GTP gamma S inhibits early c-myc protein accumulation but not DNA synthesis in Swiss 3T3 fibroblasts

Quiescent Swiss 3T3 fibroblasts stimulated with epidermal growth factor and insulin showed large transient increases in c-myc mRNA and c-myc protein accumulation which were maximal at about 2 h after addition of the co-mitogens. When the cells were loaded with 0.1 mM of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) by transient permeabilisation immediately before mitogenic stimulation, the increase in c-myc mRNA was similar to that observed in unloaded cells but the corresponding c-myc protein peak was reduced by at least 95%. The GTP gamma S completely blocked incorporation of [35S]methionine into cell proteins for 3-4 h after addition of the mitogens, but not thereafter, and caused a delay in the subsequent onset of DNA synthesis by the same period. The data show that less than 5% of the early increase in c-myc protein normally observed after mitogenic stimulation is required for its obligatory role in the progression of cells to S phase implied by other evidence.     

17β-Estradiol Directly Lowers Mitochondrial Membrane Microviscosity and Improves Bioenergetic Function in Skeletal Muscle

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Osseointegration of preformed polymethylmethacrylate craniofacial prostheses coated with bone marrow-impregnated poly (DL-lactic-co-glycolic acid) foam.

Osseointegration of bone marrow-PLGA-coated, preformed polymethylmethacrylate cranioplasties offers the possibility of reducing: operative time, periimplant seroma and infection, metallic fixation, and periprosthetic resorption following surgical skull remodeling. These alloplastic materials are FDA-approved but previously have not been used together to promote cranioplasty incorporation. The objective of this study was to determine whether the use of PLGA foam coating improves host osseointegration of preformed, textured, polymethylmethacrylate prosthetic cranioplasties. A critical-sized cranial defect was created in two groups of 10 and one group of three rabbits. The defect was filled with either a textured, preformed polymethylmethacrylate disc or a textured, preformed polymethylmethacrylate disc coated with poly (DL-lactic-co-glycolic acid). Both implants were immersed in autologous bone marrow for 20 minutes before implantation. Half of each group of 10 were killed at 3 weeks, and the remainder at 6 weeks. A third group of three rabbits with excised periosteum was evaluated at 6 weeks. Histologic analysis of the discs determined relative amounts of cancellous bone formation adjacent to the prostheses. Woven trabecular bone was present at each host bone to implant perimeter interface at 3 weeks, with fine fibrous capsular formation around the implants. Thicker, lamellar trabeculae were present at 6 weeks with an increased fibrous layer surrounding both types of implants. Bone formed on the superficial and deep implant surfaces in a noncontiguous fashion. Two of five measures showed that total bone formation was significantly greater in the PLGA-coated implants. Polymethylmethacrylate discs coated with bone marrow-impregnated PLGA foam demonstrate increased bone formation at 3 and 6 weeks as compared with non-coated preformed polymethylmethacrylate discs. Only implants with preserved periosteum showed bone formation away from the host-implant interface (centrally) on the superficial surface at 6 weeks.     

Structural conservation of the pores of calcium-activated and voltage-gated potassium channels determined by a sea anemone toxin.

The structurally defined sea anemone peptide toxins ShK and BgK potently block the intermediate conductance, Ca(2+)-activated potassium channel IKCa1, a well recognized therapeutic target present in erythrocytes, human T-lymphocytes, and the colon. The well characterized voltage-gated Kv1.3 channel in human T-lymphocytes is also blocked by both peptides, although ShK has a approximately 1,000-fold greater affinity for Kv1.3 than IKCa1. To gain insight into the architecture of the toxin receptor in IKCa1, we used alanine-scanning in combination with mutant cycle analyses to map the ShK-IKCa1 interface, and compared it with the ShK-Kv1.3 interaction surface. ShK uses the same five core residues, all clustered around the critical Lys(22), to interact with IKCa1 and Kv1.3, although it relies on a larger number of contacts to stabilize its weaker interactions with IKCa1 than with Kv1.3. The toxin binds to IKCa1 in a region corresponding to the external vestibule of Kv1.3, and the turret and outer pore of the structurally defined bacterial potassium channel, KcsA. Based on the NMR structure of ShK, we deduce the toxin receptor in IKCa1 to have x-y dimensions of approximately 22 A, a diameter of approximately 31 A, and a depth of approximately 8 A; we estimate that the ion selectivity lies approximately 13 A below the outer lip of the toxin receptor. These dimensions are in good agreement with those of the KcsA channel determined from its crystal structure, and the inferred structure of Kv1.3 based on mapping with scorpion toxins. Thus, these distantly related channels exhibit architectural similarities in the outer pore region. This information could facilitate development of specific and potent modulators of the therapeutically important IKCa1 channel.     

The search for an appropriate end-of-life formula for the purpose of the European Commission Environmental Footprint initiative

Purpose

This paper explains in details the rationale behind the choice of the end-of-life allocation approach in the European Commission Product Environmental Footprint (PEF) and Organisational Environmental Footprint (OEF) methods. The end-of-life allocation formula in the PEF/OEF methods aims at enabling the assessment of all end-of-life scenarios possible, including recycling, reuse, incineration (with heat recovery) and disposal for both open- and closed-loop systems in a consistent and reproducible way. It presents how the formula builds on existing standards and how and why it deviates from them.

Methods

Various end-of-life allocation approaches and formulas, mainly taken not only from/based on existing environmental impact assessment methods and/or standards but also one original linearly degressive approach, were analysed against a predetermined set of criteria, reflecting the overall aim of the PEF/OEF methods. This set of criteria is physical realism, distribution of burdens and benefits in a product cascade system and applicability. Besides the qualitative analysis, the various formulas were implemented for several products and for different scenarios regarding recycled content and recyclability to check the robustness of the outcomes, exemplary expressed for the Global Warming Potential impact category.

Results and discussion

As reaching physical realism was impossible at both the product and overall product cascade system level by any of the end-of-life approaches analysed, one of both had to be prioritised. The paper explains in details why a product level approach was preferred in the context of the PEF/OEF methods. In consequence, allocation of the end-of-life processes which are related to more than one product in a product cascade system is needed and should be carefully considered as it has a major influence on the results and decision taking.

Conclusions

A formula taking into account the number of recycling cycles of a material was identified as preferred to reach physical realism and to allocate burdens and benefits of repeatedly recycling of a material over the different products in a product cascade system. However, this approach was not selected for the PEF/OEF methods as data on the number of recycling cycles was insufficiently available (for the time being) for all products on the market and hence fails the criterion of “applicability”. This explains why, instead, a formula based on the 50:50 approach—allocating shared end-of-life processes equally between the previous and subsequent product—was selected for the PEF/OEF methods.

     

SIRT3 interacts with the daf-16 homolog FOXO3a in the mitochondria, as well as increases FOXO3a dependent gene expression

Cellular longevity is a complex process relevant to age-related diseases including but not limited to chronic illness such as diabetes and metabolic syndromes. Two gene families have been shown to play a role in the genetic regulation of longevity; the Sirtuin and FOXO families. It is also established that nuclear Sirtuins interact with and under specific cellular conditions regulate the activity of FOXO gene family proteins. Thus, we hypothesize that a mitochondrial Sirtuin (SIRT3) might also interact with and regulate the activity of the FOXO proteins. To address this we used HCT116 cells overexpressing either wild-type or a catalytically inactive dominant negative SIRT3. For the first time we establish that FOXO3a is also a mitochondrial protein and forms a physical interaction with SIRT3 in mitochondria. Overexpression of a wild-type SIRT3 gene increase FOXO3a DNA-binding activity as well as FOXO3a dependent gene expression. Biochemical analysis of HCT116 cells over expressing the deacetylation mutant, as compared to wild-type SIRT3 gene, demonstrated an overall oxidized intracellular environment, as monitored by increase in intracellular superoxide and oxidized glutathione levels. As such, we propose that SIRT3 and FOXO3a comprise a potential mitochondrial signaling cascade response pathway.