Hydrophilic anilinogeranyl diphosphate prenyl analogues are Ras function inhibitors

Sequential processing of H-Ras by protein farnesyl transferase (FTase), Ras converting enzyme (Rce1), and protein-S-isoprenylcysteine O-methyltransferase (Icmt) to give H-Ras C-terminal farnesyl-S-cysteine methyl ester is required for appropriate H-Ras membrane localization and function, including activation of the mitogen-activated protein kinase (MAPK) cascade. We employed a Xenopus laevis oocyte whole-cell model system to examine whether anilinogeranyl diphosphate analogues of similar shape and size, but with a hydrophobicity different from that of the FTase substrate farnesyl diphosphate (FPP), could ablate biological function of H-Ras. Analysis of oocyte maturation kinetics following microinjection of in vitro analogue-modified H-Ras into isoprenoid-depleted oocytes revealed that analogues with a hydrophobicity near that of FPP supported H-Ras biological function, while the analogues p-nitroanilinogeranyl diphosphate (p-NO2-AGPP), p-cyanoanilinogeranyl diphosphate (p-CN-AGPP), and isoxazolaminogeranyl diphosphate (Isox-GPP) with hydrophobicities 2-5 orders of magnitude lower than that of FPP did not. We found that although H-Ras modified with FPP analogues p-NO2-AGPP, p-CN-AGPP, and Isox-GPP was an efficient substrate for C-terminal postprenylation processing by Rce1 and Icmt, co-injection of H-Ras with analogues p-NO2-AGPP, p-CN-AGPP, or Isox-GPP could not activate MAPK. We propose that H-Ras biological function requires a minimum lipophilicity of the prenyl group to allow important interactions downstream of the C-terminal processed H-Ras protein. The hydrophilic FPP analogues p-NO2-AGPP, p-CN-AGPP, and Isox-GPP are H-Ras function inhibitors (RFIs) and serve as lead compounds for a unique class of potential anticancer therapeutics.     

Sequence variation in nuclear ribosomal small subunit,internal transcribed spacer and large subunit regions of Rhizophagus irregularis and Gigaspora margarita is high and isolate‐dependent

Arbuscular mycorrhizal (AM) fungi are known to exhibit high intra‐organism genetic variation. However, information about intra‐ vs. interspecific variation among the genes commonly used in diversity surveys is limited. Here, the nuclear small subunit (SSU) rRNA gene, internal transcribed spacer (ITS) region and large subunit (LSU) rRNA gene portions were sequenced from 3 to 5 individual spores from each of two isolates of Rhizophagus irregularis and Gigaspora margarita. A total of 1482 Sanger sequences (0.5 Mb) from 239 clones were obtained, spanning ~4370 bp of the ribosomal operon when concatenated. Intrasporal and intra‐isolate sequence variation was high for all three regions even though variant numbers were not exhausted by sequencing 12–40 clones per isolate. Intra‐isolate nucleotide variation levels followed the expected order of ITS > LSU > SSU, but the values were strongly dependent on isolate identity. Single nucleotide polymorphism (SNP) densities over 4 SNP/kb in the ribosomal operon were detected in all four isolates. Automated operational taxonomic unit picking within the sequence set of known identity overestimated species richness with almost all cut‐off levels, markers and isolates. Average intraspecific sequence similarity values were 99%, 96% and 94% for amplicons in SSU, LSU and ITS, respectively. The suitability of the central part of the SSU as a marker for AM fungal community surveys was further supported by its level of nucleotide variation, which is similar to that of the ITS region; its alignability across the entire phylum; its appropriate length for next‐generation sequencing; and its ease of amplification in single‐step PCR.     

Mechanisms of amino acid-mediated lifespan extension in Caenorhabditis elegans

Background

Little is known about the role of amino acids in cellular signaling pathways, especially as it pertains to pathways that regulate the rate of aging. However, it has been shown that methionine or tryptophan restriction extends lifespan in higher eukaryotes and increased proline or tryptophan levels increase longevity in C. elegans. In addition, leucine strongly activates the TOR signaling pathway, which when inhibited increases lifespan.

Results

Therefore each of the 20 proteogenic amino acids was individually supplemented to C. elegans and the effects on lifespan were determined. All amino acids except phenylalanine and aspartate extended lifespan at least to a small extent at one or more of the 3 concentrations tested with serine and proline showing the largest effects. 11 of the amino acids were less potent at higher doses, while 5 even decreased lifespan. Serine, proline, or histidine-mediated lifespan extension was greatly inhibited in eat-2 worms, a model of dietary restriction, in daf-16/FOXO, sir-2.1, rsks-1 (ribosomal S6 kinase), gcn-2, and aak-2 (AMPK) longevity pathway mutants, and in bec-1 autophagy-defective knockdown worms. 8 of 10 longevity-promoting amino acids tested activated a SKN-1/Nrf2 reporter strain, while serine and histidine were the only amino acids from those to activate a hypoxia-inducible factor (HIF-1) reporter strain. Thermotolerance was increased by proline or tryptophan supplementation, while tryptophan-mediated lifespan extension was independent of DAF-16/FOXO and SKN-1/Nrf2 signaling, but tryptophan and several related pyridine-containing compounds induced the mitochondrial unfolded protein response and an ER stress response. High glucose levels or mutations affecting electron transport chain (ETC) function inhibited amino acid-mediated lifespan extension suggesting that metabolism plays an important role. Providing many other cellular metabolites to C. elegans also increased longevity suggesting that anaplerosis of tricarboxylic acid (TCA) cycle substrates likely plays a role in lifespan extension.

Conclusions

Supplementation of C. elegans with 18 of the 20 individual amino acids extended lifespan, but lifespan often decreased with increasing concentration suggesting hormesis. Lifespan extension appears to be caused by altered mitochondrial TCA cycle metabolism and respiratory substrate utilization resulting in the activation of the DAF-16/FOXO and SKN-1/Nrf2 stress response pathways.

Electronic supplementary material

The online version of this article (doi:10.1186/s12863-015-0167-2) contains supplementary material, which is available to authorized users.     

Mitophagy is required for mitochondrial biogenesis and myogenic differentiation of C2C12 myoblasts

Myogenesis is a crucial process governing skeletal muscle development and homeostasis. Differentiation of primitive myoblasts into mature myotubes requires a metabolic switch to support the increased energetic demand of contractile muscle. Skeletal myoblasts specifically shift from a highly glycolytic state to relying predominantly on oxidative phosphorylation (OXPHOS) upon differentiation. We have found that this phenomenon requires dramatic remodeling of the mitochondrial network involving both mitochondrial clearance and biogenesis. During early myogenic differentiation, autophagy is robustly upregulated and this coincides with DNM1L/DRP1 (dynamin 1-like)-mediated fragmentation and subsequent removal of mitochondria via SQSTM1 (sequestosome 1)-mediated mitophagy. Mitochondria are then repopulated via PPARGC1A/PGC-1α (peroxisome proliferator-activated receptor gamma, coactivator 1 alpha)-mediated biogenesis. Mitochondrial fusion protein OPA1 (optic atrophy 1 [autosomal dominant]) is then briskly upregulated, resulting in the reformation of mitochondrial networks. The final product is a myotube replete with new mitochondria. Respirometry reveals that the constituents of these newly established mitochondrial networks are better primed for OXPHOS and are more tightly coupled than those in myoblasts. Additionally, we have found that suppressing autophagy with various inhibitors during differentiation interferes with myogenic differentiation. Together these data highlight the integral role of autophagy and mitophagy in myogenic differentiation.     

Microfossils,a Key to Unravel Cold-Water Carbonate Mound Evolution through Time: Evidence from the Eastern Alboran Sea

Cold-water coral (CWC) ecosystems occur worldwide and play a major role in the ocean''s carbonate budget and atmospheric CO₂ balance since the Danian (~65 m.y. ago). However their temporal and spatial evolution against climatic and oceanographic variability is still unclear. For the first time, we combine the main macrofaunal components of a sediment core from a CWC mound of the Melilla Mounds Field in the Eastern Alboran Sea with the associated microfauna and we highlight the importance of foraminifera and ostracods as indicators of CWC mound evolution in the paleorecord. Abundances of macrofauna along the core reveal alternating periods dominated by distinct CWC taxa (mostly Lophelia pertusa, Madrepora oculata) that correspond to major shifts in foraminiferal and ostracod assemblages. The period dominated by M. oculata coincides with a period characterized by increased export of refractory organic matter to the seafloor and rather unstable oceanographic conditions at the benthic boundary layer with periodically decreased water energy and oxygenation, variable bottom water temperature/density and increased sediment flow. The microfaunal and geochemical data strongly suggest that M. oculata and in particular Dendrophylliidae show a higher tolerance to environmental changes than L. pertusa. Finally, we show evidence for sustained CWC growth during the Alleröd-Younger-Dryas in the Eastern Alboran Sea and that this period corresponds to stable benthic conditions with cold/dense and well oxygenated bottom waters, high fluxes of labile organic matter and relatively strong bottom currents     

Tissue-specific regulation by ecdysone: Distinct patterns of Eip28/29 expression are controlled by different ecdysone response elements

The Eip28/29 gene of Drosophila is an example of a tissue- and stage-specific ecdysone-responsive gene. Its diverse patterns of expression during the third larval instar and a synopsis of those patterns in terms of expression groups have been reported previously. Here we have studied the expression (in transgenic flies) of reporter genes controlled by Eip28/29-derived flanking DNA. During the middle and late third instar, most tissues exhibit normal expression patterns when controlled by one of two classes of regulatory sequences. Class A sequences include only 657 Np of 5′ flanking DNA from Eip28/29. Class B sequences include an extended 3′ flanking region and a minimal (≤93 Np) 5′ flanking region. The class B sequences include all those elements known to be important for ecdvsone induction in cultured cells. They are sufficient to direct the normal premetamorphic induction of Eip28/29 in the lymph glands, hemocytes, proventriculus, and Malpighian tubules. This is consistent with our suggestion that Kc cells are derived from embryonic hematopoietic cells. It is remarkable that the epidermis requires only class A sequences. These are sufficient to up-regulate expression at medinstar and to down-regulate expression at metamorphosis. It follows that the epidermis uses EcREs distinct from those that function in Kc cells. It is possible that the Upstream EcRE, which is nearly silent in Kc cells, is active in the epidermis. © 1994 Wiley-Liss, Inc.     

Valproic Acid Causes Proteasomal Degradation of DICER and Influences miRNA Expression

Valproic acid (VPA) is a commonly used drug to treat epilepsy and bipolar disorders. Known properties of VPA are inhibitions of histone deacetylases and activation of extracellular signal regulated kinases (ERK), which cannot fully explain VPA’s clinical features. We found that VPA induces the proteasomal degradation of DICER, a key protein in the generation of micro RNAs. Unexpectedly, the concentration of several micro RNAs increases after VPA treatment, which is caused by the upregulation of their hosting genes prior to DICER degradation. The data suggest that a loss of DICER protein and changes in micro RNA concentration contributes to the clinical properties of VPA. VPA can be used experimentally to down regulate DICER protein levels, which likely reflects a natural regulation of DICER.     

Exploring the Mechanism of Biocatalyst Inhibition in Microbial Desulfurization

Microbial desulfurization, or biodesulfurization (BDS), of fuels is a promising technology because it can desulfurize compounds that are recalcitrant to the current standard technology in the oil industry. One of the obstacles to the commercialization of BDS is the reduction in biocatalyst activity concomitant with the accumulation of the end product, 2-hydroxybiphenyl (HBP), during the process. BDS experiments were performed by incubating Rhodococcus erythropolis IGTS8 resting-cell suspensions with hexadecane at 0.50 (vol/vol) containing 10 mM dibenzothiophene. The resin Dowex Optipore SD-2 was added to the BDS experiments at resin concentrations of 0, 10, or 50 g resin/liter total volume. The HBP concentration within the cytoplasm was estimated to decrease from 1,100 to 260 μM with increasing resin concentration. Despite this finding, productivity did not increase with the resin concentration. This led us to focus on the susceptibility of the desulfurization enzymes toward HBP. Dose-response experiments were performed to identify major inhibitory interactions in the most common BDS pathway, the 4S pathway. HBP was responsible for three of the four major inhibitory interactions identified. The concentrations of HBP that led to a 50% reduction in the enzymes'' activities (IC₅₀s) for DszA, DszB, and DszC were measured to be 60 ± 5 μM, 110 ± 10 μM, and 50 ± 5 μM, respectively. The fact that the IC₅₀s for HBP are all significantly lower than the cytoplasmic HBP concentration suggests that the inhibition of the desulfurization enzymes by HBP is responsible for the observed reduction in biocatalyst activity concomitant with HBP generation.     

Synthesis of acyloxymethyl ester prodrugs of the transferable protein farnesyl transferase substrate farnesyl methylenediphosphonate

Three isoprenoid diphosphate analogues of farnesyl diphosphate (FPP) where the diphosphate has been replaced by methylene diphosphonate and the negative charges masked by frangible pivaloyloxymethyl (POM) esters were prepared. Farnesyl methylenediphosphonate is a sub-micromolar substrate for protein farnesyl transferase. The tripivaloyloxymethyl esters of isoprenoid methylenediphosphonate have significantly increased lipophilicity and may act as important farnesyl diphosphate prodrugs.     

Centrin in Giardia lamblia - ultrastructural localization

Giardia lamblia is a multiflagellar parasite and one of the earliest diverging eukaryotic cells. It possesses a complex cytoskeleton based on different groups of microtubular structures - a ventral adhesive disc, four pairs of flagella, a median body and funis. Centrin is an important member of the EF-hand family of calcium-binding proteins, and it is known to show calcium-sensitive contractile behaviour. In the present study, we performed an ultrastructural localization of centrin in G. lamblia using several monoclonal antibodies to centrin. Microtubular structures such as the basal bodies, all the flagella axonemes, the adhesive disc, funis, and the median bodies presented positive labelling to centrin. In addition, the dense rods also demonstrated positive labelling. These results show that centrin is located in key positions related to microtubules. The role of centrin in these dynamic regions is discussed.