Rapid fluorescencein situ hybridization on interphasic nuclei to discriminate between homozygous and heterozygous transgenic mice

Homozygous and heterozygous transgenic mice of the Tg152 line overexpressing the human copper/zinc superoxide dismutase (hSOD-1) were rapidly differentiated by fluorescencein situ hybridization (FISH) using intérphase lymphocyte nuclei. We have devised a simple and fast method for preparing interphase nuclei with very small quantities of whole mouse blood, avoiding several steps of the classical FISH technique. Lymphocyte separation and cell culture were not required. This technique provides an excellent tool for the unambiguous detection of homozygous and heterozygous transgenic mice in a litter. It can be used to check young animals since 2 l of whole blood is sufficient. We also show that in this transgenic line numerous copies of the hSOD-1 transgene are. integrated at a single autosomal locus, in tandem head-to-tail organization     

Change in glycosylation of chicken transferrin glycans biosynthesized during embryogenesis and primary culture of embryo hepatocytes

Transferrins were isolated by immunoaffinity chromato-graphyfrom chicken serum, chicken embryo serum and from the culturemedium of chicken embryo hepatocytes in primary culture. Theglycovariants of these three transferrins were separated byion-exchange chromatography using a fast protein liquid chromatography(FPLC) system. The structures of the oligosaccharide-alditolsreleased by hydrazinolysis from the glycovariants were comparedafter analysis by a combination of methanolysis, methylatlonanalysis and ¹H-NMR spectroscopy. In the three transferrinsanalysed, the oligosaccharides were of the bian-tennary N-acetyllactosaminictype, having several prominent features. In particular, theembryo serum transferrin glycan differed from that of chickenserum transferrin by the presence of a bisecting N-acetylglucosamine,suggesting a developmental change in glycosylation. The glycanstructure of the transferrin secreted by the embryo hepatocytesin primary culture was marked by the presence of fucose (l-6)linked to the core N-acetylglucosamine, suggesting that expressionof the fucosyltransferase activity is dependent on cell cultureconditions. Moreover, comparative analysis of chicken serumtransferrin and ovotransferrin glycans reinforces the idea thatthe glycosylation of two identical poly-peptide chains is organspecific. chicken embryogenesis embryo hepatocytes glycosylation transferrin     

Deletion of the Fission Yeast Homologue of Human Insulinase Reveals a TORC1-Dependent Pathway Mediating Resistance to Proteotoxic Stress

Insulin Degrading Enzyme (IDE) is a protease conserved through evolution with a role in diabetes and Alzheimer''s disease. The reason underlying its ubiquitous expression including cells lacking identified IDE substrates remains unknown. Here we show that the fission yeast IDE homologue (Iph1) modulates cellular sensitivity to endoplasmic reticulum (ER) stress in a manner dependent on TORC1 (Target of Rapamycin Complex 1). Reduced sensitivity to tunicamycin was associated with a smaller number of cells undergoing apoptosis. Wild type levels of tunicamycin sensitivity were restored in iph1 null cells when the TORC1 complex was inhibited by rapamycin or by heat inactivation of the Tor2 kinase. Although Iph1 cleaved hallmark IDE substrates including insulin efficiently, its role in the ER stress response was independent of its catalytic activity since expression of inactive Iph1 restored normal sensitivity. Importantly, wild type as well as inactive human IDE complemented gene-invalidated yeast cells when expressed at the genomic locus under the control of iph1⁺ promoter. These results suggest that IDE has a previously unknown function unrelated to substrate cleavage, which links sensitivity to ER stress to a pro-survival role of the TORC1 pathway.     

DNA replication in phage P4: characterization of replicon II

The genetic element P4 propagates in its host Escherichia coli both as a satellite phage and as a plasmid. Two partially overlapping replicons coexist, namely replicon I and replicon II. The former is composed of two sites, ori1 and crr, and depends on P4 alpha gene product for replication. The P4 alpha protein has primase and helicase activities, and binds specifically to both ori1 and crr. Replicon II is composed of two sites, ori2 and crr, and its replication also depends on P4 alpha primase and helicase activities. In replicon II, the alpha protein binds only crr. Here we show that for replicon II the relative orientation of ori2 and crr is essential for replication to occur. Furthermore we delimit ori2 to a 22 bp region (6234-6255), internal to the alpha gene, sufficient for replicon II replication. We mutagenized this region and identified two mutants, which carry one and two base substitutions, respectively, that prevent replicon II replication. In electrophoretic mobility shift experiments of ori2, ori1, and crr DNA fragments with E. coli extracts, ori2 was not shifted, whereas both ori1 and crr were specifically bound, suggesting that other host protein(s), beside P4 alpha, are able to bind to these cis essential regions. Apparently, no binding to ori2 could be identified, thus suggesting that neither alpha nor other bacterial proteins specifically bind to this region.     

Protein interactions within the Set1 complex and their roles in the regulation of histone 3 lysine 4 methylation

Set1 is the catalytic subunit and the central component of the evolutionarily conserved Set1 complex (Set1C) that methylates histone 3 lysine 4 (H3K4). Here we have determined protein/protein interactions within the complex and related the substructure to function. The loss of individual Set1C subunits differentially affects Set1 stability, complex integrity, global H3K4 methylation, and distribution of H3K4 methylation along active genes. The complex requires Set1, Swd1, and Swd3 for integrity, and Set1 amount is greatly reduced in the absence of the Swd1-Swd3 heterodimer. Bre2 and Sdc1 also form a heteromeric subunit, which requires the SET domain for interaction with the complex, and Sdc1 strongly interacts with itself. Inactivation of either Bre2 or Sdc1 has very similar effects. Neither is required for complex integrity, and their removal results in an increase of H3K4 mono- and dimethylation and a severe decrease of trimethylation at the 5' end of active coding regions but a decrease of H3K4 dimethylation at the 3' end of coding regions. Cells lacking Spp1 have a reduced amount of Set1 and retain a fraction of trimethylated H3K4, whereas cells lacking Shg1 show slightly elevated levels of both di- and trimethylation. Set1C associates with both serine 5- and serine 2-phosphorylated forms of polymerase II, indicating that the association persists to the 3' end of transcribed genes. Taken together, our results suggest that Set1C subunits stimulate Set1 catalytic activity all along active genes.     

Analysis of the Escherichia coli RNA degradosome composition by a proteomic approach

The RNA degradosome is a bacterial protein machine devoted to RNA degradation and processing. In Escherichia coli it is typically composed of the endoribonuclease RNase E, which also serves as a scaffold for the other components, the exoribonuclease PNPase, the RNA helicase RhlB, and enolase. Several other proteins have been found associated to the core complex. However, it remains unclear in most cases whether such proteins are occasional contaminants or specific components, and which is their function. To facilitate the analysis of the RNA degradosome composition under different physiological and genetic conditions we set up a simplified preparation procedure based on the affinity purification of FLAG epitope-tagged RNase E coupled to Multidimensional Protein Identification Technology (MudPIT) for the rapid and quantitative identification of the different components. By this proteomic approach, we show that the chaperone protein DnaK, previously identified as a "minor component" of the degradosome, associates with abnormal complexes under stressful conditions such as overexpression of RNase E, low temperature, and in the absence of PNPase; however, DnaK does not seem to be essential for RNA degradosome structure nor for its assembly. In addition, we show that normalized score values obtain by MudPIT analysis may be taken as quantitative estimates of the relative protein abundance in different degradosome preparations.     

Palmitic acid follows a different metabolic pathway than oleic acid in human skeletal muscle cells; lower lipolysis rate despite an increased level of adipose triglyceride lipase

Development of insulin resistance is positively associated with dietary saturated fatty acids and negatively associated with monounsaturated fatty acids. To clarify aspects of this difference we have compared the metabolism of oleic (OA, monounsaturated) and palmitic acids (PA, saturated) in human myotubes. Human myotubes were treated with 100μM OA or PA and the metabolism of [(14)C]-labeled fatty acid was studied. We observed that PA had a lower lipolysis rate than OA, despite a more than two-fold higher protein level of adipose triglyceride lipase after 24h incubation with PA. PA was less incorporated into triacylglycerol and more incorporated into phospholipids after 24h. Supporting this, incubation with compounds modifying lipolysis and reesterification pathways suggested a less influenced PA than OA metabolism. In addition, PA showed a lower accumulation than OA, though PA was oxidized to a relatively higher extent than OA. Gene set enrichment analysis revealed that 24h of PA treatment upregulated lipogenesis and fatty acid β-oxidation and downregulated oxidative phosphorylation compared to OA. The differences in lipid accumulation and lipolysis between OA and PA were eliminated in combination with eicosapentaenoic acid (polyunsaturated fatty acid). In conclusion, this study reveals that the two most abundant fatty acids in our diet are partitioned toward different metabolic pathways in muscle cells, and this may be relevant to understand the link between dietary fat and skeletal muscle insulin resistance.     

The Escherichia coli Lpt Transenvelope Protein Complex for Lipopolysaccharide Export Is Assembled via Conserved Structurally Homologous Domains

Lipopolysaccharide is a major glycolipid component in the outer leaflet of the outer membrane (OM), a peculiar permeability barrier of Gram-negative bacteria that prevents many toxic compounds from entering the cell. Lipopolysaccharide transport (Lpt) across the periplasmic space and its assembly at the Escherichia coli cell surface are carried out by a transenvelope complex of seven essential Lpt proteins spanning the inner membrane (LptBCFG), the periplasm (LptA), and the OM (LptDE), which appears to operate as a unique machinery. LptC is an essential inner membrane-anchored protein with a large periplasm-protruding domain. LptC binds the inner membrane LptBFG ABC transporter and interacts with the periplasmic protein LptA. However, its role in lipopolysaccharide transport is unclear. Here we show that LptC lacking the transmembrane region is viable and can bind the LptBFG inner membrane complex; thus, the essential LptC functions are located in the periplasmic domain. In addition, we characterize two previously described inactive single mutations at two conserved glycines (G56V and G153R, respectively) of the LptC periplasmic domain, showing that neither mutant is able to assemble the transenvelope machinery. However, while LptCG56V failed to copurify any Lpt component, LptCG153R was able to interact with the inner membrane protein complex LptBFG. Overall, our data further support the model whereby the bridge connecting the inner and outer membranes would be based on the conserved structurally homologous jellyroll domain shared by five out of the seven Lpt components.     

Reversal of age-related oxidative stress prevents hippocampal synaptic plasticity deficits by protecting D-serine-dependent NMDA receptor activation

Oxidative stress (OS) resulting from an imbalance between antioxidant defenses and the intracellular accumulation of reactive oxygen species (ROS) contributes to age-related memory deficits. While impaired synaptic plasticity in neuronal networks is thought to underlie cognitive deficits during aging, whether this process is targeted by OS and what the mechanisms involved are still remain open questions. In this study, we investigated the age-related effects of the reducing agent N-acetyl-L-cysteine (L-NAC) on the activation of the N-methyl-D-aspartate receptor (NMDA-R) by its co-agonist D-serine, because alterations in this mechanism contribute greatly to synaptic plasticity deficits in aged animals. Long-term dietary supplementation with L-NAC prevented oxidative damage in the hippocampus of aged rats. Electrophysiological recordings in the CA1 of hippocampal slices indicated that NMDA-R-mediated synaptic potentials and theta-burst-induced long-term potentiation (LTP) were depressed in aged animals, deficits that could be reversed by exogenous D-serine. Chronic treatment with L-NAC, but not acute application of the reducing agent, restored potent D-serine-dependent NMDA-R activation and LTP induction in aged rats. In addition, it is also revealed that the age-related decrease in D-serine levels and in the expression of the synthesizing enzyme serine racemase, which underlies the decrease in NMDA-R activation by the amino acid, was rescued by long-term dietary treatment with L-NAC. Our results indicate that protecting redox status in aged animals could prevent injury to the cellular mechanisms underlying cognitive aging, in part by maintaining potent NMDA-R activation through the D-serine-dependent pathway.