Vitamin E alleviates non-alcoholic fatty liver disease in phosphatidylethanolamine N-methyltransferase deficient mice

Phosphatidylethanolamine N-methyltransferase (PEMT) converts phosphatidylethanolamine (PE) to phosphatidylcholine (PC), mainly in the liver. Pemt^?/? mice are protected from high-fat diet (HFD)-induced obesity and insulin resistance, but develop severe non-alcoholic fatty liver disease (NAFLD) when fed a HFD, mostly due to impaired VLDL secretion. Oxidative stress is thought to be an essential factor in the progression from simple steatosis to steatohepatitis. Vitamin E is an antioxidant that has been clinically used to improve NAFLD pathology. Our aim was to determine whether supplementation of the diet with vitamin E could attenuate HFD-induced hepatic steatosis and its progression to NASH in Pemt^?/? mice. Treatment with vitamin E (0.5?g/kg) for 3?weeks improved VLDL-TG secretion and normalized cholesterol metabolism, but failed to reduce hepatic TG content. Moreover, vitamin E treatment was able to reduce hepatic oxidative stress, inflammation and fibrosis. We also observed abnormal ceramide metabolism in Pemt^?/? mice fed a HFD, with elevation of ceramides and other sphingolipids and higher expression of mRNAs for acid ceramidase (Asah1) and ceramide kinase (Cerk). Interestingly, vitamin E supplementation restored Asah1 and Cerk mRNA and sphingolipid levels. Together this study shows that vitamin E treatment efficiently prevented the progression from simple steatosis to steatohepatitis in mice lacking PEMT.     

Unconventional myosins at the crossroad of signal transduction and cytoskeleton remodeling

The cytoplasm of eukaryotic cells is a complex milieu and unraveling how its unique cytoarchitecture is achieved and maintained is a central theme in modern cell biology. The actin cytoskeleton is essential for the maintenance of cell shape and locomotion, and also provides tracks for active intracellular transport. Myosins, the actin-dependent motor proteins form a superfamily of at least 15 structural classes and have been identified in a wide variety of organisms, making the presence of actin and myosins a hallmark feature of eukaryotes. Direct connections of myosins to a variety of cellular tasks are now emerging, such as in cytokinesis, phagocytosis, endocytosis, polarized secretion and exocytosis, axonal transport. Recent studies reveal that myosins also play an essential role in many aspects of signal transduction and neurosensation.     

Swelling activation of transport pathways in erythrocytes: effects of Cl-, ionic strength, and volume changes

If swelling of acell is induced by a decrease in external medium tonicity, theregulatory response is more complex than if swelling of similarmagnitude is due to salt uptake. The present results provide anexplanation. In fish erythrocytes, two distinct transport pathways wereswelling activated: a channel of broad specificity and aK⁺-Clcotransporter. Each was activated by a specific signal: the channel bya decrease in intracellular ionic strength and theK⁺-Clcotransporter by cell enlargement. A decrease in ionic strength alsoaffectedK⁺-Clcotransport activity, but by acting as a negative modulator of thecotransport. Thus cells swollen by salt accumulation respond byactivating exclusively theK⁺-Clcotransport, leading to aCl-dependentK⁺ loss. By contrast, cellsswollen by electrolyte dilution respond by activating both pathways,leading to a reduced loss of electrolytes and a large loss of taurine.Thus two swelling-sensitive pathways, differently regulated, wouldallow control of the ionic composition of a cell exposed to differentvolume perturbations.

     

Trapping radioactive carbon dioxide during cellular metabolic assays under standard culture conditions: description of a unique gas-capturing device

Measurement of carbon dioxide levels has been employed to follow cellular metabolic reactions for quite some time. By radio-labeling substrate molecules and evaluating the radioactivity levels of the carbon dioxide released, insight into metabolic pathways can be gleaned. Currently, no carbon dioxide capturing device is available that can be used with large volume cell monolayers growing under standard conditions within a regular commercially available culture flask. In this note we describe a simple device for collecting radio-labeled carbon dioxide from a standard culture flask. The device is independent of the culture flask, but can be attached for metabolic measurements allowing cells to be grown under standard conditions prior to study. The presented design permits convenient transfer of the device between flasks without contaminating or disturbing cells growing within the flasks. Data are presented demonstrating the reproducibility of measurements made with multiple devices with different substrate concentrations and varying periods of time, ranging up to 3 h.     

Milking of microalgae

The low productivity of algal cultures in the production of high-value compounds is the most significant bottleneck for commercialization of this technology. Cultures in which cell mass is reused for continuous production are proposed as a solution to overcome this problem. Recently, a method was developed in which beta-carotene was harvested from the microalga Dunaliella salina grown in a two-phase bioreactor. This raises the question of whether this technique could also be used in the mass production of secondary metabolites. Understanding the mechanism of the milking process and its relationship to the product formation pathway should reveal whether other products can be milked from various species of microalgae.     

Autoclave method for rapid preparation of bacterial PCR-template DNA

An autoclave method for preparing bacterial DNA for PCR template is presented, it eliminates the use of detergents, organic solvents, and mechanical cellular disruption approaches, thereby significantly reducing processing time and costs while increasing reproducibility. Bacteria are lysed by rapid heating and depressurization in an autoclave. The lysate, cleared by microcentrifugation, was either used directly in the PCR reaction, or concentrated by ultrafiltration. This approach was compared with seven established methods of DNA template preparation from four bacterial sources which included boiling Triton X-100 and SDS, bead beating, lysozyme/proteinase K, and CTAB lysis method components. Bacteria examined were Enterococcus and Escherichia coli, a natural marine bacterial community and an Antarctic cyanobacterial-mat. DNAs were tested for their suitability as PCR templates by repetitive element random amplified polymorphic DNA (RAPD) and denaturing gradient gel electrophoresis (DGGE) analysis. The autoclave method produced PCR amplifiable template comparable or superior to the other methods, with greater reproducibility, much shorter processing time, and at a significantly lower cost.     

Domain movements of elongation factor eEF2 and the eukaryotic 80S ribosome facilitate tRNA translocation

An 11.7-A-resolution cryo-EM map of the yeast 80S.eEF2 complex in the presence of the antibiotic sordarin was interpreted in molecular terms, revealing large conformational changes within eEF2 and the 80S ribosome, including a rearrangement of the functionally important ribosomal intersubunit bridges. Sordarin positions domain III of eEF2 so that it can interact with the sarcin-ricin loop of 25S rRNA and protein rpS23 (S12p). This particular conformation explains the inhibitory action of sordarin and suggests that eEF2 is stalled on the 80S ribosome in a conformation that has similarities with the GTPase activation state. A ratchet-like subunit rearrangement (RSR) occurs in the 80S.eEF2.sordarin complex that, in contrast to Escherichia coli 70S ribosomes, is also present in vacant 80S ribosomes. A model is suggested, according to which the RSR is part of a mechanism for moving the tRNAs during the translocation reaction.     

Stem cells: cross-talk and developmental programs

The thesis advanced in this essay is that stem cells-particularly those in the nervous system-are components in a series of inborn 'programs' that not only ensure normal development, but persist throughout life so as to maintain homeostasis in the face of perturbations-both small and great. These programs encode what has come to be called 'plasticity'. The stem cell is one of the repositories of this plasticity. This review examines the evidence that interaction between the neural stem cell (as a prototypical somatic stem cell) and the developing or injured brain is a dynamic, complex, ongoing reciprocal set of interactions where both entities are constantly in flux. We suggest that this interaction can be viewed almost from a 'systems biology' vantage point. We further advance the notion that clones of exogenous stem cells in transplantation paradigms may not only be viewed for their therapeutic potential, but also as biological tools for 'interrogating' the normal or abnormal central nervous system environment, indicating what salient cues (among the many present) are actually guiding the expression of these 'programs'; in other words, using the stem cell as a 'reporter cell'. Based on this type of analysis, we suggest some of the relevant molecular pathways responsible for this 'cross-talk' which, in turn, lead to proliferation, migration, cell genesis, trophic support, protection, guidance, detoxification, rescue, etc. This type of developmental insight, we propose, is required for the development of therapeutic strategies for neurodegenerative disease and other nervous system afflictions in humans. Understanding the relevant molecular pathways of stem cell repair phenotype should be a priority, in our view, for the entire stem cell field.