Chronic rapamycin treatment or lack of S6K1 does not reduce ribosome activity in vivo

Reducing activity of the mTORC1/S6K1 pathway has been shown to extend lifespan in both vertebrate and invertebrate models. For instance, both pharmacological inhibition of mTORC1 with the drug rapamycin or S6K1 knockout extends lifespan in mice. Since studies with invertebrate models suggest that reducing translational activity can increase lifespan, we reasoned that the benefits of decreased mTORC1 or S6K1 activity might be due, at least in part, to a reduction of general translational activity. Here, we report that mice given a single dose of rapamycin have reduced translational activity, while mice receiving multiple injections of rapamycin over 4 weeks show no difference in translational activity compared with vehicle-injected controls. Furthermore, mice lacking S6K1 have no difference in global translational activity compared with wild-type littermates as measured by the percentage of ribosomes that are active in multiple tissues. Translational activity is reduced in S6K1-knockout mice following single injection of rapamycin, demonstrating that rapamycin’s effects on translation can occur independently of S6K1. Taken together, these data suggest that benefits of chronic rapamycin treatment or lack of S6K1 are dissociable from potential benefits of reduced translational activity, instead pointing to a model whereby changes in translation of specific subsets of mRNAs and/or translation-independent effects of reduced mTOR signaling underlie the longevity benefits.     

Biosynthesis of lysosomal hydrolases: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution

By in vitro translation of mRNA’s isolated from free and membrane-bound polysomes, direct evidence was obtained for the synthesis of two lysosomal hydrolases, β-glucuronidase of the rat preputial gland and cathespin D of mouse spleen, on polysomes bound to rough endoplasmic reticulum (ER) membranes. When the mRNA’s for these two proteins were translated in the presence of microsomal membranes, the in vitro synthesized polypeptides were cotranslationally glycosylated and transferred into the microsomal lumen. Polypeptides synthesized in the absence of microsomal membranes were approximately 2,000 daltons larger than the respective unglycosylated microsomal polypeptides found after short times of labeling in cultured rat liver cells treated with tunicamycin. This strongly suggests that nascent chains of the lysosomal enzymes bear transient amino terminal signals which determine synthesis on bound polysomes and are removed during the cotranslational insertion of the polypeptides into the ER membranes. In the line of cultured rat liver cells used for this work, newly synthesized lysosomal hydrolases showed a dual destination; approximately 60 percent of the microsomal polypeptides detected after short times of labeling were subsequently processed proteolytically to lower molecular weight forms characteristic of the mature enzymes. The remainder was secreted from the cells without further proteolytic processing. As previously observed by other investigations in cultured fibroblasts (A. Gonzalez-Noriega, J.H. Grubbs, V. Talkad, and W.S. Sly, 1980, J Cell Biol. 85: 839-852; A. Hasilik and E.F. Neufeld, 1980, J. Biol. Chem., 255:4937-4945.) the lysosomotropic amine chloroquine prevented the proteolytic maturation of newly synthesized hydrolases and enhanced their section. In addition, unglycosylated hydrolases synthesized in cells treated with tunicamycin were exclusively exported from the cells without undergoing proteolytic processing. These results support the notions that modified sugar residues serve as sorting out signals which address the hydrolases to their lysosomal destination and that final proteolytic cleavage of hydrolase precursors take place within lysosome itself. Structural differences in the carbohydrate chains of intracellular and secreted precursors of cathespin D were detected from their differential sensitivity to digestion with endoglycosidases H and D. These observations suggest that the hydrolases exported into the medium follow the normal secretory route and that some of their oligosaccharides are subject to modifications known to affect many secretory glycoproteins during their passage through the Golgi apparatus.     

Noisy-threshold control of cell death

Background  

Cellular responses to death-promoting stimuli typically proceed through a differentiated multistage process, involving a lag phase, extensive death, and potential adaptation. Deregulation of this chain of events is at the root of many diseases. Improper adaptation is particularly important because it allows cell sub-populations to survive even in the continuous presence of death conditions, which results, among others, in the eventual failure of many targeted anticancer therapies.     

Molecular evolution of P transposable elements in the genus Drosophila. III. The melanogaster species group

Phylogenetic relationships were determined for 76 partial P-element sequences from 14 species of the melanogaster species group within the Drosophila subgenus Sophophora. These results are examined in the context of the phylogeny of the species from which the sequences were isolated. Sequences from the P-element family fall into distinct subfamilies, or clades, which are often characteristic for particular species subgroups. When examined locally among closely related species, the evolution of P elements is characterized by vertical transmission, whereby the P-element phylogeny traces the species phylogeny. On a broader scale, however, the P-element phylogeny is not congruent with the species phylogeny. One feature of P-element evolution in the melanogaster group is the presence of more than one P-element subfamily, differing by as much as 36%, in the genomes of some species. Thus, P elements from several individual species are not monophyletic, and a likely explanation for the incongruence between P-element and species phylogenies is provided by the comparison of paralogous sequences. In certain instances, horizontal transfer seems to be a valid alternative explanation for lack of congruence between species and P-element phylogenies. The canonical P-element subfamily, which represents the active, autonomous transposable element, is restricted to D. melanogaster. Thus, its origin clearly lies outside of the melanogaster species group, consistent with the earlier conclusion of recent horizontal transfer.      

Effects of arsenate (AS5+) on growth and production of glutathione (GSH) and phytochelatins (PCS) in Chlorella vulgaris

The effect of arsenate (As5+) on growth and chlorophyll a production in Chlorella vulgaris, its removal by C. vulgaris and the role of glutathione (GSH) and phytochelatins (PCs) were investigated. C. vulgaris was tolerant to As5+ at up to 200 mg/L and was capable of consistently removing around 70% of the As5+ present in growth media over a wide range of exposure concentrations. Spectral analysis revealed that PCs and their arsenic-combined complexes were absent, indicating that the high bioaccumulation and tolerance to arsenic observed was not due to intracellular chelation. In contrast, GSH was found in all samples ranging from 0.8 mg/L in the control to 6.5 mg/L in media containing 200 mg/L As5+ suggesting that GSH plays a more prominent role in the detoxification of As5+ in C. vulgaris than PC. At concentrations below 100 mg/L cell surface binding and other mechanisms may play the primary role in As5+ detoxification, whereas above this concentration As5+ begins to accumulate inside the algal cells and activates a number of intracellular cell defense mechanisms, such as increased production of GSH. The overall findings complement field studies which suggest C. vulgaris as an increasingly promising low cost As phytoremediation method for developing countries.     

Competition of As and other Group 15 elements for surface binding sites of an extremophilic Acidomyces acidophilus isolated from a historical tin mining site

An arsenic-resistant fungal strain, designated WKC-1, was isolated from a waste roaster pile in a historical tin mine in Cornwall, UK and successfully identified to be Acidomyces acidophilus using matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS) proteomic-based biotyping approach. WKC-1 showed considerable resistance to As⁵⁺ and Sb⁵⁺ where the minimal inhibitory concentration (MIC) were 22500 and 100 mg L⁻¹, respectively, on Czapex-Dox Agar (CDA) medium; it was substantially more resistant to As⁵⁺ than the reference strains CBS 335.97 and CCF 4251. In a modified CDA medium containing 0.02 mg L⁻¹ phosphate, WKC-1 was able to remove 70.30% of As⁵⁺ (100 mg L⁻¹). Sorption experiment showed that the maximum capacity of As⁵⁺ uptake was 170.82 mg g⁻¹ dry biomass as predicted by the Langmuir model. The presence of Sb⁵⁺ reduced the As⁵⁺ uptake by nearly 40%. Based on the Fourier-transform infrared spectroscopy (FT-IR) analysis, we propose that Sb is competing with As for these sorption sites: OH, NH, CH, SO₃ and PO₄ on the fungal cell surface. To our knowledge, this is the first report on the impact of other Group 15 elements on the biosorption of As⁵⁺ in Acidomyces acidophilus.

     

Monoclonal antibody to dengue capsid protein: Its application in dengue studies

Dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) are considered the most important arthropod-borne viral diseases in terms of morbidity and mortality. The emergency and severity of dengue (Den) infections increase the necessity of an early, quick and effective dengue laboratory diagnostic. Viral isolation is considered a gold standard for diagnosis of dengue infection using monoclonal antibodies (mAbs) as a tool for determining serotype specificity. Alternatives have been used to improve sensitivity and time to dengue diagnosis. Based on the early expression of dengue C protein in the life cycle, we focused our study on the application of an anti-dengue 2 virus capsid protein mAb in dengue diagnosis. The kinetic expression of dengue-2 capsid in mosquito cells and its immuno-localization in experimentally infected suckling albin Swiss (OF-1) mice brain tissues was established. The results demonstrate the possible utility of this mAb in early dengue diagnosis versus traditional isolation. In addition, a preliminary study of an enzyme immunoassay method using 8H8 mAb for specific detection of dengue C protein antigen was performed, making possible recombinant C protein quantification. The results suggest that detection of dengue capsid protein could be useful in the diagnosis of early dengue infection.Key words: monoclonal antibodies, capsid protein, dengue virus, diagnosis, immunoassays     

Evaluation of a vermicompost and leachates on <i>Solidago x hybrida</i> and organic carbon mineralization under aerobic incubations

In the floriculture region of Tenancingo in the State of Mexico, the application of stabilized organic matter, such as vermicompost and leachates, contributes to improve the quality of the soil and plant nutrition. However, it is important to know the chemical composition of a vermicompost and the mineralization process. This is because the amount and speed of nutrient release which will be available to the crop will depend on that knowledge. The objective of the study was to evaluate the effect of the application of a vermicompost and leachates on various quantitative variables of Solidago x hybrid, and the mineralization of organic carbon under aerobic incubations. Vermicompost (74 and 36 g/kg soil), leachates (5 and 10 L/kg soil), 0.33 g/kg soil of chemical fertilizer Ca (NO₃)₂, and not treated soil (control) were applied under greenhouse conditions to evaluate their effects on plant growth variables. Mixtures of 100 g of soil with vermicompost and leachates were made in the laboratory which were incubated during 9 weeks to obtain the potentially mineralizable organic carbon (Corg PM) and the rate of mineralization (k) after adjusting an exponential model. In the greenhouse experiment there were no statistical differences after applying vermicompost and leachates on the quantitative variables (number of stems per plant, diameter of the panicle, fresh weight, plant length and stem diameter) with respect to the control (p>0.05). The effect among the applied doses was evident only for variables such as fresh weight, panicle length and stem diameter with respect to the control. In incubated soils, k values ranged between 0.209-0.325 C mg/kg soil/week. Only with the application of leachates in high doses two pools of organic matter were shown: one soluble labile (102.9 mg/kg soil) and the other hydrolysable (819 mg/kg soil). The soluble, labile fraction favored nutrient availability immediately after its application to the soil. However, a single pool of hydrolysable organic C (987-1074 mg/kg soil) was found when vermicompost was applied. It was associated with a release of organic matter during crop development, and to a possible stimulation of microbial activity. High values of electrical conductivity in vermicompost and leachates (8.2-11.7 mS/m) suggest a moderate application of both products.