Estimating spatially distributed SOC sequestration potentials of sustainable land management practices in Ethiopia.

The sustainable land management program (SLMP) of Ethiopia aims to improve livelihoods and create resilient communities and landscape to climate change. Soil organic carbon (SOC) sequestration is one of the key co-benefits of the SLMP. The objective of this study was to estimate the spatial dynamics of SOC in 2010 and 2018 (before and after SLMP) and identify the SOC sequestration hotspots at landscape scale in four selected SLMP watersheds in the Ethiopian highlands. The specific objectives were to: 1) comparatively evaluate SOC sequestration estimation model building strategies using either a single watershed, a combined dataset from all watersheds, and leave-one-watershed-out using Random Forest (RF) model; 2) map SOC stock of 2010 and 2018 to estimate amount of SOC sequestration and potential; 3) evaluate the impacts of SLM practices on SOC in four SLMP watersheds. A total of 397 auger composite samples from the topsoil (0-20 cm depth) were collected in 2010, and the same number of samples were collected from the same locations in 2018. We used simple statistics to assess the SOC change between the two periods, and machine learning models to predict SOC stock spatially. The study showed that statistically significant variation (P < 0.05) of SOC was observed between the two years in two watersheds (Gafera and Adi Tsegora) whereas the differences were not significant in the other two watersheds (Yesir and Azugashuba). Comparative analysis of model-setups shows that a combined dataset from all the four watersheds to train and test RF outperform the other two strategies (a single watershed alone and a leave-one-watershed-out to train and test RF) during the testing dataset. Thus, this approach was used to predict SOC stock before (2010) and after (2018) land management interventions and to derive the SOC sequestration maps. We estimated the sequestrated, achievable and target level of SOC stock spatially in the four watersheds. We assessed the impact of SLM practices, specifically bunds, terraces, biological and various forms of tillage practices on SOC using partial dependency algorithms of prediction models. No tillage (NT) increased SOC in all watersheds. The combination of physical and biological interventions ("bunds + vegetations" or "terraces + vegetations") resulted in the highest SOC stock, followed by the biological intervention. The achievable SOC stock analysis showed that further SOC stock sequestration of up to 13.7 Mg C ha--1 may be possible in the Adi Tsegora, 15.8 Mg C ha-1 in Gafera, 33.2 Mg C ha-1 in Azuga suba and 34.7 Mg C ha-1 in Yesir watersheds.

Predictions of Cu, Zn, and Cd Concentrations in Soil Using Portable X-Ray Fluorescence Measurements.

Portable X-ray fluorescence (PXRF) measurements on 1520 soil samples were used to create national prediction models for copper (Cu), zinc (Zn), and cadmium (Cd) concentrations in agricultural soil. The models were validated at both national and farm scales. Multiple linear regression (MLR), random forest (RF), and multivariate adaptive regression spline (MARS) models were created and compared. National scale cross-validation of the models gave the following R2 values for predictions of Cu (R2 = 0.63), Zn (R2 = 0.92), and Cd (R2 = 0.70) concentrations. Independent validation at the farm scale revealed that Zn predictions were relatively successful regardless of the model used (R2 > 0.90), showing that a simple MLR model can be sufficient for certain predictions. However, predictions at the farm scale revealed that the non-linear models, especially MARS, were more accurate than MLR for Cu (R2 = 0.94) and Cd (R2 = 0.80). These results show that multivariate modelling can compensate for some of the shortcomings of the PXRF device (e.g., high limits of detection for certain elements and some elements not being directly measurable), making PXRF sensors capable of predicting elemental concentrations in soil at comparable levels of accuracy to conventional laboratory analyses.

Performance Evaluation of Proximal Sensors for Soil Assessment in Smallholder Farms in Embu County, Kenya.

Four proximal soil sensors were tested at four smallholder farms in Embu County, Kenya: a portable X-ray fluorescence sensor (PXRF), a mobile phone application for soil color determination by photography, a dual-depth electromagnetic induction (EMI) sensor, and a LED-based soil optical reflectance sensor. Measurements were made at 32-43 locations at each site. Topsoil samples were analyzed for plant-available nutrients (N, P, K, Mg, Ca, S, B, Mn, Zn, Cu, and Fe), pH, total nitrogen (TN) and total carbon (TC), soil texture, cation exchange capacity (CEC), and exchangeable aluminum (Al). Multivariate prediction models of each of the lab-analyzed soil properties were parameterized for 576 sensor-variable combinations. Prediction models for K, N, Ca and S, B, Zn, Mn, Fe, TC, Al, and CEC met the setup criteria for functional, robust, and accurate models. The PXRF sensor was the sensor most often included in successful models. We concluded that the combination of a PXRF and a portable soil reflectance sensor is a promising combination of handheld soil sensors for the development of in situ soil assessments as a field-based alternative or complement to laboratory measurements.

Acidic preparations of lysed platelets upregulate proliferative pathways in osteoblast-like cells as demonstrated by genome-wide microarray analysis.

Platelets contain numerous growth factors essential for wound and fracture healing. We investigated the gene expression in human osteoblast-like cells stimulated with lysed platelets prepared in acidic, neutral, or alkaline buffers. Lysed platelets prepared in buffers at pH 5.4, 7.4, and 7.9, were added after neutralization to hFOB 1.19 cells. Genome-wide microarray analysis was performed using the Affymetrix GeneChip 7G Scanner. Biometric, cluster, and pathway analyses were performed with GeneSpring GX. Biometric analyses demonstrated that 53 genes were differentially regulated (p ≤ 0.005, ≥2-fold increase). Pathway analysis revealed 10 significant pathways of which eight are common ones regulating bone formation and cancer growth. Eleven genes were selected for quantitative real-time polymerase chain reaction (PCR) based on the microarray analysis of the lysed platelets prepared in the pH 5.4 experiments. In conclusion, acidic preparations of lysed platelet concentrates release factors essential for cell proliferation and particularly cell metabolism under hypoxic conditions. The genetic response from these factors was dominated by genes associated with the same pathways observed in bone formation and cancer growth. Activation of TGF-ß in the acidic preparation could be a stimulatory key factor of cell proliferation. These results support the hypothesis that acidification of platelets modifies the stimulatory response of mesenchymal cells in vitro, which is analogous with the observed milieu of a low pH present in wound and fracture sites, as well as in growing tumors.

Soil resources influence spatial patterns of denitrifying communities at scales compatible with land management.

Knowing spatial patterns of functional microbial guilds can increase our understanding of the relationships between microbial community ecology and ecosystem functions. Using geostatistical modeling to map spatial patterns, we explored the distribution of the community structure, size, and activity of one functional group in N cycling, the denitrifiers, in relation to 23 soil parameters over a 44-ha farm divided into one organic and one integrated crop production system. The denitrifiers were targeted by the nirS and nirK genes that encode the two mutually exclusive types of nitrite reductases, the cd(1) heme-type and copper reductases, respectively. The spatial pattern of the denitrification activity genes was reflected by the maps of the abundances of nir genes. For the community structure, only the maps of the nirS community were related to the activity. The activity was correlated with nitrate and dissolved organic nitrogen and carbon, whereas the gene pools for denitrification, in terms of size and composition, were influenced by the soil structure. For the nirS community, pH and soil nutrients were also important in shaping the community. The only unique parameter related to the nirK community was the soil Cu content. However, the spatial pattern of the nirK denitrifiers corresponded to the division of the farm into the two cropping systems. The different community patterns, together with the spatial distribution of the nirS/nirK abundance ratio, suggest habitat selection on the nirS- and nirK-type denitrifiers. Our findings constitute a first step in identifying niches for denitrifiers at scales relevant to land management.

Distinct parts of leukotriene C(4) synthase interact with 5-lipoxygenase and 5-lipoxygenase activating protein.

Leukotriene C(4) is a potent inflammatory mediator formed from arachidonic acid and glutathione. 5-Lipoxygenase (5-LO), 5-lipoxygenase activating protein (FLAP) and leukotriene C(4) synthase (LTC(4)S) participate in its biosynthesis. We report evidence that LTC(4)S interacts in vitro with both FLAP and 5-LO and that these interactions involve distinct parts of LTC(4)S. FLAP bound to the N-terminal part/first hydrophobic region of LTC(4)S. This part did not bind 5-LO which bound to the second hydrophilic loop of LTC(4)S. Fluorescent FLAP- and LTC(4)S-fusion proteins co-localized at the nuclear envelope. Furthermore, GFP-FLAP and GFP-LTC(4)S co-localized with a fluorescent ER marker. In resting HEK293/T or COS-7 cells GFP-5-LO was found mainly in the nuclear matrix. Upon stimulation with calcium ionophore, GFP-5-LO translocated to the nuclear envelope allowing it to interact with FLAP and LTC(4)S. Direct interaction of 5-LO and LTC(4)S in ionophore-stimulated (but not un-stimulated) cells was demonstrated by BRET using GFP-5-LO and Rluc-LTC(4)S.

Fetal hepatic expression of 5-lipoxygenase activating protein is confined to colonizing hematopoietic cells.

Leukotriene C(4) is a potent inflammatory mediator formed from arachidonic acid and glutathione. 5-Lipoxygenase (5-LO), 5-lipoxygenase activating protein (FLAP) and leukotriene C(4) synthase (LTC(4)S) participate in its biosynthesis. We report evidence from insitu hybridization experiments that FLAP mRNA is abundantly expressed in fetal mouse liver from e11.5 until delivery. In contrast very little or no FLAP mRNA was detected in adult liver. The fetal expression in liver was not uniform but occurred in patches. Cells from e15.5 livers were fractionated by fluorescence activated cell sorting into hepatocytes and other CD45(-) cells and CD45(+) hematopoietic cells. The latter were further separated into immature (Lin(-)) and mature (Lin(+)) cells and analyzed for FLAP mRNA content by quantitative RT-PCR. FLAP mRNA expression was confined to CD45(+) cells and the mature cells had approximately 4-fold higher FLAP mRNA levels compared to the immature cells.

Leukotriene C4 synthase promoter driven expression of GFP reveals cell specificity.

Leukotriene C(4) synthase is a key enzyme in leukotriene biosynthesis. Its gene has been cloned and mapped to mouse chromosome 11. Expression occurs in cells of myeloid origin and also in the choroid plexus, the hypothalamus and the medial eminence of mouse brain. In this study a vector that expresses enhanced green fluorescent protein (eGFP) under the control of the mouse leukotriene C(4) synthase promoter was constructed and used to study promoter activity in different cell lines. Specific eGFP expression was observed in human monocytic leukemia (THP-1) and rat basophilic leukemia (RBL-1) myeloid cells which both express leukotriene C(4) synthase, but not in human embryonic kidney (HEK293/T) epithelial cells which do not express this enzyme. In the myeloid cells, but not in the epithelial cells, we observed that the leukotriene C(4) synthase promoter activity was stimulated by 12-O-tetradecanoylphorbol-13-acetate and all-trans-retinoic acid. In contrast dimethyl sulfoxide did not affect promoter activity.

Redox-signaling transmitted in trans to neighboring cells by melanoma-derived TNF-containing exosomes.

Hydrogen peroxide is known to be involved in redox signaling pathways that regulate normal processes and disease progression, including cytokine signaling, oxidative stress, and cancer. In studies on immune surveillance against cancer, hydrogen peroxide was found to disrupt cytotoxic T-cell function, thus contributing to tumor escape. In this study, secretion of TNF-containing vesicles of rab9+ endosomal origin, termed exosomes, was investigated using GFP-TNF constructs. We observed a polarized intracellular trafficking and apical secretion of TNF-positive nanovesicles. Cell-to-cell transfer of TNF was observed in exosomes in real-time microscopy, occurring separate from the melanin/melanosome compartment. Exosomes were prepared by ultracentrifugation or immunoisolation on anti-beta2-microglobulin magnetic beads. TNF as well as TNF receptors 1 and 2 were present in the exosomes as determined by Western blot, flow cytometry, and deconvolution microscopy. The functional significance of melanoma-derived exosomes was established by their signaling competence with ability to generate significantly higher ROS levels in T cells compared with sham exosomes (P=0.0006). In conclusion, we report here, for the first time, that TNF is found in tumor cell-derived exosomes and that these exosomes transmit redox signaling in trans to neighboring cells. The results are of importance for a better understanding of tumor escape mechanisms.

Predicting deoxynivalenol in oats under conditions representing Scandinavian production regions.

Deoxynivalenol (DON) in cereals, produced by Fusarium fungi, cause poisoning in humans and animals. Fusarium infections in cereals are favoured by humid conditions. Host species are susceptible mainly during the anthesis stage. Infections are also positively correlated with a regional history of Fusarium infections, frequent cereal production and non-tillage field management practices. Here, previously developed process-based models based on relative air humidity, rain and temperature conditions, Fusarium sporulation, host phenology and mycelium growth in host tissue were adapted and tested on oats. Model outputs were used to calculate risk indices. Statistical multivariate models, where independent variables were constructed from weather data, were also developed. Regressions of the risk indices obtained against DON concentrations in field experiments on oats in Sweden and Norway 2012-14 had coefficient of determination values (R2) between 0.84 and 0.88. Regressions of the same indices against DON concentrations in oat samples averaged for 11 × 11 km grids in farmers' fields in Sweden 2012-14 resulted in R2 values between 0.27 and 0.41 for randomly selected grids and between 0.31 and 0.62 for grids with average DON concentration above 1000 µg kg-1 grain in the previous year. When data from all three years were evaluated together, a cross-validated statistical partial least squares model resulted in R2 = 0.70 and a standard error of cross-validation (SECV) = 522 µg kg-1 grain for the period 1 April-28 August in the construction of independent variables and R2 = 0.54 and SECV = 647 µg kg-1 grain for 1 April-23 June. Factors that were not accounted for in this study probably explain large parts of the variation in DON among samples and make further model development necessary before these models can be used practically. DON prediction in oats could potentially be improved by combining weather-based risk index outputs with agronomic factors.

Platelet-induced growth of human fibroblasts is associated with an increased expression of 5-lipoxygenase.

Proliferation of fibroblasts is vital for adequate wound healing but is probably also involved in different hyperproliferative disorders such as atherosclerosis and cancer. The regeneration of tissue usually starts with coagulation, involving release of mitogenic and inflammatory factors from activated platelets. This study focuses on the role of eicosanoids in the proliferative effects of platelets on human fibroblasts. We show that the phospholipase A (2) inhibitor 7,7-dimethyl-5,8-eicosadienoic acid (DMDA), the combined cyclooxygenase (COX) and lipoxygenase (LOX) inhibitor 5,8,11,14-eicosatetraynoic acid (ETYA) and the LOX inhibitor 5,8,11-eicosatriynoic acid (ETI) block the platelet-induced proliferation of serum starved subconfluent human fibroblasts. Anti-proliferative effects were also obtained by specific inhibition of 5-LOX with 5,6-dehydro arachidonic acid (5,6-dAA), whereas the 12-LOX inhibitor cinnamyl-3,4-dihydroxy- a -cyanocinnamate (CDC) did not affect the platelet-stimulated growth of fibroblasts. The expression of 5-LOX was analyzed by reverse-transcriptase-mediated PCR (RT-PCR), Western blotting and HPLC. 5-LOX message and protein was detected in fibroblasts but not in platelets. Incubation with platelets markedly increased, already after one hour, the expression of 5-LOX in the fibroblast culture. The increased 5-LOX activity was associated with an elevated level of the 5-LOX metabolite 5-hydroxyeicosatetraenoic acid (5-HETE) reaching its maximum after 1 - 2 hours of co-incubation of fibroblasts and platelets. The 5-HETE production was reduced by the inhibitors DMDA, ETYA and ETI. In conclusion, this study suggests that platelet-stimulated proliferation of fibroblasts is mediated by an increased 5-LOX activity, which supports recent findings indicating a crucial role for this enzyme in proliferative disorders such as atherosclerosis.

PPAR-gamma response element activity in intact primary human adipocytes: effects of fatty acids.

OBJECTIVE: We studied the activity and regulation of the peroxisome proliferator-activated receptor-gamma response element (PPRE) in primary human adipocytes. METHODS: We transfected primary human adipocytes with a plasmid-encoding firefly luciferase cDNA under control of a PPRE from the acyl-coenzyme A oxidase gene by using our newly developed electroporation-based method. Several fatty acids were added to the fat cells to study potential activation of peroxisome proliferator-activated receptor-gamma. RESULTS: Cells responded maximally to 5 microM of rosiglitazone at a 5.1 +/- 1.4-fold over basal increase in luciferase activity. There was a positive correlation between body mass index and the response to 5 microM of rosiglitazone (r = 0.36, P = 0.03). Patients with type 2 diabetes had similar basal PPRE activity but responded more strongly to 5 microM of rosiglitazone than did non-diabetic subjects (10.2 +/- 5-fold and 5.4 +/- 1-fold over basal increase, respectively, P < 0.0001). Among saturated fatty acids, lauric acid was without effect, but 10 microM of palmitic or stearic acid increased PPRE activity 20% to 35% above basal levels. Monounsaturated palmitoleic acid at 1 microM induced a PPRE transcriptional activity that corresponded to half the therapeutic levels of rosiglitazone. CONCLUSION: Adipocytes from obese subjects and patients with type 2 diabetes responded particularly strongly to the effect of rosiglitazone on PPRE. Because fatty acids in the diet can affect the transcriptional activity of peroxisome proliferator-activated receptor-gamma over decades, the stimulation induced by stearic and palmitoleic acids can affect insulin sensitivity and, hence, cardiovascular morbidity and mortality in humans.

Leukotriene C4 synthase homo-oligomers detected in living cells by bioluminescence resonance energy transfer.

Leukotrienes (LTs) are biologically active compounds derived from arachidonic acid which have important pathophysiological roles in asthma and inflammation. The cysteinyl leukotriene LTC(4) and its metabolites LTD(4) and LTE(4) stimulate bronchoconstriction, airway mucous formation and generalized edema formation. LTC(4) is formed by addition of glutathione to LTA(4), catalyzed by the integral membrane protein, LTC(4) synthase (LTCS). We now report the use of bioluminescence resonance energy transfer (BRET) to demonstrate that LTCS forms homo-oligomers in living cells. Fusion proteins of LTCS and Renilla luciferase (Rluc) and a variant of green fluorescent protein (GFP), respectively, were prepared. High BRET signals were recorded in transiently transfected human embryonic kidney (HEK 293) cells co-expressing Rluc/LTCS and GFP/LTCS. Homo-oligomer formation in living cells was verified by co-transfection of a plasmid expressing non-chimeric LTCS. This resulted in dose-dependent attenuation of the BRET signal. Additional evidence for oligomer formation was obtained in cell-free assays using glutathione S-transferase (GST) pull-down assay. To map interaction domains for oligomerization, GFP/LTCS fusion proteins were prepared with truncated variants of LTCS. The results obtained identified a C-terminal domain (amino acids 114-150) sufficient for oligomerization of LTCS. Another, centrally located, interaction domain appeared to exist between amino acids 57-88. The functional significance of LTCS homo-oligomer formation is currently being investigated.

Novel prostaglandin D(2)-derived activators of peroxisome proliferator-activated receptor-gamma are formed in macrophage cell cultures.

Incubation of RAW 264.7 murine macrophages with 9,15-dihydroxy-11-oxo-, (5Z,9alpha,13E,15(S))-Prosta-5,13-dien-1-oic acid [prostaglandin D(2) (PGD(2))] induced formation of considerable peroxisome proliferator-activated receptor-gamma (PPARgamma) activity [Nature 391 (1998) 79]. Because PGD(2) itself is a poor PPARgamma ligand, we incubated RAW 264.7 macrophage cultures with prostaglandin D(2) for 24 h and studied the ability of the metabolites formed to activate PPARgamma. PGD(2) products were extracted and fractionated by reverse phase high-performance liquid chromatography. Chemical identification was achieved by UV spectroscopy, gas-liquid chromatography/mass spectrometry and chemical syntheses of reference compounds. PGD(2) was converted to eight products, six of which were identified. Ligand-induced interaction of PPARgamma with steroid receptor coactivator-1 was determined by glutathione-S-transferase pull-down assays and PPARgamma activation was investigated by transient transfection of RAW 264.7 macrophages. In addition to the previously known ligand 11-oxo-(5Z,9,12E,14Z)-Prosta-5,9,12,14-tetraen-1-oic acid (15-deoxy-delta(12,14)-PGJ(2)), a novel PPARgamma ligand and activator viz. 9-hydroxy-11-oxo-, (5Z,9alpha,12E,14Z)-Prosta-5,12,14-trien-1-oic acid (15-deoxy-delta(12,14)-PGD(2)) was identified. The biological significance of these results is currently under investigation.

An LXXLL motif in nuclear receptor corepressor mediates ligand-induced repression of the thyroid stimulating hormone-beta gene.

Nuclear receptor corepressor (N-CoR) regulates gene expression through interaction with DNA-bound nuclear receptors, recruiting multicomponent repressor complexes to the sites of target genes. We recently reported the presence of an LXXLL motif in N-CoR, and showed that this motif interacts in vitro and in vivo with retinoic acid receptor alpha (RARalpha) and thyroid hormone receptor beta (TRbeta). Transient transfection experiments now suggest that TRbeta and N-CoR act synergistically and may both be required for ligand-induced repression from the negative TR response element in the thyroid stimulating hormone-beta (TSHbeta) gene promoter. Mutation of the LXXLL motif in N-CoR abolished ligand-induced repression at this response element. Furthermore, in vitro binding of N-CoR to a complex between TRbeta and the negative TR response element was strictly ligand-dependent. We conclude that N-CoR and TRbeta cooperate in the regulation of the TSHbeta gene and that the ligand-dependent repression is mediated by the LXXLL motif in N-CoR.

Secondary lymphoid organ chemokines are elevated in the cerebrospinal fluid during central nervous system inflammation.

Secondary lymphoid organ chemokines have been implicated in chronic inflammation. Their expression in the central nervous system (CNS) has not been studied. Here, levels of secondary lymphoid organ chemokines CCL19 (Exodus-3, MIP-3beta), CCL21 (Exodus-2, 6Ckine, SLC) and CXCL12 (SDF-1alpha) were analysed by ELISA in cerebrospinal fluid (CSF) and plasma from patients with multiple sclerosis (MS); acute optic neuritis (ON) with oligoclonal IgG in the CSF (i.e., first bout of MS); acute ON without oligoclonal IgG (non-MS-type ON); other inflammatory neurological diseases (OIND); and non-inflammatory neurological diseases (NIND). NIND CSF contained CCL19 and CXCL12, while CCL21 was not detected. Intrathecal production of CCL19 and CCL21 was elevated in MS, MS-type ON, and OIND, but not in non-MS-type ON. In MS, CSF levels of CCL19 weakly correlated with CSF cell counts. Intrathecal production of CXCL12 was elevated only in OIND. The role of elevated CCL19 and CCL21 in MS could be retention of mature dendritic cells (DC) in the CNS, recruitment of nai;ve T cells and activated B cells, as well as de novo formation of secondary lymphoid structures in MS plaques.

Effect of glatiramer acetate (Copaxone) on CD4+CD25high T regulatory cells and their IL-10 production in multiple sclerosis.

CD4(+)CD25(high) T regulatory (Tr) cells, representing high IL-2 receptor alpha chain expressing cells, have been shown to inhibit proliferation and cytokine secretion by CD4(+) T cells that are assumed to represent important effector cells in auto-aggressive immunity. Tr cells may therefore be considered of importance in the pathogenesis of multiple sclerosis (MS). Glatiramer acetate (GA; Copaxone) is approved as a disease-modulating agent that ameliorates the course of MS. The goal of this study was to examine in vitro effects of GA on Tr cells from MS patients subgrouped according to treatment without or with disease-modulating drugs, and healthy controls (HC). Three-colour flow cytometry was used to investigate in vitro influence of GA, and of the encephalitogenic myelin basic protein (MBP) peptide 83-89 as control, on the blood Tr cell proportion and on their functionally important cell surface molecules CD45RO, CD69, CD95 and HLA-DR, and on intracellular CTLA-4 and IL-10. Irrespective of exposure to GA or MBP((83-99)), levels of blood Tr cells expressing HLA-DR remained low in untreated MS patients and HC compared to the three treated MS patient groups. In vitro exposure to GA resulted in elevated levels of IL-10 producing Tr cells in all MS patient groups irrespective of receiving treatment as well as in HC. Exposure to GA or MBP((83-99)) had no effects on levels of Tr cells expressing other above-mentioned molecules. We conclude that GA induces elevated IL-10 production by Tr cells that is uniform and independent of ongoing MS treatment with IFN-beta or GA or IFN-beta+GA.

Expression of a mutant IRS inhibits metabolic and mitogenic signalling of insulin in human adipocytes.

Adipose tissue is a primary target of insulin, but knowledge about insulin signalling in human adipocytes is limited. We developed an electroporation technique for transfection of primary human adipocytes with a transfection efficiency of 15% +/- 5 (mean +/- S.D.). Human adipocytes were co-transfected with a mutant of IRS-3 (all four potential PI3-kinase binding motifs mutated: IRS-3F4) and HA-tagged protein kinase B (HA-PKB/Akt). HA-PKB/Akt was immunoprecipitated from cell lysates with anti-HA antibodies, resolved with SDS-PAGE, and immunoblotted with phospho-specific antibodies. We found that IRS-3F4 blocked insulin stimulation of HA-PKB/Akt phosphorylation and in further analyses also translocation of recombinant HA-tagged glucose transporter to the plasma membrane. IRS-3F4 also blocked insulin-induced activation of the transcription factor Elk-1. Our results demonstrate the critical importance of IRS for metabolic as well as mitogenic signalling by insulin. This method for transfection of primary human adipocytes will be useful for studying insulin signalling in human adipocytes with molecular biological techniques.

The nuclear receptor corepressor (N-CoR) modulates basal and activated transcription of genes controlled by retinoic acid.

Variation in cell morphology and function is caused by differentiation. In myeloid differentiation, retinoid signaling, acting through heterodimers consisting of retinoic acid receptor and retinoid X receptor (RAR/RXR) plays a crucial part. The RAR/RXR heterodimers bind to naturally occurring response elements in the promoter regions of target genes, deciding whether the gene is to be transcribed or not. In the absence of the RAR-specific ligand all trans retinoic acid, RAR/RXR heterodimers are associated with the nuclear receptor corepressor N-CoR or the related SMRT. Here we show, using Western, far-Western and Northern blot techniques, that when the human monocytic cell line THP-1 is allowed to differentiate into macrophage-like cells the expression of N-CoR is down-regulated both at the protein and at the mRNA level. To investigate how this affects the transcriptional activity of retinoic acid response element (RARE)-controlled genes, we performed transient transfection experiments in THP-1 and CV-1 cells. The results indicate that N-CoR functions not merely as a repressor of basal transcription, but rather as a modulator of both basal and ligand-activated transcription of genes controlled by RAR/RXR heterodimers in a dose-dependent manner.

Functional analyses of an LXXLL motif in nuclear receptor corepressor (N-CoR).

Transcriptional repression is a major regulatory mechanism in cell differentiation, organogenesis, and oncogenesis. Two repressors of ligand-dependent transcription factors, nuclear receptor corepressor (N-CoR) and the related protein SMRT were identified as a silencing mediator for thyroid hormone receptor beta and as a silencing mediator for retinoic acid and thyroid hormone receptors, respectively. Nuclear receptor coactivators such as steroid receptor coactivator-1 (SRC-1) contain multiple LXXLL motifs, which are essential and sufficient for its ligand-dependent interaction with nuclear receptors. N-CoR also has an LXXLL motif, located between repressor domains 1 and 2, and conserved between mouse and man. In contrast, SMRT lacks this motif. This paper describes functional implications of the LXXLL motif in N-CoR. A 57-amino acid portion of N-CoR containing the LDNLL sequence (N-CoR(LDNLL)) fused to GST interacted with retinoic acid receptor alpha (RARalpha) and thyroid hormone receptor beta (TRbeta) in vitro. Similarly, [(35)S-methionine]N-CoR(LDNLL) interacted with a RARalpha fusion protein. N-CoR(LDNLL) also bound to RARalpha in vivo as determined in mammalian one-hybrid system in transfected CV-1 cells and by two-hybrid assays in bacteria. The interaction with RARalpha in vitro and in vivo was specific as determined by mutation of the sequence LDNLL to LDNAA. Our data suggest that the LDNLL motif in N-CoR has functional significance because it mediates interaction with nuclear receptors such as RARalpha and TRbeta.