Rapid induction of pluripotency genes after exposure of human somatic cells to mouse ES cell extracts

The expression of 4 pluripotency genes (Oct4, Sox2, c-Myc and Klf4) in mouse embryonic fibroblasts can reprogramme them to a pluripotent state. We have investigated the expression of these pluripotency genes when human somatic 293T cells are permeabilized and incubated in extracts of mouse embryonic stem (ES) cells. Expression of all 4 genes was induced over 1–8 h. Gene expression was associated with loss of repressive histone H3 modifications and increased recruitment of RNA polymerase II at the promoters. Lamin A/C, which is typically found only in differentiated cells, was also removed from the nuclei. When 293T cells were returned to culture after exposure to ES cell extract, the expression of the pluripotency genes continued to rise over the following 48 h of culture, suggesting that long-term reprogramming of gene expression had been induced. This provides a methodology for studying the de-differentiation of somatic cells that can potentially lead to an efficient way of reprogramming somatic cells to a pluripotent state without genetically altering them.     

Structure and synthesis of polyisoprenoids used in N-glycosylation across the three domains of life

N-linked protein glycosylation was originally thought to be specific to eukaryotes, but evidence of this post-translational modification has now been discovered across all domains of life: Eucarya, Bacteria, and Archaea. In all cases, the glycans are first assembled in a step-wise manner on a polyisoprenoid carrier lipid. At some stage of lipid-linked oligosaccharide synthesis, the glycan is flipped across a membrane. Subsequently, the completed glycan is transferred to specific asparagine residues on the protein of interest. Interestingly, though the N-glycosylation pathway seems to be conserved, the biosynthetic pathways of the polyisoprenoid carriers, the specific structures of the carriers, and the glycan residues added to the carriers vary widely. In this review we will elucidate how organisms in each basic domain of life synthesize the polyisoprenoids that they utilize for N-linked glycosylation and briefly discuss the subsequent modifications of the lipid to generate a lipid-linked oligosaccharide.     

Characterization of a thy1.2 GFP transgenic mouse reveals a tissue-specific organization of the spinal dorsal horn

In this study, transgenic mice in which membrane-linked enhanced green fluorescent protein (mGFP) is expressed from the Thy1.2 promoter were used. In these mice, a subpopulation of small to medium sized DRG neurons double stained for IB4 but not for CGRP. Most of the peripheral terminals traversed the dermis and ramify within the epidermis and form superficial terminals. Within the spinal cord, these afferents terminated exclusively within the substantia gelatinosa (SG). A second fibre type in the skin also expressed mGFP, and formed club-shaped endings towards the bases of hairs. Injury to the sciatic nerve resulted in mGFP loss from the SG ipsilateral to the nerve injury, but also in the corresponding region contralaterally. Together, these findings reveal the specificity of connectivity of a defined subpopulation of DRG sensory neurons innervating the epidermis and this will facilitate analysis of their physiological functions.     

Insights into the function of PsbR protein in Arabidopsis thaliana

The functional state of the Photosystem (PS) II complex in Arabidopsis psbR T-DNA insertion mutant was studied. The ΔPsbR thylakoids showed about 34% less oxygen evolution than WT, which correlates with the amounts of PSII estimated from Y_D^ox radical EPR signal. The increased time constant of the slow phase of flash fluorescence (FF)-relaxation and upshift in the peak position of the main TL-bands, both in the presence and in the absence of DCMU, confirmed that the S₂Q_A⁻ and S₂Q_B⁻ charge recombinations were stabilized in ΔPsbR thylakoids. Furthermore, the higher amount of dark oxidized Cyt-b559 and the increased proportion of fluorescence, which did not decay during the 100s time span of the measurement thus indicating higher amount of Y_D⁺Q_A⁻ recombination, pointed to the donor side modifications in ΔPsbR. EPR measurements revealed that S₁-to-S₂-transition and S₂-state multiline signal were not affected by mutation. The fast phase of the FF-relaxation in the absence of DCMU was significantly slowed down with concomitant decrease in the relative amplitude of this phase, indicating a modification in Q_A to Q_B electron transfer in ΔPsbR thylakoids. It is concluded that the lack of the PsbR protein modifies both the donor and the acceptor side of the PSII complex.     

Action mechanism of tachyplesin I and effects of PEGylation

PEGylation of protein and peptide drugs is frequently used to improve in vivo efficacy. We investigated the action mechanism of tachyplesin I, a membrane-acting cyclic antimicrobial peptide from Tachypleus tridentatus and the effects of PEGylation on the mechanism. The PEGylated peptide induced the leakage of calcein from egg yolk l-α-phosphatidylglycerol/egg yolk l-α-phosphatidylcholine large unilamellar vesicles similarly to the parent peptide. Both peptides induced lipid flip-flop coupled to leakage and was translocated into the inner leaflet of the bilayer, indicating that tachyplesin I forms a toroidal pore and that PEGylation did not alter the basic mechanism of membrane permeabilization of the parent peptide. Despite their similar activities against model membranes, the peptides showed very different biological activities. The cytotoxicity of tachyplesin I was greatly reduced by PEGylation, although the antimicrobial activity was significantly weakened. We investigated the enhancement of the permeability of inner membranes induced by the peptides. Our results suggested that outer membranes and peptidoglycan layers play an inhibitory role in the permeation of the PEG moiety. Furthermore, a reduction in DNA binding by PEGylation may also contribute to the weak activity of the PEGylated peptide.     

Invariant chain modulates HLA class II protein recycling and peptide presentation in nonprofessional antigen presenting cells

The expression of MHC class II molecules and the invariant chain (Ii) chaperone, is coordinately regulated in professional antigen presenting cells (APC). Ii facilitates class II subunit folding as well as transit and retention in mature endosomal compartments rich in antigenic peptides in these APC. Yet, in nonprofessional APC such as tumors, fibroblasts and endocrine tissues, the expression of class II subunits and Ii may be uncoupled. Studies of nonprofessional APC indicate class II molecules access antigenic peptides by distinct, but poorly defined pathways in the absence of Ii. Here, investigations demonstrate that nonprofessional APC such as human fibroblasts lacking Ii internalize antigenic peptides prior to the binding of these ligands to recycling class II molecules. By contrast, fibroblast lines expressing Ii favor exogenous peptides binding directly to cell surface class II molecules without a need for ligand internalization. Endocytosis of class II molecules was enhanced in cells lacking Ii compared with Ii-expressing APC. These results suggest enhanced reliance on the endocytic recycling pathway for functional class II presentation in nonprofessional APC.     

Association of dystrobrevin and regulatory subunit of protein kinase A: a new role for dystrobrevin as a scaffold for signaling proteins

The dystrophin-related and -associated protein dystrobrevin is a component of the dystrophin-associated protein complex, which directly links the cytoskeleton to the extracellular matrix. It is now thought that this complex also serves as a dynamic scaffold for signaling proteins, and dystrobrevin may play a role in this context. Since dystrobrevin involvement in signaling pathways seems to be dependent on its interaction with other proteins, we sought new insights and performed a two-hybrid screen of a mouse brain cDNA library using beta-dystrobrevin, the isoform expressed in non-muscle tissues, as bait. Among the positive clones characterized after the screen, one encodes the regulatory subunit RIalpha of the cAMP-dependent protein kinase A (PKA). We confirmed the interaction by in vitro and in vivo association assays, and mapped the binding site of beta-dystrobrevin on RIalpha to the amino-terminal region encompassing the dimerization/docking domain of PKA regulatory subunit. We also found that the domain of interaction for RIalpha is contained in the amino-terminal region of beta-dystrobrevin. We obtained evidence that beta-dystrobrevin also interacts directly with RIIbeta, and that not only beta-dystrobrevin but also alpha-dystrobrevin interacts with PKA regulatory subunits. We show that both alpha and beta-dystrobrevin are specific phosphorylation substrates for PKA and that protein phosphatase 2A (PP2A) is associated with dystrobrevins. Our results suggest a new role for dystrobrevin as a scaffold protein that may play a role in different cellular processes involving PKA signaling.     

Microbial production of 4-hydroxybenzylidene acetone, the direct precursor of raspberry ketone

AIMS: To investigate the enzymatic aldol reaction between acetone as a donor and 4-hydroxybenzaldehyde as a receptor to generate 4-(4-hydroxyphenyl)-but-3-ene-2-one or 4-hydroxybenzylidene acetone, the direct precursor of 4-(4-hydroxyphenyl)-butan-2-one or raspberry ketone, using different species of filamentous fungi and bacteria. METHODS AND RESULTS: Different classes of micro-organisms were tested in a medium containing mainly acetone and 4-hydoxybenzaldehyde. Of the micro-organisms tested, only bacteria were able to synthesize significant amounts of 4-hydroxybenzylidene acetone, ranging from 15 to 160 mg l(-1) after 21 h of bioconversion, as a function of the bacteria tested. CONCLUSIONS: The biological production of 4-hydroxybenzylidene acetone has been described with bacteria possessing 2-deoxyribose-5-phosphate aldolase (DERA, EC 4.1.2.4). This result suggests that DERA is involved in the catalytic aldolization of precursors for the production of 4-hydroxybenzylidene acetone. SIGNIFICANCE AND IMPACT OF THE STUDY: Raspberry ketone or frambinone represents a total market value of between euro6 million and euro10 million. The possibility of producing its direct precursor through a simple process using bacteria is of considerable interest to the flavour market and the food industry as a whole. This paper broadens the spectrum for the use of aldolase to achieve the biological synthesis of compounds of interest.     

Entamoeba histolytica: ADP-ribosylation of secreted glyceraldehyde-3-phosphate dehydrogenase

In addition to its classic glycolytic role, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been implicated in many activities unrelated to glycolysis, such as membrane fusion, binding to host proteins and signal transduction. GAPDH can be the target of several modifications that allow incorporation to membranes and possible regulation of its activity; among these modifications is mono-ADP-ribosylation. This post-translational modification is important for the regulation of many cellular processes and is the mechanism of action of several bacterial toxins. In a previous study, we observed the extracellular ADP-ribosylation of a 37-kDa ameba protein. We report here that GAPDH and cysteine synthase A are the main ADP-ribosylated proteins in Entamoeba histolytica extracellular medium, GAPDH is secreted from ameba at 37 degrees C in a time-dependent manner, and its enzymatic activity is not inhibited by ADP-ribosylation. Extracellular GAPDH from ameba may play an important role in the survival of this human pathogen or in interaction with host molecules, as occurs in other organisms.