Functional characterisation of the regulation of CAAT enhancer binding protein alpha by GSK-3 phosphorylation of Threonines 222/226

Background  

Glycogen Synthase Kinase-3 (GSK3) activity is repressed following insulin treatment of cells. Pharmacological inhibition of GSK3 mimics the effect of insulin on Phosphoenolpyruvate Carboxykinase (PEPCK), Glucose-6 Phosphatase (G6Pase) and IGF binding protein-1 (IGFBP1) gene expression. CAAT/enhancer binding protein alpha (C/EBPα) regulates these gene promoters in liver and is phosphorylated on two residues (T222/T226) by GSK3, although the functional outcome of the phosphorylation has not been established. We aimed to establish whether CEBPα is a link between GSK3 and these gene promoters.     

Adeno-associated virus Rep78 binds to E2-responsive element 1 of bovine papillomavirus type 1

Adeno-associated virus type-2 (AAV-2) is a helper-dependent parvovirus that has been implicated in the inhibition of replication and oncogenic transformation of bovine papillomavirus type-1 (BPV-1) and other transforming DNA viruses. Previous studies have suggested that the Rep78 protein of AAV-2 is a key player mediating this effect. In this report we have analyzed the effect of AAV-2 Rep78 protein on the regulation of gene expression of a reporter gene under the control of the long control region (LCR) of BPV-1. Our results show that Rep78 is capable of down-regulating the promoter activity of the LCR in vivo in tissue culture cells. Inhibition of LCR activity in vivo suggested the need for Rep78 to bind to a region of the LCR promoter spanning the E2-responsive elements of BPV-1. This observation was further confirmed in vitro with gel shift assays showing specific binding of Rep78 to DNA oligonucleotides containing E2-responsive element 1 (E2RE1) sequences of BPV-1 LCR. Our results expand the understanding of the mechanism of trans-regulation mediated by Rep78 and involving this protein and DNA sequences with complex secondary structure.     

Long-range RNA-RNA interaction between the 5' nontranslated region and the core-coding sequences of hepatitis C virus modulates the IRES-dependent translation

Hepatitis C virus (HCV) is a positive-sense RNA virus approximately 9600 bases long. An internal ribosomal entry site (IRES) spans the 5' nontranslated region, which is the most conserved and highly structured region of the HCV genome. In this study, we demonstrate that nucleotides 428-442 of the HCV core-coding sequence anneal to nucleotides 24-38 of the 5'NTR, and that this RNA-RNA interaction modulates IRES-dependent translation in rabbit reticulocyte lysate and in HepG2 cells. The inclusion of the core-coding sequence (nucleotides 428-442) significantly suppressed the translational efficiency directed by HCV IRES in dicistronic reporter systems, and this suppression was relieved by site-directed mutations that blocked the long-range interaction between nucleotides 24-38 and 428-442. These findings suggest that the long-range interaction between the HCV 5'NTR and the core-coding nucleotide sequence down-regulate cap-independent translation via HCV IRES. The modulation of protein synthesis by long-range RNA-RNA interaction may play a role in the regulation of viral gene expression.     

Regional pulmonary blood flow in dogs by 4D-X-ray CT.

ECG-triggered computed tomography (CT) was used during passage of iodinated contrast to determine regional pulmonary blood flow (PBF) in anesthetized prone/supine dogs. PBF was evaluated as a function of height within the lung (supine and prone) as a function of various normalization methods: raw unit volume data (PBFraw) or PBF normalized to regional fraction air (PBFair), fractional non-air (PBFgm), or relative number of alveoli (PBFalv). The coefficient of variation of PBFraw, PBFair, PBFalv, and PBFgm ranged between 30 and 50% in both lungs and both body postures. The position of maximal flow along the height of the lung (MFP) was calculated for PBFraw, PBFair, PBFalv, and PBFgm. Only PBFgm showed a significantly different MFP height supine vs. prone (whole lung: 2.60 +/- 1.08 cm supine vs. 5.08 +/- 1.61 cm prone, P < 0.01). Mean slopes (ml/min/gm water content/cm) of PBFgm were steeper supine vs. prone in the right (RL) but not left lung (LL) (RL: -0.65 +/- 0.29 supine vs. -0.26 +/- 0.25 prone, P < 0.02; LL: -0.47 +/- 0.21 supine vs. -0.32 +/- 0.26 prone, P > 0.10). Mean slopes of PBFgm vs. vertical lung height were not different prone vs. supine above this vertical height of MFP (VMFP), but PBFgm slopes were steeper in the supine position below the VMFP in the RL. We conclude that PBFgm distribution was posture dependent in RL but not LL. Support of the heart may play a role. We demonstrate that normalization factors can lead to differing attributions of gravitational effects on PBF heterogeneity.     

Multiple duplications of yeast hexose transport genes in response to selection in a glucose-limited environment

When microbes evolve in a continuous, nutrient-limited environment, natural selection can be predicted to favor genetic changes that give cells greater access to limiting substrate. We analyzed a population of baker's yeast that underwent 450 generations of glucose-limited growth. Relative to the strain used as the inoculum, the predominant cell type at the end of this experiment sustains growth at significantly lower steady-state glucose concentrations and demonstrates markedly enhanced cell yield per mole glucose, significantly enhanced high-affinity glucose transport, and greater relative fitness in pairwise competition. These changes are correlated with increased levels of mRNA hybridizing to probe generated from the hexose transport locus HXT6. Further analysis of the evolved strain reveals the existence of multiple tandem duplications involving two highly similar, high- affinity hexose transport loci, HXT6 and HXT7. Selection appears to have favored changes that result in the formation of more than three chimeric genes derived from the upstream promoter of the HXT7 gene and the coding sequence of HXT6. We propose a genetic mechanism to account for these changes and speculate as to their adaptive significance in the context of gene duplication as a common response of microorganisms to nutrient limitation.      

Acceptor specificity of the human leukocyte alpha3 fucosyltransferase: role of FucT-VII in the generation of selectin ligands

The alpha3 fucosyltransferase, FucT-VII, is one of the key glycosyltransferases involved in the biosynthesis of the sialyl Lewis X (sLex) antigen on human leukocytes. The sialyl Lewis X antigen (NeuAcalpha(2-3)Galbeta(1-4)[Fucalpha(1-3)]GlcNAc-R) is an essential component of the recruitment of leukocytes to sites of inflammation, mediating the primary interaction between circulating leukocytes and activated endothelium. In order to characterize the enzymatic properties of the leukocyte alpha3 fucosyltransferase FucT-VII, the enzyme has been expressed in Trichoplusia ni insect cells. The enzyme is capable of synthesizing both sLexand sialyl-dimeric-Lexstructures in vitro , from 3'-sialyl-lacNAc and VIM-2 structures, respectively, with only low levels of fucose transfer observed to neutral or 3'-sulfated acceptors. Studies using fucosylated NeuAcalpha(2-3)-(Galbeta(1- 4)GlcNAc)3-Me acceptors demonstrate that FucT-VII is able to synthesize both di-fucosylated and tri-fucosylated structures from mono- fucosylated precursors, but preferentially fucosylates the distal GlcNAc within a polylactosamine chain. Furthermore, the rate of fucosylation of the internal GlcNAc residues is reduced once fucose has been added to the distal GlcNAc. These results indicate that FucT-VII is capable of generating complex selectin ligands, in vitro , however the order of fucose addition to the lactosamine chain affects the rate of selectin ligand synthesis.      

Role of orthophosphate and other factors in the regulation of starch formation in leaves and isolated chloroplasts

Starch synthesis in leaves was increased by phosphate starvation or by treatments which decreased cytoplasmic orthophosphate levels (such as mannose feeding). Usually less than 30% of the total carbon fixed during CO₂ assimilation was incorporated into starch in spinach (Spinacia oleracea L.), spinach beet (Beta vulgaris), and tobacco (Nicotiana tabacum) leaves.