top of page


Public·98 members

Fusion 360 2008 Activation [BETTER]

Fusion 360 2008 Activation >

I am having trouble with the product activation. So every time I start autodesk newly, the activation license pops up. So when I connect online, it says congratulations! your product has been activated. But when I restart the program, again the pop up comes up asking for activation. I have tried do it manually by getting a resquest code and it accepted my activation offline as well but the same problem persists after restart. Please help.

Online license transfer is the process of moving your software activation information from one installation of your software to another via the Internet using the License Transfer Utility. Here are some notes about the process:

Note: If you no longer have access to the computer where your software was installed or already made system changes and your software is no longer activated, you cannot use the License Transfer Utility for reactivation. See the Product Reactivation FAQs for information about activating a new installation.

Transferring your software license helps you avoid activation limits and errors that could occur with standard reactivation of software that has already been through the online activation process. Here are some common scenarios in which you may want to transfer your license:

Note: Some Autodesk software is activated by signing into an Autodesk Account when the software is launched and can be used on any computer. This type of sign-in activation does not require license transfer to work on a different computer.

Brown adipose tissue (BAT) mitochondria thermogenesis is regulated by uncoupling protein 1 (UCP 1), GDP and fatty acids. In this report, we observed fusion of the endoplasmic reticulum (ER) membrane with the mitochondrial outer membrane of rats BAT. Ca2+-ATPase (SERCA 1) was identified by immunoelectron microscopy in both ER and mitochondria. This finding led us to test the Ca2+ effect in BAT mitochondria thermogenesis. We found that Ca2+ increased the rate of respiration and heat production measured with a microcalorimeter both in coupled and uncoupled mitochondria, but had no effect on the rate of ATP synthesis. The Ca2+ concentration needed for half-maximal activation varied between 0.08 and 0.11 µM. The activation of respiration was less pronounced than that of heat production. Heat production and ATP synthesis were inhibited by rotenone and KCN.

Liver mitochondria have no UCP1 and during respiration synthesize a large amount of ATP, produce little heat, GDP had no effect on mitochondria coupling, Ca2+ strongly inhibited ATP synthesis and had little or no effect on the small amount of heat released. These finding indicate that Ca2+ activation of thermogenesis may be a specific feature of BAT mitochondria not found in other mitochondria such as liver.

BAT cells did contain a large number of mitochondria and an extended ER network that surrounded mitochondria, the nucleus and the cell lipid deposits (Fig. 1). The shape and diameter of the ER varied, ranging from straight neat tubules to large and convoluted structures. Protruding from the ER there were globular structures (Figs. 2 and 3). In the vicinity of mitochondria, these protrusions enter in contact with the outer mitochondrial membrane (Fig. 3). The images of Figs. 3, 4, 5 suggest that, after establishing contact, the ER projections propitiate the fusion of the ER membrane with the mitochondrial outer membrane. Immunolabeling with monoclonal anti-SERCA 1 antibodies and gold-labeled goat anti-mouse IgG revealed the presence of SERCA 1 in the ER, ER projections and in mitochondrial cristae (Figs. 2, 6 and 7a). These images raise the possibility that, in addition to lipids and Ca2+, SERCA 1 (Figs. 4 and 6) could also be transferred from the ER to mitochondria via MAM. Immunolabeling was clearly seen in isolated mitochondria and vesicles isolated from the ER by differential centrifugation (Fig. 7). In isolated mitochondria, we observed that some of them retain ER attached to the outer membrane (Fig. 8), indicating that the fusion between the two structures can be strong enough to resist tissue homogenization and centrifugation in a Percoll gradient.

The mechanism by which Ca2+ released in the cell during adrenergic stimulation activates BAT thermogenesis is not yet clear. The finding of SERCA 1 in BAT mitochondria (Fig. 7) raises the possibility that the activation by Ca2+ is somehow related to the mitochondrial SERCA 1. Therefore, in the following experiments, we tested the effects of GDP, Ca2+, and lipids in BAT isolated mitochondria. As a control, some of the experiments performed with BAT mitochondria were repeated with liver mitochondria which has UCP2 [23], [24] but does not contain UCP 1 (Fig. 9). The aim was to verify if the effects observed with BAT were specific of this tissue or if they could also be observed in other tissues containing different UCP isoforms such as liver mitochondria. Initially we measured the effects of GDP and Ca2+, in the formation of an electrochemical membrane potential (ΔΨ). BAT mitochondria were not able to form a Δψ after the single addition of the respiratory substrates pyruvate and malate [Fig. 10). Removal of lipids with excess fatty free serum albumin (faf-BSA) promoted the formation of a ΔΨ which was further enhanced by GDP. The same profile was observed if GDP was added before faf-BSA (data not shown). BAT ΔΨ formation was not altered by Ca2+ concentrations varying from 0.1 up to 2.0 µM, (data not shown). Different from BAT, in liver mitochondria a ΔΨ was formed after the addition of respiratory substrate without the need of adding either faf-BSA or GDP (Fig 10 inset). In both BAT and in liver mitochondria, the ΔΨ formed was collapsed by the proton ionophore FCCP. In conclusion, GDP promote a significant ΔΨ increase in BAT but had no measurable effect in liver mitochondria.

BAT mitochondria become coupled when faf-BSA and GDP were included in the assay medium. In these mitochondria the rates of respiration and heat production were decreased, and the rate of ATP synthesis rose to high values (Table 1). Similar to uncoupled mitochondria, Ca2+ enhanced the rate of heat production and respiration. Although in coupled mitochondria the rate of heat production was several folds slower than that measured in uncoupled mitochondria, the percent of activation promoted by Ca2+ was similar in the two conditions, 60.3% in uncoupled and 54.1% in coupled mitochondria. In coupled mitochondria Ca2+ had a discrete effect on the rate of respiration and no effect on the ATP synthesis rate.

To date, the establishment of a physical connection between the ER and mitochondria in BAT was not previously described. In these cells, the globular ER structures touch the mitochondria. The two structures are apparently pulled together, propitiating the fusion of mitochondrial and ER membranes (Figs. 3, 4, 5). This is different from what was observed in striated muscle where there seems to be no membrane fusion. Small tubular units (tethers) hold the mitochondria and ER together, and communication between the two sub cellular compartments would then be mediated by the tethering structures [3], [4], [6]. A link between BAT and skeletal muscle has been recently reported by Seale et al. [31]. These authors found that the transcriptional regulator PRDM 16 controls a bidirectional differentiation between skeletal myoblasts and brown adipocytes.

It has been proposed that the Ca2+ entering the mitochondria through MAM would activate bioenergetics because Ca2+ can activate enzymes in the tricarboxylic cycle, namely α-ketoglutarate and isocitrate dehydrogenase [4]. Acceleration of the tricarboxylic cycle would ultimately lead to an activation of both ATP synthesis and heat production. In favor of this possibility is the finding that in uncoupled mitochondria, a small amount of oligomycin-insensitive ATP was synthesized in the presence of Ca2+ (Table 1), and, during the tricarboxylic cycle, one GTP is synthesized from GDP and Pi. The GTP synthesized would then be transformed in to ATP. Against this possibility is the finding that Ca2+ activated only the heat production rate and had no effect on the rate of ATP synthesis. If the effect of Ca2+ would be derived from activation of the tricarboxylic cycle, then it would be expected that in coupled mitochondria, heat and ATP synthesis would be equally activated.

BAT was extracted from rats and reduced to three 1-mm pieces, whereas mitochondria from BAT were isolated by differential centrifugation. For routine transmission electron microscopy, samples were fixed in 2.5% glutaraldehyde (v/v) and 5 mM CaCl2 in 0.1 M cacodylate buffer (pH 7.2). The pieces were then washed in phosphate buffer saline (PBS) and post-fixed for 60 min in 1% OsO4 in cacodylate buffer containing 5 mM CaCl2 and 0.8% potassium ferricyanide. After washes in PBS, the material was dehydrated in acetone and embedded in Epon. Ultra-thin (70 nm) sections were stained with uranyl acetate and lead citrate and observed with a JEOL 1210 electron microscope. This procedure allows for high-quality images, but it is not adequate for immunoelectron microscopy because it impairs antibody diffusion through the resin [16].

Samples were fixed in 0.7% glutaraldehyde (v/v), 0.1% picric acid, 1% sucrose, 2% paraformaldehyde and 5 mM CaCl2 in 0.1 M cacodylate buffer (pH 7.2), dehydrated in ethanol and embedded in Unicryl (Ted Pella, USA). Ultra-thin sections were collected in nickel grids with 300 mesh and quenched in 50 mM NH4Cl for 30 min. Afterwards, the samples were incubated in the presence of monoclonal anti-Serca-1 antibody (clone IIH11, Affinity BioReagents, Inc., Brazil). After several washes in PBS-1% albumin, sections were incubated in the presence of 10-nm gold-labeled goat anti-mouse IgG (BB International, UK), washed, and observed with a JEOL 1210 electron microscope. This method allows for an adequate diffusion of the antibody but it decreases the preservation of the material due to light fixation, and, therefore, it decreases the quality of the image [16]. 153554b96e


Welcome to the group! You can connect with other members, ge...


  • Adrian Teo
  • John White
    John White
  • Crackps Store
    Crackps Store
  • Zs Cracked
    Zs Cracked
  • Crack deck
    Crack deck
Group Page: Groups_SingleGroup
bottom of page