Comorbidity and genotype modify receptor expression in human alcoholics

Chronic alcohol misuse leads to both widespread and localized damage in human cerebral cortex. The latter, as neuronal loss, is marked in superior frontal cortex (SFC) but milder in primary motor cortex (PMC) and elsewhere. Quantitative morphometry by Harper et al showed that neuronal loss is greater in alcoholics with comorbidity (Wernicke Korsakoff syndrome, liver cirrhosis). Previous work revealed a paradox: the marked differences in GABAA receptor density, pharmacology, and expression between alcoholics without cormorbidity and controls are muted or absent in cirrhotic alcoholics. This concurs with work by the Butterworth group on hepatic encephalopathy cases — most of whom had an alcoholic ætiology — who show only minor differences from controls. Glutamate receptor differences are muted in many autopsy studies, though we have evidence that NMDA site pharmacology may vary in cirrhotic alcoholics. Here we used Real-Time PCR normalized to GAPDH deltaCT to quantify NMDA NR1, NR2A and NR2B subunit expression in SFC and PMC samples obtained at autopsy from alcoholics with and without comorbid cirrhosis and matched controls. Overall subunit transcript expression was signifi cantly lower in alcoholic cirrhotics than in either of the other groups (F2,42 = 12.942, P < 0.001). The effect was most marked for the NR1 subunit; males differed from females, particularly in SFC. The data suggest that if excitotoxicity mediates neuronal loss in SFC, it may be implemented differently: passively in uncomplicated alcoholics, by altered GABAergic transmission; actively in cirrhotic alcoholics, by altered glutamatergic transmission. We also subdivided cases on a panel of genetic markers. Different genotypes interacted with NMDA and GABAA pharmacology and expression. Cirrhotic and uncomplicated alcoholics may differ pathogenically because of inherent characteristics in addition to possible neurotoxic sequelæ to the liver damage.

Measurement and management of genotype-environment interaction (GxE) for the improvement of rainfed lowland rice yield in Cambodia

The magnitude and nature of genotype-by-environment interactions (G×E) for grain yield (GY) and days to flower (DTF) in Cambodia were examined using a random population of 34 genotypes taken from the Cambodian rice improvement program. These genotypes were evaluated in multi-environment trials (MET) conducted across three years (2000 to 2002) and eight locations in the rainfed lowlands. The G×E interaction was partitioned into components attributed to genotype-by-location (G×L), genotype-by-year (G×Y) and genotype-by-location-by-year (G×L×Y) interactions. The G×L×Y interaction was the largest component of variance for GY. The G×L interaction was also significant and comparable in size to the genotypic component (G). The G×Y interaction was small and non significant. A major factor contributing to the large G×L×Y interactions for GY was the genotypic variation for DTF in combination with environmental variation for the timing and intensity of drought. Some of the interactions for GY associated with timing of plant development and exposure to drought were repeatable across the environments enabling the identification of three-target populations of environments (TPE) for consideration in the breeding program. Four genotypes were selected for wide adaptation in the rainfed lowlands in Cambodia.

Rho and smooth muscle cell phenotype

Smooth muscle cell (SMC) phenotypic modulation from the mature ’contractile’ to a less differentiated ’synthetic’ phenotype involves not only altered expression but also a reorganisation of contractile and cytoskeletal proteins. Objective: To investigate the role of RhoA, a known regulator of the actin cytoskeleton, in SMC phenotypic regulation. Methods: Rho transcription (RT-PCR), expression (Western analysis) and activation (membrane translocation or Rho ’pull-down’ assay) was investigated in cultured rabbit aortic SMC during phenotypic modulation, and under the influence of known SM-regulatory proteins (thrombin, heparin and TGF- β). Rho’s effect on cell morphology was examined by transient transfection of ’synthetic’ state SMC with either constitutively active Rho (Val14RhoA) or its inhibitor, C3 transferase. Results: RhoA transcription was elevated in the first 3 days of primary culture, and protein expression peaked at 2 days post-confluence when SMC return to a more ’contractile’ state. However, RhoA showed augmented activation at three time-points in primary culture: the transition point when SMCs enter logarithmic growth and are highly motile, upon reaching quiescence, and when they return to a more ’contractile’ state. Thrombin, heparin and TGF-β activated RhoA in ’synthetic’ state SMCs. Transfection with Val14RhoA caused a dramatic decrease in SMC size and a reorganization of cytoskeletal proteins, reminiscent of the ’contractile’ phenotype. Specific inhibition of endogenous Rho by C3 transferase resulted in an almost complete loss of contractile proteins. Conclusion: These data indicate that Rho is an important determining factor of SMC functional state.

Transcriptome analysis of a respiratory Saccharomyces cerevisiae strain suggests the expression of its phenotype is glucose insensitive and predominantly controlled by Hap4, Cat8 and Mig1

BACKGROUND: We previously described the first respiratory Saccharomyces cerevisiae strain, KOY.TM6*P, by integrating the gene encoding a chimeric hexose transporter, Tm6*, into the genome of an hxt null yeast. Subsequently we transferred this respiratory phenotype in the presence of up to 50 g/L glucose to a yeast strain, V5 hxt1-7Delta, in which only HXT1-7 had been deleted. In this study, we compared the transcriptome of the resultant strain, V5.TM6*P, with that of its wild-type parent, V5, at different glucose concentrations. RESULTS: cDNA array analyses revealed that alterations in gene expression that occur when transitioning from a respiro-fermentative (V5) to a respiratory (V5.TM6*P) strain, are very similar to those in cells undergoing a diauxic shift. We also undertook an analysis of transcription factor binding sites in our dataset by examining previously-published biological data for Hap4 (in complex with Hap2, 3, 5), Cat8 and Mig1, and used this in combination with verified binding consensus sequences to identify genes likely to be regulated by one or more of these. Of the induced genes in our dataset, 77% had binding sites for the Hap complex, with 72% having at least two. In addition, 13% were found to have a binding site for Cat8 and 21% had a binding site for Mig1. Unexpectedly, both the up- and down-regulation of many of the genes in our dataset had a clear glucose dependence in the parent V5 strain that was not present in V5.TM6*P. This indicates that the relief of glucose repression is already operable at much higher glucose concentrations than is widely accepted and suggests that glucose sensing might occur inside the cell. CONCLUSION: Our dataset gives a remarkably complete view of the involvement of genes in the TCA cycle, glyoxylate cycle and respiratory chain in the expression of the phenotype of V5.TM6*P. Furthermore, 88% of the transcriptional response of the induced genes in our dataset can be related to the potential activities of just three proteins: Hap4, Cat8 and Mig1. Overall, our data support genetic remodelling in V5.TM6*P consistent with a respiratory metabolism which is insensitive to external glucose concentrations.

Palmitate induces insulin resistance and a pro-atherogenic phenotype in monocytes

The present study investigated the effect of the two most abundant FFA in plasma – palmitate and oleate – on insulin sensitivity and vascular function (monocyte phenotype and adhesion to endothelium) using in vitro cell culture models and Wistar rats. Palmitate at 300µM for 6h induced insulin resistance in THP-1 monocytes and L6 monocytes. The ceramide synthesis pathway partly accounted for the palmitate-induced insulin resistance in THP-1 monocytes but not for L6 myotubes. Oleate treatment did not induce insulin resistance in either cell type and co-incubation with oleate protected cells from palmitate-induced insulin resistance. Palmitate at 300µN for 24h significantly increased cell surface CD11b and CD36 expression in U937 monocytes. The increase in CD11b and CD36 expression was effectively inhibited by Fumonisin B1, an inhibitor of ceramide synthesis. Oleate treatment did not show any effect on CD11b and CD36 expression and co-incubation with oleate antagonised the effect of palmitate on CD11b and CD36 expression in U937 monocytes. The increase in CD11b expression did not affect U937 monocyte adhesion to ICAM-1. Treating Wistar rats with palmitate for 6h caused a transient delay in glucose disposal and an increase in adhesion of U937 monocytes to the aortic endothelium, particularly at bifurcations. In conclusion, the present study demonstrates that the saturated free fatty acid palmitate induces insulin resistance and a pro-atherogenic phenotype for monocytes, whereas the unsaturated free fatty acid oleate does not. In vivo studies also confirmed that palmitate induces insulin resistance and an increase in monocyte adhesion to aorta.

Does metabolic reprogramming underpin age-associated changes in T cell phenotype and function?

T cells are required for an effective adaptive immune response. The principal function of T cells is to promote efficient removal of foreign material by identifying and mounting a specific response to nonself. A decline in T cell function in aging is thought to contribute to reduced response to infection and vaccination and an increase in autoimmunity. This may in part be due to the age-related decrease in naïve CD4+ T cells and increase in antigen-experienced CD4+ T cells, loss of redox homeostasis, and impaired metabolic switching. Switching between subsets is triggered by the integration of extracellular signals sensed through surface receptors and the activation of discrete intracellular metabolic pathways. This article explores how metabolic programming and loss of redox homeostasis during aging may contribute to age-associated changes in T cell phenotype and function. © 2014 Elsevier Inc.

Maternal periconceptional and gestational low protein diet affects mouse offspring growth, cardiovascular and adipose phenotype at 1 year of age

Human and animal studies have revealed a strong association between periconceptional environmental factors, such as poor maternal diet, and an increased propensity for cardiovascular and metabolic disease in adult offspring. Previously, we reported cardiovascular and physiological effects of maternal low protein diet (LPD) fed during discrete periods of periconceptional development on 6-month-old mouse offspring. Here, we extend the analysis in 1 year aging offspring, evaluating mechanisms regulating growth and adiposity. Isocaloric LPD (9% casein) or normal protein diet (18% casein; NPD) was fed to female MF-1 mice either exclusively during oocyte maturation (for 3.5 days prior to mating; Egg-LPD, Egg-NPD, respectively), throughout gestation (LPD, NPD) or exclusively during preimplantation development (for 3.5 days post mating; Emb-LPD). LPD and Emb-LPD female offspring were significantly lighter and heavier than NPD females respectively for up to 52 weeks. Egg-LPD, LPD and Emb-LPD offspring displayed significantly elevated systolic blood pressure at 52 weeks compared to respective controls (Egg-NPD, NPD). LPD females had significantly reduced inguinal and retroperitoneal fat pad: body weight ratios compared to NPD females. Expression of the insulin receptor (Insr) and insulin-like growth factor I receptor (Igf1r) in retroperitoneal fat was significantly elevated in Emb-LPD females (P&0.05), whilst Emb-LPD males displayed significantly decreased expression of the mitochondrial uncoupling protein 1 (Ucp1) gene compared to NPD offspring. LPD females displayed significantly increased expression of Ucp1 in interscapular brown adipose tissue when compared to NPD offspring. Our results demonstrate that aging offspring body weight, cardiovascular and adiposity homeostasis can be programmed by maternal periconceptional nutrition. These adverse outcomes further exemplify the criticality of dietary behaviour around the time of conception on long-term offspring health. © 2011 Watkins et al.