A validated antibody panel for the characterization of tau post-translational modifications

Background

Tau is a microtubule-binding protein, which is subject to various post-translational modifications (PTMs) including phosphorylation, methylation, acetylation, glycosylation, nitration, sumoylation and truncation. Aberrant PTMs such as hyperphosphorylation result in tau aggregation and the formation of neurofibrillary tangles, which are a hallmark of Alzheimer’s disease (AD). In order to study the importance of PTMs on tau function, antibodies raised against specific modification sites are widely used. However, quality control of these antibodies is lacking and their specificity for particular modifications is often unclear.

Methods

In this study, we first designed an online tool called ‘TauPTM’, which enables the visualization of PTMs and their interactions on human tau. Using TauPTM, we next searched for commercially available antibodies against tau PTMs and characterized their specificity by peptide array, immunoblotting, electrochemiluminescence ELISA and immunofluorescence technologies.

Results

We demonstrate that commercially available antibodies can show a significant lack of specificity, and PTM-specific antibodies in particular often recognize non-modified versions of the protein. In addition, detection may be hindered by other PTMs in close vicinity, complicating the interpretation of results. Finally, we compiled a panel of specific antibodies and show that they are useful to detect PTM-modified endogenous tau in hiPSC-derived neurons and mouse brains.

Conclusion

This study has created a platform to reliably and robustly detect changes in localization and abundance of post-translationally modified tau in health and disease. A web-based version of TauPTM is fully available at http://www.tauptm.org.

     

Effects of L-carnitine L-tartrate supplementation on muscle oxygenation responses to resistance exercise

Previous research has shown that L-carnitine L-tartrate (LCLT) supplementation beneficially affects markers of hypoxic stress following resistance exercise. However, the mechanism of this response is unclear. Therefore, the primary purpose of this study was to determine the effects of LCLT supplementation on muscle tissue oxygenation during and after multiple sets of squat exercise. Nine healthy, previously resistance-trained men (25.2 +/- 6.years, 91.2 +/- 10.2 kg, 180.2 +/- 6.3 cm) ingested 2 g.d of LCLT or an identical placebo for 23 days in a randomized, balanced, crossover, double-blind, placebo-controlled, repeated-measures study design. On day 21, forearm muscle oxygenation was measured during and after an upper arm occlusion protocol using near infrared spectroscopy (NIRS), which measures the balance of oxygen delivery in relation to oxygen consumption. On day 22, subjects performed 5 sets of 15 to 20 repetitions of squat exercise with corresponding measures of thigh muscle oxygenation, via NIRS, and serial blood draws. Compared to the placebo trial, muscle oxygenation was reduced in the LCLT trial during upper arm occlusion and following each set of resistance exercise. Despite reduced oxygenation, plasma malondealdehyde, a marker of membrane damage, was attenuated during the LCLT trial. There were no differences between trials in the vasoactive substance prostacyclin. In conclusion, because oxygen delivery was occluded during the forearm protocol, it is proposed that enhanced oxygen consumption mediated the reduced muscle oxygenation during the LCLT trial. Enhanced oxygen consumption would explain why hypoxic stress was attenuated with LCLT supplementation.     

Effects of stretching on upper-body muscular performance

The purpose of this investigation was to examine the influence of upper-body static stretching and dynamic stretching on upper-body muscular performance. Eleven healthy men, who were National Collegiate Athletic Association Division I track and field athletes (age, 19.6 +/- 1.7 years; body mass, 93.7 +/- 13.8 kg; height, 183.6 +/- 4.6 cm; bench press 1 repetition maximum [1RM], 106.2 +/- 23.0 kg), participated in this study. Over 4 sessions, subjects participated in 4 different stretching protocols (i.e., no stretching, static stretching, dynamic stretching, and combined static and dynamic stretching) in a balanced randomized order followed by 4 tests: 30% of 1 RM bench throw, isometric bench press, overhead medicine ball throw, and lateral medicine ball throw. Depending on the exercise, test peak power (Pmax), peak force (Fmax), peak acceleration (Amax), peak velocity (Vmax), and peak displacement (Dmax) were measured. There were no differences among stretch trials for Pmax, Fmax, Amax, Vmax, or Dmax for the bench throw or for Fmax for the isometric bench press. For the overhead medicine ball throw, there were no differences among stretch trials for Vmax or Dmax. For the lateral medicine ball throw, there was no difference in Vmax among stretch trials; however, Dmax was significantly larger (p 相似文献   

Vaccine-induced cellular immune responses reduce plasma viral concentrations after repeated low-dose challenge with pathogenic simian immunodeficiency virus SIVmac239

The goal of an AIDS vaccine regimen designed to induce cellular immune responses should be to reduce the viral set point and preserve memory CD4 lymphocytes. Here we investigated whether vaccine-induced cellular immunity in the absence of any Env-specific antibodies can control viral replication following multiple low-dose challenges with the highly pathogenic SIVmac239 isolate. Eight Mamu-A*01-positive Indian rhesus macaques were vaccinated with simian immunodeficiency virus (SIV) gag, tat, rev, and nef using a DNA prime-adenovirus boost strategy. Peak viremia (P = 0.007) and the chronic phase set point (P = 0.0192) were significantly decreased in the vaccinated cohort, out to 1 year postinfection. Loss of CD4(+) memory populations was also ameliorated in vaccinated animals. Interestingly, only one of the eight vaccinees developed Env-specific neutralizing antibodies after infection. The control observed was significantly improved over that observed in animals vaccinated with SIV gag only. Vaccine-induced cellular immune responses can, therefore, exert a measure of control over replication of the AIDS virus in the complete absence of neutralizing antibody and give us hope that a vaccine designed to induce cellular immune responses might control viral replication.     

Prohibitins Are Required for Cancer Cell Proliferation and Adhesion

Prohibitin 1 (PHB1) is a highly conserved protein that together with its homologue prohibitin 2 (PHB2) mainly localizes to the inner mitochondrial membrane. Although it was originally identified by its ability to inhibit G1/S progression in human fibroblasts, its role as tumor suppressor is debated. To determine the function of prohibitins in maintaining cell homeostasis, we generated cancer cell lines expressing prohibitin-directed shRNAs. We show that prohibitin proteins are necessary for the proliferation of cancer cells. Down-regulation of prohibitin expression drastically reduced the rate of cell division. Furthermore, mitochondrial morphology was not affected, but loss of prohibitins did lead to the degradation of the fusion protein OPA1 and, in certain cancer cell lines, to a reduced capability to exhibit anchorage-independent growth. These cancer cells also exhibited reduced adhesion to the extracellular matrix. Taken together, these observations suggest prohibitins play a crucial role in adhesion processes in the cell and thereby sustaining cancer cell propagation and survival.     

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The degradation of collagens, the most abundant proteins of the extracellular matrix, is involved in numerous physiological and pathological conditions including cancer invasion. An important turnover pathway involves cellular internalization and degradation of large, soluble collagen fragments, generated by initial cleavage of the insoluble collagen fibers. We have previously observed that in primary mouse fibroblasts, this endocytosis of collagen fragments is dependent on the receptor urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180. Others have identified additional mechanisms of collagen uptake, with different associated receptors, in other cell types. These receptors include β1-integrins, being responsible for collagen phagocytosis, and the mannose receptor. We have now utilized a newly developed monoclonal antibody against uPARAP/Endo180, which down-regulates the receptor protein level on treated cells, to examine the role of uPARAP/Endo180 as a mediator of collagen internalization by a wide range of cultured cell types. With the exception of macrophages, all cells that proved capable of efficient collagen internalization were of mesenchymal origin and all of these utilized uPARAP/Endo180 for their collagen uptake process. Macrophages internalized collagen in a process mediated by the mannose receptor, a protein belonging to the same protein family as uPARAP/Endo180. β1-Integrins were found not to be involved in the endocytosis of soluble collagen, irrespectively of whether this was mediated by uPARAP/Endo180 or the mannose receptor. This further distinguishes these pathways from the phagocytic uptake of particulate collagen.     

Diversity of culturable phyllosphere bacteria on beech and oak: the effects of lepidopterous larvae

The community composition of epiphytic heterotrophic bacteria on leaves of beech and oak, which were either damaged by lepidopterous larvae or remained undamaged, was investigated. In addition, the ability of these bacteria to utilize inorganic nitrogen was studied. The bacteria were isolated on nutrient agar and systematically identified with biochemical and physiological tests. Rarefaction plots and the Shannon-Wiener function revealed that species diversity was significantly higher on leaves of damaged beech compared to undamaged leaves, but no differences were found on leaves of oak. The portion of bacterial isolates showing a strong response to ammonia and nitrate was significantly larger on leaves of oak than on those of beech. Furthermore, significantly more isolates with a high capability to assimilate both nitrogen compounds were found on leaves attacked by the folivorous larvae compared to those not attacked on oak. It is suggested that the changes in the microbial community in response to folivorous insects might affect the extent of nutrient cycling exceeding eventually the scale of a leaf.     

X-ray structures of two xanthine inhibitors bound to PEPCK and N-3 modifications of substituted 1,8-dibenzylxanthines

The analysis of the X-ray structures of two xanthine inhibitors bound to PEPCK and a comparison to the X-ray structure of GTP bound to PEPCK are reported. The SAR at N-1, N-7 and developing SAR at C-8 are consistent with information gained from the X-ray structures of compounds 1 and 2 bound to PEPCK. Representative N-3 modifications of compound 2 that led to the discovery of 3-cyclopropylmethyl and its carboxy analogue as optimal N-3 groups are presented.