Taking the diet of cows into consideration in designing payments to reduce enteric methane emissions on dairy farms.

Enteric fermentation from dairy cows is a major source of methane. Significantly and rapidly reducing those emissions would be a powerful lever to mitigate climate change. For a given productivity level, introducing fodder with high sources of n-3 content, such as grass or linseed, in the feed ration of dairy cows both improves the milk nutritional profile and reduces enteric methane emissions per liter. Changing cows' diet may represent additional costs for dairy farmers and calls for the implementation of payments for environmental services to support the transition. This paper analyzes 2 design elements influencing the effectiveness of a payment conditioned toward the reduction of enteric methane emissions: (1) the choice of emission indicator capturing the effect of farmers' practices, and (2) the payment amount relative to the additional milk production costs incurred. Using representative farm-level economic data from the French farm accountancy data network, we compare enteric methane emissions per liter of milk calculated with an Intergovernmental Panel on Climate Change Tier 2 method, to baseline emissions from a Tier 3 method accounting for diet effects. We also quantify the additional milk production costs of integrating more grass in the fodder systems by estimating variable cost functions for different dairy systems in France. Our results show the relevance of using an emission indicator sensitive to diet effects, and that the significance and direction of the additional costs for producing milk with a diet containing more grass differ according to the production basin and the current share of grasslands in the fodder crop rotation. We emphasize the importance of developing payments for environmental services with well-defined environmental indicators accounting for the technical problems addressed, and the need to better characterize heterogeneous funding requirements for supporting a large-scale adoption of more environment-friendly practices by farmers.

Review: Associations among goods, impacts and ecosystem services provided by livestock farming.

Livestock is a major driver in most rural landscapes and economics, but it also polarises debate over its environmental impacts, animal welfare and human health. Conversely, the various services that livestock farming systems provide to society are often overlooked and have rarely been quantified. The aim of analysing bundles of services is to chart the coexistence and interactions between the various services and impacts provided by livestock farming, and to identify sets of ecosystem services (ES) that appear together repeatedly across sites and through time. We review three types of approaches that analyse associations among impacts and services from local to global scales: (i) detecting ES associations at system or landscape scale, (ii) identifying and mapping bundles of ES and impacts and (iii) exploring potential drivers using prospective scenarios. At a local scale, farming practices interact with landscape heterogeneity in a multi-scale process to shape grassland biodiversity and ES. Production and various ES provided by grasslands to farmers, such as soil fertility, biological regulations and erosion control, benefit to some extent from the functional diversity of grassland species, and length of pasture phase in the crop rotation. Mapping ES from the landscape up to the EU-wide scale reveals a frequent trade-off between livestock production on one side and regulating and cultural services on the other. Maps allow the identification of target areas with higher ecological value or greater sensitivity to risks. Using two key factors (livestock density and the proportion of permanent grassland within utilised agricultural area), we identified six types of European livestock production areas characterised by contrasted bundles of services and impacts. Livestock management also appeared to be a key driver of bundles of services in prospective scenarios. These scenarios simulate a breakaway from current production, legislation (e.g. the use of food waste to fatten pigs) and consumption trends (e.g. halving animal protein consumption across Europe). Overall, strategies that combine a reduction of inputs, of the use of crops from arable land to feed livestock, of food waste and of meat consumption deliver a more sustainable food future. Livestock as part of this sustainable future requires further enhancement, quantification and communication of the services provided by livestock farming to society, which calls for the following: (i) a better targeting of public support, (ii) more precise quantification of bundles of services and (iii) better information to consumers and assessment of their willingness to pay for these services.

The JNK binding domain of islet-brain 1 inhibits IL-1 induced JNK activity and apoptosis but not the transcription of key proapoptotic or protective genes in insulin-secreting cell lines.

The stress-activated protein kinase c-Jun NH2-terminal kinase (JNK) is a central signal for interleukin-1beta (IL-1beta)-induced apoptosis in insulin-producing beta-cells. The cell-permeable peptide inhibitor of JNK (JNKI1), that introduces the JNK binding domain (JBD) of the scaffold protein islet-brain 1 (IB1) inside cells, effectively prevents beta-cell death caused by this cytokine. To define the molecular targets of JNK involved in cytokine-induced beta-cell apoptosis we investigated whether JNKI1 or stable expression of JBD affected the expression of selected pro- and anti-apoptotic genes induced in rat (RIN-5AH-T2B) and mouse (betaTC3) insulinoma cells exposed to IL-1beta. Inhibition of JNK significantly reduced phosphorylation of the specific JNK substrate c-Jun (p<0.05), IL-1beta-induced apoptosis (p<0.001), and IL-1beta-mediated c-fos gene expression. However, neither JNKI1 nor JBD did influence IL-1beta-induced NO synthesis or iNOS expression or the transcription of the genes encoding mitochondrial manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase rho (GSTrho), heat shock protein (HSP) 70, IL-1beta-converting enzyme (ICE), caspase-3, apoptosis-inducing factor (AIF), Bcl-2 or Bcl-xL. We suggest that the anti-apoptotic effect of JNK inhibition by JBD is independent of the transcription of major pro- and anti-apoptotic genes, but may be exerted at the translational or posttranslational level.

Destruction of conditional insulinoma cell lines in NOD mice: a role for autoimmunity.

AIMS/HYPOTHESIS: betaTC-tet (H2(k)) is a conditional insulinoma cell line derived from transgenic mice expressing a tetracycline-regulated oncogene. Transgenic expression of several proteins implicated in the apoptotic pathways increase the resistance of betaTC-tet cells in vitro. We tested in vivo the sensitivity of the cells to rejection and the protective effect of genetic alterations in NOD mice. METHODS: betaTC-tet cells and genetically engineered lines expressing Bcl-2 (CDM3D), a dominant negative mutant of MyD88 or SOCS-1 were transplanted in diabetic female NOD mice or in male NOD mice with diabetes induced by high-dose streptozotocin. Survival of functional cell grafts in NOD-scid mice was also analyzed after transfer of splenocytes from diabetic NOD mice. Autoreactive T-cell hybridomas and splenocytes from diabetic NOD mice were stimulated by betaTC-tet cells. RESULTS: betaTC-tet cells and genetically engineered cell lines were all similarly rejected in diabetic NOD mice and in NOD-scid mice after splenocyte transfer. In 3- to 6-week-old male NOD mice treated with high-dose streptozotocin, the cells temporarily survived, in contrast with C57BL/6 mice treated with high-dose streptozotocin (indefinite survival) and untreated 3- to 6-week-old male NOD mice (rejection). The protective effect of high-dose streptozotocin was lost in older male NOD mice. betaTC-tet cells did not stimulate autoreactive T-cell hybridomas, but induced IL-2 secretion by splenocytes from diabetic NOD mice. CONCLUSION/INTERPRETATION: The autoimmune process seems to play an important role in the destruction of betaTC-tet cells in NOD mice. Genetic manipulations intended at increasing the resistance of beta cells were inefficient. Similar approaches should be tested in vivo as well as in vitro. High dose streptozotocin influences immune rejection and should be used with caution.

SOCS-1 protein prevents Janus Kinase/STAT-dependent inhibition of beta cell insulin gene transcription and secretion in response to interferon-gamma.

In the pathogenesis of type I diabetes mellitus, activated leukocytes infiltrate pancreatic islets and induce beta cell dysfunction and destruction. Interferon (IFN)-gamma, tumor necrosis factor-alpha and interleukin (IL)-1 beta play important, although not completely defined, roles in these mechanisms. Here, using the highly differentiated beta Tc-Tet insulin-secreting cell line, we showed that IFN-gamma dose- and time-dependently suppressed insulin synthesis and glucose-stimulated secretion. As described previously IFN-gamma, in combination with IL-1 beta, also induces inducible NO synthase expression and apoptosis (Dupraz, P., Cottet, S., Hamburger, F., Dolci, W., Felley-Bosco, E., and Thorens, B. (2000) J. Biol. Chem. 275, 37672--37678). To assess the role of the Janus kinase/signal transducer and activator of transcription (STAT) pathway in IFN-gamma intracellular signaling, we stably overexpressed SOCS-1 (suppressor of cytokine signaling-1) in the beta cell line. We demonstrated that SOCS-1 suppressed cytokine-induced STAT-1 phosphorylation and increased cellular accumulation. This was accompanied by a suppression of the effect of IFN-gamma on: (i) reduction in insulin promoter-luciferase reporter gene transcription, (ii) decrease in insulin mRNA and peptide content, and (iii) suppression of glucose-stimulated insulin secretion. Furthermore, SOCS-1 also suppressed the cellular effects that require the combined presence of IL-1 beta and IFN-gamma: induction of nitric oxide production and apoptosis. Together our data demonstrate that IFN-gamma is responsible for the cytokine-induced defect in insulin gene expression and secretion and that this effect can be completely blocked by constitutive inhibition of the Janus kinase/STAT pathway.

Engineering tolerance into transplanted beta cell lines.

In this paper we explore the possibility of improving, by genetic engineering, the resistance of insulin-secreting cells to the metabolic and inflammatory stresses that are anticipated to limit their function and survival when encapsulated and transplanted in a type 1 diabetic environment. We show that transfer of the Bcl-2 antiapoptotic gene, and of genes specifically interfering with cytokine intracellular signaling pathways, greatly improves resistance of the cells to metabolic limitations and inflammatory stresses.

Dominant negative MyD88 proteins inhibit interleukin-1beta /interferon-gamma -mediated induction of nuclear factor kappa B-dependent nitrite production and apoptosis in beta cells.

Insulin-dependent diabetes mellitus is an autoimmune disease in which pancreatic islet beta cells are destroyed by a combination of immunological and inflammatory mechanisms. In particular, cytokine-induced production of nitric oxide has been shown to correlate with beta cell apoptosis and/or inhibition of insulin secretion. In the present study, we investigated whether the interleukin (IL)-1beta intracellular signal transduction pathway could be blocked by overexpression of dominant negative forms of the IL-1 receptor interacting protein MyD88. We show that overexpression of the Toll domain or the lpr mutant of MyD88 in betaTc-Tet cells decreased nuclear factor kappaB (NF-kappaB) activation upon IL-1beta and IL-1beta/interferon (IFN)-gamma stimulation. Inducible nitric oxide synthase mRNA accumulation and nitrite production, which required the simultaneous presence of IL-1beta and IFN-gamma, were also suppressed by approximately 70%, and these cells were more resistant to cytokine-induced apoptosis as compared with parental cells. The decrease in glucose-stimulated insulin secretion induced by IL-1beta and IFN-gamma was however not prevented. This was because these dysfunctions were induced by IFN-gamma alone, which decreased cellular insulin content and stimulated insulin exocytosis. These results demonstrate that IL-1beta is involved in inducible nitric oxide synthase gene expression and induction of apoptosis in mouse beta cells but does not contribute to impaired glucose-stimulated insulin secretion. Furthermore, our data show that IL-1beta cellular actions can be blocked by expression of MyD88 dominant negative proteins and, finally, that cytokine-induced beta cell secretory dysfunctions are due to the action of IFN-gamma.

Glucose uptake, utilization, and signaling in GLUT2-null islets.

We previously reported that pancreatic islet beta-cells from GLUT2-null mice lost the first phase but preserved the second phase of glucose-stimulated insulin secretion (GSIS). Furthermore, we showed that the remaining secretory activity required glucose uptake and metabolism because it can be blocked by inhibition of oxidative phosphorylation. Here, we extend these previous studies by analyzing, in GLUT2-null islets, glucose transporter isoforms and glucokinase expression and by measuring glucose usage, GSIS, and glucose-stimulated insulin mRNA biosynthesis. We show that in the absence of GLUT2, no compensatory expression of either GLUT1 or GLUT3 is observed and that glucokinase is expressed at normal levels. Glucose usage by isolated islets was increased between 1 and 6 mmol/l glucose but was not further increased between 6 and 20 mmol/l glucose. Parallel GSIS measurements showed that insulin secretion was not stimulated between 2.8 and 6 mmol/l glucose but was increased by >4-fold between 6 and 20 mmol/l glucose. Stimulation by glucose of total protein and insulin biosynthesis was also markedly impaired in the absence of GLUT2. Finally, we re-expressed GLUT2 in GLUT2-null beta-cells using recombinant lentiviruses and demonstrated a restoration of normal GSIS. Together, these data show that in the absence of GLUT2, glucose can still be taken up by beta-cells, albeit at a low rate, and that this transport activity is unlikely to be attributed to GLUT1 or GLUT3. This uptake activity, however, is limiting for normal glucose utilization and signaling to secretion and translation. These data further demonstrate the key role of GLUT2 in murine beta-cells for glucose signaling to insulin secretion and biosynthesis.

Lentivirus-mediated Bcl-2 expression in betaTC-tet cells improves resistance to hypoxia and cytokine-induced apoptosis while preserving in vitro and in vivo control of insulin secretion.

betaTC-tet cells are conditionally immortalized pancreatic beta cells which can confer long-term correction of hyperglycemia when transplanted in syngeneic streptozocin diabetic mice. The use of these cells for control of type I diabetes in humans will require their encapsulation and transplantation in non-native sites where relative hypoxia and cytokines may threaten their survival. In this study we genetically engineered betaTC-tet cells with the anti-apoptotic gene Bcl-2 using new lentiviral vectors and showed that it protected this cell line against apoptosis induced by hypoxia, staurosporine and a mixture of cytokines (IL-1beta, IFN-gamma and TNF-alpha). We further demonstrated that Bcl-2 expression permitted growth at higher cell density and with shorter doubling time. Expression of Bcl-2, however, did not inter- fere either with the intrinsic mechanism of growth arrest present in the betaTC-tet cells or with their normal glucose dose-dependent insulin secretory activity. Furthermore, Bcl-2 expressing betaTC-tet cells retained their capacity to secrete insulin under mild hypoxia. Finally, transplantation of these cells under the kidney capsule of streptozocin diabetic C3H mice corrected hyperglycemia for several months. These results demonstrate that the murine betaTC-tet cell line can be genetically modified to improve its resistance against different stress-induced apoptosis while preserving its normal physiological function. These modified cells represent an improved source for cell transplantation therapy of type I diabetes.

The murine AIDS virus Gag precursor protein binds to the SH3 domain of c-Abl.

The Pr60gag protein of the murine AIDS (MAIDS) defective virus promotes the proliferation of the infected target B cells and is responsible for inducing a severe immunodeficiency disease. Using the yeast two-hybrid system, we identified the SH3 domain of c-Abl as interacting with the proline-rich p12 domain of Pr60gag. The two proteins were shown to associate in vitro and in vivo in MAIDS virus-infected B cells. Overexpression of Pr60(gag) in these cells led to a detectable increase of the levels of c-Abl protein and to its translocation at the membrane. These results suggest that this viral protein serves as a docking site for signaling molecules and that c-Abl may be involved in the proliferation of infected B cells.

Analysis of deletions and thermosensitive mutations in Rous sarcoma virus gag protein p10.

Rous sarcoma virus protein p10 is a gag component of the virion present in stoichiometric amount but of unknown function. To characterize this protein, a series of mutants of p10 with linker insertions or deletions was generated by site-directed mutagenesis of a cloned proviral DNA. The deletions and two of the linkers insertions, which disrupted proline pairs, reduced the yield of virus particles upon transfection. These two linker insertion mutants were moreover thermosensitive for this phenotype, producing fewer virus particles at 41 degrees C than at 36 degrees C. Examination of the intracellular viral proteins demonstrated that for all mutants, the amount of gag precursor was similar to the wild-type level. Moreover, the amount of mature gag CA that could be detected by this analysis was similar between each of the mutants and the wild type. This finding suggests that the transport of gag to the membrane and the initial stages of maturation were not affected by the mutations. The virus particles contained normal amounts of active reverse transcriptase, showing that the gag-pol polyprotein was incorporated and cleaved properly. Viral RNA was quantitatively and qualitatively similar in mutant and wild-type virions. However, the infectivity of the mutants virions differed; one of the thermosensitive linker insertions that had no effect on particle production at 36 degrees C was nevertheless noninfectious at that temperature. Together, these data suggest that the p10 protein is involved in a late steps of virus maturation, possibly budding, and perhaps also in an early event of viral infection.

Specificity of Rous sarcoma virus nucleocapsid protein in genomic RNA packaging.

Site-directed mutagenesis has shown that the nucleocapsid (NC) protein of Rous sarcoma virus (RSV) is required for packaging and dimerization of viral RNA. However, it has not been possible to demonstrate, in vivo or in vitro, specific binding of viral RNA sequences by NC. To determine whether specific packaging of viral RNA is mediated by NC in vivo, we have constructed RSV mutants carrying sequences of Moloney murine leukemia virus (MoMuLV). Either the NC coding region alone, the psi RNA packaging sequence, or both the NC and psi sequences of MoMuLV were substituted for the corresponding regions of a full-length RSV clone to yield chimeric plasmid pAPrcMNC, pAPrc psi M, or pAPrcM psi M, respectively. In addition, a mutant of RSV in which the NC is completely deleted was tested as a control. Upon transfection, each of the chimeric mutants produced viral particles containing processed core proteins but were noninfectious. Thus, MoMuLV NC can replace RSV NC functionally in the assembly and release of mature virions but not in infectivity. Surprisingly, the full-deletion mutant showed a strong block in virus release, suggesting that NC is involved in virus assembly. Mutant PrcMNC packaged 50- to 100-fold less RSV RNA than did the wild type; in cotransfection experiments, MoMuLV RNA was preferentially packaged. This result suggests that the specific recognition of viral RNA during virus assembly involves, at least in part, the NC protein.

Point mutations in the proximal Cys-His box of Rous sarcoma virus nucleocapsid protein.

To extend our previous studies of the function of the Cys-His box of Rous sarcoma virus NC protein, we have constructed a series of point mutations of the conserved or nonconserved amino acids of the proximal Cys-His box and a one-amino-acid deletion. All mutants were characterized for production of viral proteins and particles, for packaging and maturation of viral RNA, for reverse transcriptase activity, and for infectivity. Our results indicated the following. (i) Mutations affecting the strictly conserved amino acids cysteine 21, cysteine 24, and histidine 29 were lethal; only the mutant His-29----Pro was still able to package viral RNA, most of it in an immature form. (ii) Mutation of the highly conserved glycine 28 to valine reduced viral RNA packaging by 90% and infectivity 30-fold, whereas mutant Gly-28----Ala was fully infectious. This suggests a steric hindrance limit at this position. (iii) Shortening the distance between cysteine 24 and histidine 29 by deleting one amino acid abolished the maturation of viral RNA and yielded noninfectious particles. (iv) Substitution of tyrosine 22 by serine lowered viral RNA packaging efficiency and yielded particles that were 400-fold less infectious; double mutant Tyr-22Thr-23----SerSer had the same infectivity as Tyr-22----Ser, whereas mutant Thr-23----Ser was fully infectious. (v) Changing glutamine 33 to a charged glutamate residue did not affect virus infectivity. Similarities and differences between our avian mutants and those in murine retroviruses are discussed.

Ryanodine binding to sarcoplasmic reticulum membrane; comparison between cardiac and skeletal muscle.

[3H]Ryanodine binding to skeletal muscle and cardiac sarcoplasmic reticulum (SR) vesicles was compared under experimental conditions known to inhibit or stimulate Ca2+ release. In the skeletal muscle SR, ryanodine binds to a single class of high-affinity sites (Kd of 11.3 nM). In cardiac SR vesicles, more than one class of binding sites is observed (Kd values of 3.6 and 28.1 nM). Ryanodine binding to skeletal muscle SR vesicles requires high concentrations of NaCl, whereas binding of the drug to cardiac SR is only slightly influenced by ionic strength. In the presence of 5'-adenylyl imidodiphosphate (p[NH]ppA), increased pH, and micromolar concentration of Ca2+ (which all induce Ca2+ release from SR) binding of ryanodine to SR is significantly increased in skeletal muscle, while being unchanged in cardiac muscle. Ryanodine binding to skeletal but not to cardiac muscle SR is inhibited in the presence of high Ca2+ or Mg2+ concentrations (all known to inhibit Ca2+ release from skeletal muscle SR). Ruthenium red or dicyclohexylcarbodiimide modification of cardiac and skeletal muscle SR inhibit Ca2+ release and ryanodine binding in both skeletal and cardiac membranes. These results indicate that significant differences exist in the properties of ryanodine binding to skeletal or cardiac muscle SR. Our data suggest that ryanodine binds preferably to site(s) which are accessible only when the Ca2+ release channel is in the open state.