Estrogenic regulation of skeletal muscle proteome: a study of premenopausal women and postmenopausal MZ cotwins discordant for hormonal therapy

Female middle age is characterized by a decline in skeletal muscle mass and performance, predisposing women to sarcopenia, functional limitations, and metabolic dysfunction as they age. Menopausal loss of ovarian function leading to low circulating level of 17β‐estradiol has been suggested as a contributing factor to aging‐related muscle deterioration. However, the underlying molecular mechanisms remain largely unknown and thus far androgens have been considered as a major anabolic hormone for skeletal muscle. We utilized muscle samples from 24 pre‐ and postmenopausal women to establish proteome‐wide profiles, associated with the difference in age (30–34 years old vs. 54–62 years old), menopausal status (premenopausal vs. postmenopausal), and use of hormone replacement therapy (HRT; user vs. nonuser). None of the premenopausal women used hormonal medication while the postmenopausal women were monozygotic (MZ) cotwin pairs of whom the other sister was current HRT user or the other had never used HRT. Label‐free proteomic analyses resulted in the quantification of 797 muscle proteins of which 145 proteins were for the first time associated with female aging using proteomics. Furthermore, we identified 17β‐estradiol as a potential upstream regulator of the observed differences in muscle energy pathways. These findings pinpoint the underlying molecular mechanisms of the metabolic dysfunction accruing upon menopause, thus having implications for understanding the complex functional interactions between female reproductive hormones and health.     

Is coproporphyrin III a copper-acquisition compound in Paracoccus denitrificans?

Paracoccus denitrificans is a soil bacterium which can respire aerobically and also denitrify if oxygen is absent. Both processes are highly dependent on copper enzymes and copper is therefore likely to be an essential trace element for the bacterium. If copper is not easily available, a copper-acquisition mechanism would be highly beneficial. In this paper, we have addressed the question of whether Paracoccus secretes a copper-acquisition compound functionally analogous to that found in some methanotrophs. Bacteria were grown both in copper-containing and copper-deficient denitrification media, cells were removed by centrifugation and the supernatant was analysed using chromatography and spectroscopy. Bacterial growth yield in the absence of copper was 70-80% of that in the copper-containing medium. A notable difference between the two culture conditions was that spent copper-deficient medium was pigmented, whereas the copper-containing medium was not. Spectrophotometry indicated that a red compound with an absorption maximum at 405 nm was produced under copper-limited conditions. In addition to the strong 405 nm maximum, the visible spectrum of the purified red molecule had weaker maxima at 535 nm and 570 nm, features typical of metallated tetrapyrroles. Mass spectrometry showed that the purified pigment had a molecular mass of 716.18. Moreover, the fine structure of the mass spectrum suggested the presence of zinc and was consistent with the chemical formula of C(36)H(36)N(4)O(8)Zn. The presence of zinc was also demonstrated using inductively coupled plasma atomic emission spectroscopy. Fragmentation analysis with mass spectrometry showed the release of consecutive 59 Da fragments, assignable to four -CH(2)-COOH moieties. Thin layer chromatography as well as NMR analysis of the C-13/N-15 labelled red pigment suggested that it is predominantly zinc coproporphyrin III with a minor fraction of metal-free coproporphyrin III. We propose that in a copper-poor environment P. denitrificans secretes coproporphyrin III for copper chelation and subsequent uptake of the bound copper into the cell. Consistent with this idea, cell yields of copper-deficient cultures grown in the presence of 1 microM copper-coproporphyrin III were 90-95% of the yields of cultures grown in the normal copper-containing media. Coproporphyrin III may work as a copper-acquisition compound in P. denitrificans.     

EC144, a synthetic inhibitor of heat shock protein 90, blocks innate and adaptive immune responses in models of inflammation and autoimmunity

Heat shock protein 90 (Hsp90) is a molecular chaperone involved in folding and stabilizing multiple intracellular proteins that have roles in cell activation and proliferation. Many Hsp90 client proteins in tumor cells are mutated or overexpressed oncogenic proteins driving cancer cell growth, leading to the acceptance of Hsp90 as a potential therapeutic target for cancer. Because several signal transduction molecules that are dependent on Hsp90 function are also involved in activation of innate and adaptive cells of the immune system, we investigated the mechanism by which inhibiting Hsp90 leads to therapeutic efficacy in rodent models of inflammation and autoimmunity. EC144, a synthetic Hsp90 inhibitor, blocked LPS-induced TLR4 signaling in RAW 264.7 cells by inhibiting activation of ERK1/2, MEK1/2, JNK, and p38 MAPK but not NF-κB. Ex vivo LPS-stimulated CD11b(+) peritoneal exudate cells from EC144-treated mice were blocked from phosphorylating tumor progression locus 2, MEK1/2, and ERK1/2. Consequently, EC144-treated mice were resistant to LPS administration and had suppressed systemic TNF-α release. Inhibiting Hsp90 also blocked in vitro CD4(+) T cell proliferation in mouse and human MLRs. In vivo, semitherapeutic administration of EC144 blocked disease development in rat collagen-induced arthritis by suppressing the inflammatory response. In a mouse collagen-induced arthritis model, EC144 also suppressed disease development, which correlated with a suppressed Ag-specific Ab response and a block in activation of Ag-specific CD4(+) T cells. Our results describe mechanisms by which blocking Hsp90 function may be applicable to treatment of autoimmune diseases involving inflammation and activation of the adaptive immune response.     

Investigation of the steps involved in the difference of susceptibility of Ephestia kuehniella and Spodoptera littoralis to the Bacillus thuringiensis Vip3Aa16 toxin

BUPM95 is a Bacillusthuringiensis subsp. kurstaki strain producing the Vip3Aa16 toxin with an interesting insecticidal activity against the Lepidopteran larvae Ephestia kuehniella. Study of different steps in the mode of action of this Vegetative Insecticidal Protein on the Mediterranean flour moth (E. kuehniella) was carried out in the aim to investigate the origin of the higher susceptibility of this insect to Vip3Aa16 toxin compared to that of the Egyptian cotton leaf worm Spodoptera littoralis. Using E. kuehniella gut juice, protoxin proteolysis generated a major band corresponding to the active toxin and another band of about 22 kDa, whereas the activation of Vip3Aa16 by S. littoralis gut juice proteases generated less amount of the 62 kDa active form and three other proteolysis products. As demonstrated by zymogram analysis, the difference in proteolysis products was due to the variability of proteases in the two gut juices larvae. The study of the interaction of E. kuehniella BBMV with biotinylated Vip3Aa16 showed that this toxin bound to a putative receptor of 65 kDa compared to the 55 and 100 kDa receptors recognized in S. littoralis BBMV. The histopathological observations demonstrated similar damage caused by the toxin in the two larvae midguts. These results demonstrate that the step of activation, mainly, is at the origin of the difference of susceptibility of these two larvae towards B. thuringiensis Vip3Aa16 toxin.     

Emergence pattern,microhabitat choice,and population structure of the Maghribian endemic Gomphus lucasii Selys, 1849 (Odonata: Gomphidae) in northeastern Algeria

Emergence of Gomphus lucasii Selys, 1849, an unstudied Maghreb endemic, was synchronised by overwintering in the final stadium in the Seybouse River in northeastern Algeria. Regular collections revealed that half of the annual population emerged during 10 days, showing a typical ‘spring species’ emergence pattern. Sex ratio was slightly male biased (51%). Males and females did not differ in vertical stratification. Emergence support choice was not random, but rather depended on support height, body size, and daily population density. Mortality was caused mainly by ants, although deformity of tenerals and bird predation were also important factors. Gomphus lucasii has been assessed as vulnerable (International Union for Conservation of Nature (IUCN) Red List), and the information provided in our study will be helpful in future conservation efforts.     

Antigenic Differences between AS03 Adjuvanted Influenza A (H1N1) Pandemic Vaccines: Implications for Pandemrix-Associated Narcolepsy Risk

Background

Narcolepsy results from immune-mediated destruction of hypocretin secreting neurons in hypothalamus, however the triggers and disease mechanisms are poorly understood. Vaccine-attributable risk of narcolepsy reported so far with the AS03 adjuvanted H1N1 vaccination Pandemrix has been manifold compared to the AS03 adjuvanted Arepanrix, which contained differently produced H1N1 viral antigen preparation. Hence, antigenic differences and antibody response to these vaccines were investigated.

Methods and Findings

Increased circulating IgG-antibody levels to Pandemrix H1N1 antigen were found in 47 children with Pandemrix-associated narcolepsy when compared to 57 healthy children vaccinated with Pandemrix. H1N1 antigen of Arepanrix inhibited poorly these antibodies indicating antigenic difference between Arepanrix and Pandemrix. High-resolution gel electrophoresis quantitation and mass spectrometry identification analyses revealed higher amounts of structurally altered viral nucleoprotein (NP) in Pandemrix. Increased antibody levels to hemagglutinin (HA) and NP, particularly to detergent treated NP, was seen in narcolepsy. Higher levels of antibodies to NP were found in children with DQB1*06∶02 risk allele and in DQB1*06∶02 transgenic mice immunized with Pandemrix when compared to controls.

Conclusions

This work identified 1) higher amounts of structurally altered viral NP in Pandemrix than in Arepanrix, 2) detergent-induced antigenic changes of viral NP, that are recognized by antibodies from children with narcolepsy, and 3) increased antibody response to NP in association of DQB1*06∶02 risk allele of narcolepsy. These findings provide a link between Pandemrix and narcolepsy. Although detailed mechanisms of Pandemrix in narcolepsy remain elusive, our results move the focus from adjuvant(s) onto the H1N1 viral proteins.     

Digoxin Derivatives with Enhanced Selectivity for the α2 Isoform of Na,K-ATPase: EFFECTS ON INTRAOCULAR PRESSURE IN RABBITS*

In the ciliary epithelium of the eye, the pigmented cells express the α1β1 isoform of Na,K-ATPase, whereas the non-pigmented cells express mainly the α2β3 isoform of Na,K-ATPase. In principle, a Na,K-ATPase inhibitor with selectivity for α2 could effectively reduce intraocular pressure with only minimal local and systemic toxicity. Such an inhibitor could be applied topically provided it was sufficiently permeable via the cornea. Previous experiments with recombinant human α1β1, α2β1, and α3β1 isoforms showed that the classical cardiac glycoside, digoxin, is partially α2-selective and also that the trisdigitoxose moiety is responsible for isoform selectivity. This led to a prediction that modification of the third digitoxose might increase α2 selectivity. A series of perhydro-1,4-oxazepine derivatives of digoxin have been synthesized by periodate oxidation and reductive amination using a variety of R-NH₂ substituents. Several derivatives show enhanced selectivity for α2 over α1, close to 8-fold in the best case. Effects of topically applied cardiac glycosides on intraocular pressure in rabbits have been assessed by their ability to either prevent or reverse acute intraocular pressure increases induced by 4-aminopyridine or a selective agonist of the A3 adenosine receptor. Two relatively α2-selective digoxin derivatives efficiently normalize the ocular hypertension, by comparison with digoxin, digoxigenin, or ouabain. This observation is consistent with a major role of α2 in aqueous humor production and suggests that, potentially, α2-selective digoxin derivatives could be of interest as novel drugs for control of intraocular pressure.     

Early Detection of Motor Dysfunction in the SOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis (ALS) Using Home Cage Running Wheels

The SOD1^G93A mouse has been used since 1994 for preclinical testing in amyotrophic lateral sclerosis (ALS). Despite recent genetic advances in our understanding of ALS, transgenic mice expressing mutant SOD1 remain the best available, and most widely used, vertebrate model of the disease. We previously described an optimised and rapid approach for preclinical studies in the SOD1^G93A mouse. Here we describe improvements to this approach using home cage running wheels to obtain daily measurements of motor function, with minimal intervention. We show that home cage running wheels detect reductions in motor function at a similar time to the rotarod test, and that the data obtained are less variable allowing the use of smaller groups of animals to obtain satisfactory results. This approach refines use of the SOD1^G93A model, and reduces the number of animals undergoing procedures of substantial severity, two central principles of the 3Rs (replacement, reduction and refinement of animal use in research). The small group sizes and rapid timescales enable affordable large-scale therapeutic pre-screening in the SOD1^G93A mouse, as well as rapid validation of published positive effects in a second laboratory, one of the major stumbling blocks in ALS preclinical therapy development.     

Carnosol Induces ROS-Mediated Beclin1-Independent Autophagy and Apoptosis in Triple Negative Breast Cancer

Background

In this study we investigated the in vitro and in vivo anticancer effect of carnosol, a naturally occurring polyphenol, in triple negative breast cancer.

Results

We found that carnosol significantly inhibited the viability and colony growth induced G2 arrest in the triple negative MDA-MB-231. Blockade of the cell cycle was associated with increased p21/WAF1 expression and downregulation of p27. Interestingly, carnosol was found to induce beclin1-independent autophagy and apoptosis in MDA-MB-231 cells. The coexistence of both events, autophagy and apoptosis, was confirmed by electron micrography. Induction of autophagy was found to be an early event, detected within 3 h post-treatment, which subsequently led to apoptosis. Carnosol treatment also caused a dose-dependent increase in the levels of phosphorylated extracellular signal-regulated kinase 1 and 2 (pERK1/2). Moreover, we show that carnosol induced DNA damage, reduced the mitochondrial potential and triggered the activation of the intrinsic and extrinsic apoptotic pathway. Furthermore, we found that carnosol induced a dose-dependent generation of reactive oxygen species (ROS) and inhibition of ROS by tiron, a ROS scavenger, blocked the induction of autophagy and apoptosis and attenuated DNA damage. To our knowledge, this is the first report to identify the induction of autophagy by carnosol.

Conclusion

In conclusion our findings provide strong evidence that carnosol may be an alternative therapeutic candidate against the aggressive form of breast cancer and hence deserves more exploration.     

Transient N-acetylgalactosaminylation of mannosyl phosphate side chain in Paramecium primaurelia glycosylphosphatidylinositols.

The surface antigens of the free-living protozoan Paramecium primaurelia belong to the family of glycosylphosphatidylinositol (GPtdIns)-anchored proteins. Using a cell-free system prepared from P. primaurelia, we have described the structure and biosynthetic pathway for GPtdIns glycolipids. The core glycans of the polar glycolipids are modified by a mannosyl phosphate side chain. The data suggest that the mannosyl phosphate side chain is added onto the core glycan in two steps. The first step involves the phosphorylation of the GPtdIns trimannosyl conserved core glycan via an ATP-dependent kinase, prior to the addition of the mannose linked to the phosphate group. We show that dolichol phosphate mannose is the donor of all mannose residues including the mannose linked to phosphate. Furthermore, we were able to identify in vitro a hydrophilic intermediate containing an additional N-acetylgalactosamine linked to the mannosyl phosphate side chain. The addition of this purified hydrophilic radiolabelled intermediate into the cell-free system leads to a loss of the GalNAc residue and its conversion to the penultimate intermediate having only mannosyl phosphate as a side chain. Together the data indicate that the GalNAc-containing intermediate is a transitional intermediate. We suggest that the GalNAc-containing intermediate is essential for biosynthesis and maturation of GPtdIns precursors. It is hypothesized that this oligosaccharide processing in the course of GPtdIns biosynthesis is required for the translocation of GPtdIns from the cytoplasmic side of the endoplasmic reticulum to the luminal side.