitization with multiple third-party alloantigens, contributed equally to graft rejection when only the direct pathway was operative. In contrast, the indirect response was predominantly mediated by the naïve subset. Elimination of regulatory CD4(+)CD25(+) T cells enabled memory cells to reject grafts through the indirect pathway, but at a much slower tempo than for naïve cells. These findings have implications for better targeting of immunosuppression to inhibit immediate and later forms of alloimmunity.

Tenascin-W, a new marker of cancer stroma, is elevated in sera of colon and breast cancer patients.

Tenascins are extracellular matrix proteins present during the development of organisms as well as in pathological conditions. Tenascin-W, the fourth and last member of the tenascin family remains the least well-characterized one. Our study aimed to evaluate the potential significance of tenascin-W as cancer biomarker by monitoring its presence in the serum of colorectal and breast cancer patients and its expression in colorectal tumor tissues. To measure serum tenascin-W levels, a sensitive sandwich-ELISA was established. Mean tenascin-W concentration in sera of patients with nonmetastatic colorectal cancer at time of diagnosis was highly increased compared to that of healthy volunteers. A similar tendency was observed for tenascin-C in the same patient cohort. However, the increase was much more striking for tenascin-W. We also detected elevated tenascin-W levels in sera of breast cancer patients. Furthermore, we could show a prominent expression of tenascin-W in extracts from colorectal tumor tissues by immunoblot analysis, whereas tenascin-W was not detectable in the corresponding normal colon mucosa. To confirm the western blot results, we performed immunohistochemistry of frozen sections of the same patients as well as of an additional, independently chosen collection of colorectal cancer tissues. In all cases, similarly to tenascin-C, tenascin-W was detected in the tumor stroma. Our results reveal a clear association between elevated levels of tenascin-W and the presence of cancer. These results warrant further studies to evaluate the potential value of serum and tissue tenascin-W levels as diagnostic, prognostic or monitoring biomarker in colorectal, breast and possibly other solid cancers.

Transplantation of a human mammary carcinoma cell line (BT 20) into nude mice.

Cell suspensions of a human mammary carcinoma cellline (BT 20), wh en injected subcutaneously into nude athymie mice (BALB/c NujNu), produced tumor nodules at the injection site. Subsequent seriai transM plantations also gave rise to neoplastic nodules after latency periods averaging 3 weeks. The nodules displayed morphologie and functional characteristics comparable to those of the original tumor cells. Metastases, however, were not observed in any of the tumor-bearing mice.

Pseudogenes in the ENCODE regions: consensus annotation, analysis of transcription, and evolution.

Arising from either retrotransposition or genomic duplication of functional genes, pseudogenes are "genomic fossils" valuable for exploring the dynamics and evolution of genes and genomes. Pseudogene identification is an important problem in computational genomics, and is also critical for obtaining an accurate picture of a genome's structure and function. However, no consensus computational scheme for defining and detecting pseudogenes has been developed thus far. As part of the ENCyclopedia Of DNA Elements (ENCODE) project, we have compared several distinct pseudogene annotation strategies and found that different approaches and parameters often resulted in rather distinct sets of pseudogenes. We subsequently developed a consensus approach for annotating pseudogenes (derived from protein coding genes) in the ENCODE regions, resulting in 201 pseudogenes, two-thirds of which originated from retrotransposition. A survey of orthologs for these pseudogenes in 28 vertebrate genomes showed that a significant fraction ( approximately 80%) of the processed pseudogenes are primate-specific sequences, highlighting the increasing retrotransposition activity in primates. Analysis of sequence conservation and variation also demonstrated that most pseudogenes evolve neutrally, and processed pseudogenes appear to have lost their coding potential immediately or soon after their emergence. In order to explore the functional implication of pseudogene prevalence, we have extensively examined the transcriptional activity of the ENCODE pseudogenes. We performed systematic series of pseudogene-specific RACE analyses. These, together with complementary evidence derived from tiling microarrays and high throughput sequencing, demonstrated that at least a fifth of the 201 pseudogenes are transcribed in one or more cell lines or tissues.

Role of FGF and Wnt pathways in endoderm patterning and pancreas organogenesis

Summary Between gastrulation and gut tube formation, the endoderm becomes regionally specified along the anterior-posterior axis. An early sign of patterning is the expression of organ-specific genes in restricted endoderm domains. We studied the role of the fibroblast growth factor (FGF) and Wnt pathways in the establishment of the antero-posterior (A-P) axis domains. Here we report the first evidence that graded FGF4-mediated signaling establishes gut tube domains along the A-P axis in vivo from gastrulation to somitogenesis. At gastrulation, FGF4 may act cooperatively with Wnts, since both of them affect the gut tube patterning by promoting posterior and inhibiting anterior endoderm cell fate. The activity of the Wnt pathway is however time restricted, since. it does not affect patterning at somitogenesis. Our experiments point to a global mechanism that coordinates the A-P patterning of all three primary germ layers. Soon after regionalization of the gut tube, morphogenetic evidences of organogenesis appear. We focused our attention on one of these organs, the pancreas. We report a comprehensive investigation of the activity and the role of the Wnt pathway in pancreas organogenesis. We have used two mouse reporter lines to monitor canonical Wnt-pathway activity during development and after birth and demonstrate activity in early pancreatic bud, endocrine cells and in the mesenchyme. We have specifically deleted the ß-catenin .gene, a key component of the Wnt pathway, in the epithelium of the pancreas and duodenum using Pdxl -Cre mice. In agreement with Wnt pathway activity in pancreatic endocrine cells, we find a reduction in endocrine islet numbers. Our study reveals that ß-catenin deletion also affects cells in which Wnt pathway activity is not detected. Indeed, ß-catenin mutant cells have a competitive disadvantage during development that also' affects the exocrine compartment. Moreover, the conditional KO mice develop acute edematous pancreatitis perinatally due to the disruption of the epithelial structure of acini. These effects are likely to be due to the function of ß-catenin at the membrane. Résumé Entre la gastrulation et la formation du tube digestif, l'endoderme est progressivement régionalisé le long de l'axe antéropostérieur (A-P). Un des premiers signes de cette régionalisation est l'expression de gènes spécifiques à certains organes dans une région restreinte. Nous avons étudié l'implication des voies de signalisation FGF et Wnt dans l'établissement de la régionalisation A-P. Nous rapportons les premières preuves que FGF4 établit la ségrégation des domaines de l'endoderme le long de l'axe A-P in vivo de la gastrulation à la somitogenèse. Cette activité peut être menée en collaboration avec les Wnts, puisque ceux-ci influencent aussi l'endoderme en inhibant le destin antérieur et en induisant le destin postérieur des cellules. Cette activité des Wnts est perdue à la somitogenèse. Nos expériences démontrent une régionalisation coordonnée des trois feuillets germinaux le long de l'axe A-P. Peu après la régionalisation, les premiers signes morphologiques de l'organogenèse apparaissent. Nous nous sommes intéressés au rôle des Wnts dans un des dérivés de l'endoderme : le pancréas. Nous avons utilisés deux lignés de souris rapportrices de l'activité de la voie canonique des Wnts, qui montrent une activité dans le bourgeon précoce du pancréas avant la différentiation, puis plus tard dans les cellules endocrines et le mésenchyme. Nous avons utilisé la souris transgénique Pdxl -Cre pour inactiver spécifiquement le gène de la ß-caténine, un intermédiaire de la voie des Wnts, dans la région pancréatique. En accord avec l'activité de la voie de signalisation Wnt, la perte de la ßcaténine conduit à une réduction du nombre de cellules endocrines. De plus certaines cellules qui ne montrent aucune activité de la voie Wnt sont aussi affectées. En effet, les cellules ayant perdu la ß-caténine ont un désavantage compétitif face aux cellules sauvages dans un environnement mosaïque. Cette compétition résulte en l'absence de cellules déplétées en ßcaténine chez l'adulte. De plus, vers la naissance, les animaux déficients pour la ß-caténine développent une pancréatite aiguë due à la destruction de l'architecture des acini. Ceci est probablement aux fonctions d'adhésion de la ß-caténine à la membrane.

Early diagenesis of bone and tooth apatite in fluvial and marine settings: Constraints from combined oxygen isotope, nitrogen and REE analysis

Fossil bones and teeth of Late Pleistocene terrestrial mammals from Rhine River gravels (RS) and the North Sea (NS), that have been exposed to chemically and isotopically distinct diagenetic fluids (fresh water versus seawater), were investigated to study the effects of early diagenesis on biogenic apatite. Changes in phosphate oxygen isotopic composition (delta O-18(PO4)), nitrogen content (wt.% N) and rare earth element (REE) concentrations were measured along profiles within bones that have not been completely fossilized, and in skeletal tissues (bone, dentine, enamel) with different susceptibilities to diagenetic alteration. Early diagenetic changes of elemental and isotopic compositions of apatite in fossil bone are related to the loss of the stabilizing collagen matrix. The REE concentration is negatively correlated with the nitrogen content, and therefore the amount of collagen provides a sensitive proxy for early diagenetic alteration. REE patterns of RS and NS bones indicate initial fossilization in a fresh water fluid with similar REE compositions. Bones from both settings have nearly collagen-free, REE-, U-, F- and Sr-enriched altered outer rims, while the collagen-bearing bone compacta in the central part often display early diagenetic pyrite void-fillings. However, NS bones exposed to Holocene seawater have outer rim delta O-18(PO4) values that are 1.1 to 2.6 parts per thousand higher compared to the central part of the same bones (delta O-18(PO4) = 18.2 +/- 0.9 parts per thousand, n = 19). Surprisingly, even the collagen-rich bone compacta with low REE contents and apatite crystallinity seems altered, as NS tooth enamel (delta O-18(PO4) =15.0 +/- 0.3 parts per thousand, n=4) has about 3%. lower delta O-18(PO4) values, values that are also similar to those of enamel from RS teeth. Therefore, REE concentration, N content and apatite crystallinity are in this case only poor proxies for the alteration of delta O-18(PO4) values. Seawater exposure of a few years up to 8 kyr can change the delta O-18(PO4) values of the bone apatite by > 3 parts per thousand. Therefore, bones fossilized in marine settings must be treated with caution for palaeoclimatic reconstructions. However, enamel seems to preserve pristine delta O-18(PO4) values on this time scale. Using species-specific calibrations for modern mammals, a mean delta O-18(H2O) value can be reconstructed for Late Pleistocene mammalian drinking water of around -9.2 +/- 0.5 parts per thousand, which is similar to that of Late Pleistocene groundwater from central Europe. (c) 2008 Elsevier B.V. All rights reserved.