Arginine methylation regulates mitochondrial gene expression in Trypanosoma brucei through multiple effector proteins

Arginine methylation is a post-translational modification that impacts gene expression in both the cytoplasm and nucleus. Here, we demonstrate that arginine methylation also affects mitochondrial gene expression in the protozoan parasite, Trypanosoma brucei. Down-regulation of the major trypanosome type I protein arginine methyltransferase, TbPRMT1, leads to destabilization of specific mitochondrial mRNAs. We provide evidence that some of these effects are mediated by the mitochondrial RNA-binding protein, RBP16, which we previously demonstrated affects both RNA editing and stability. TbPRMT1 catalyzes methylation of RBP16 in vitro. Further, MALDI-TOF-MS analysis of RBP16 isolated from TbPRMT1-depleted cells indicates that, in vivo, TbPRMT1 modifies two of the three known methylated arginine residues in RBP16. Expression of mutated, nonmethylatable RBP16 in T. brucei has a dominant negative effect, leading to destabilization of a subset of those mRNAs affected by TbPRMT1 depletion. Our results suggest that the specificity and multifunctional nature of RBP16 are due, at least in part, to the presence of differentially methylated forms of the protein. However, some effects of TbPRMT1 depletion on mitochondrial gene expression cannot be accounted for by RBP16 action. Thus, these data implicate additional, unknown methylproteins in mitochondrial gene regulation.     

Proteomic based investigation of rhamnolipid production by Pseudomonas chlororaphis strain NRRL B-30761

We recently reported that a strain of the non-pathogenic bacterial species Pseudomonas chlororaphis was capable of producing the biosurfactant molecule, rhamnolipids. Previous to this report the organisms known to produce rhamnolipids were almost exclusively pathogens. The newly described P. chlororaphis strain produced rhamnolipids at room temperature in static minimal media, as opposed to previous reports of rhamnolipid production which occurred at elevated temperatures with mechanical agitation. The non-pathogenic nature and energy conserving production conditions make the P. chlororaphis strain an attractive candidate for commercial rhamnolipid production. However, little characterization of molecular/biochemical processes in P. chlororaphis have been reported. In order to achieve a greater understanding of the process by which P. chlororaphis produces rhamnolipids, a survey of proteins differentially expressed during rhamnolipid production was performed. Separation and measurement of the bacteria’s proteome was achieved using Beckman Coulter’s Proteome Lab PF2D packed column-based protein fractionation system. Statistical analysis of the data identified differentially expressed proteins and known orthologues of those proteins were identified using an AB 4700 Proteomics Analyzer mass spectrometer system. A list of proteins differentially expressed by P. chlororaphis strain NRRL B-30761 during rhamnolipid production was generated, and confirmed through a repetition of the entire separation process.Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

A chronology for late prehistoric Madagascar

A database has been assembled with 278 age determinations for Madagascar. Materials 14C dated include pretreated sediments and plant macrofossils from cores and excavations throughout the island, and bones, teeth, or eggshells of most of the extinct megafaunal taxa, including the giant lemurs, hippopotami, and ratites. Additional measurements come from uranium-series dates on speleothems and thermoluminescence dating of pottery. Changes documented include late Pleistocene climatic events and, in the late Holocene, the apparently human-caused transformation of the environment. Multiple lines of evidence point to the earliest human presence at ca. 2300 14C yr BP (350 cal yr BC). A decline in megafauna, inferred from a drastic decrease in spores of the coprophilous fungus Sporormiella spp. in sediments at 1720+/-40 14C yr BP (230-410 cal yr AD), is followed by large increases in charcoal particles in sediment cores, beginning in the SW part of the island, and spreading to other coasts and the interior over the next millennium. The record of human occupation is initially sparse, but shows large human populations throughout the island by the beginning of the Second Millennium AD. Dating of the "subfossil" megafauna, including pygmy hippos, elephant birds, giant tortoises, and large lemurs, demonstrates that most if not all the extinct taxa were still present on the island when humans arrived. Many taxa overlapped chronologically with humans for a millennium or more. The extinct lemurs Hadropithecus stenognathus, Pachylemur insignis, Mesopropithecus pithecoides, and Daubentonia robusta, and the elephant birds Aepyornis spp. and Mullerornis spp., were still present near the end of the First Millennium AD. Palaeopropithecus ingens, Megaladapis edwardsi, and Archaeolemur sp. (cf. edwardsi) may have survived until the middle of the Second Millennium A.D. One specimen of Hippopotamus of unknown provenance dates to the period of European colonization.     

Aged men display blunted biorhythmic variation of muscle performance and physiological responses.

Aging is known to disrupt the "biological clock" that governs physiological variables at rest. This study sought to determine whether aged men demonstrated biorhythmic variation in muscle performance during resistance exercise and physiological responses to that stimulus. Ten aged (75.6 +/- 1.6 yr; mean +/- SE) men completed an isokinetic testing protocol of knee extensors and flexors at 0800, 1200, 1600, and 2000 h. Although time of day variation in peak torque was detectable, significant (P < or = 0.05) oscillation was established only in the knee flexors at 3.14 rad/s. Heart rate, blood pressure, and rectal temperature displayed no significant variation, but trends (P < 0.10) in oscillation of postexercise blood pressure and rectal temperature were noted. Temporal patterns in biorhythmic variation of muscle performance, as well as thermal and cardiovascular measures, emulated those observed in a previous study involving young men where the magnitude of variation was sufficient to achieve statistical significance. Similar to our earlier findings in young men, however, pre- and postexercise testosterone and cortisol concentrations demonstrated significant variation among aged men. These data confirm the blunting of biorhythmic variation in muscle performance and physiological variables, except for circulating hormones, in aged men.     

Niche-specific features of the intestinal bacteroidales

By analyzing the genomic sequences of 12 Bacteroidales species, we found that all intestinal species have numerous polysaccharide biosynthesis loci, many with promoters that we demonstrate undergo DNA inversion. This feature is not conserved in the Bacteroidales order as a whole, as oral species do not share these genetic features.     

Carbon isotope effect study on orotidine 5'-monophosphate decarboxylase: support for an anionic intermediate

Orotidine 5'-monophosphate decarboxylase has been heavily examined in recent years due to its enzymatic proficiency, which provides a catalytic enhancement to a reaction rate approximately 1017 times greater than that of the nonenzymatic reaction. Several mechanisms proposed to explain this catalytic enhancement have included covalent addition, ylide or carbene formation, and most recently concerted protonation. All of these mechanisms have circumvented the formation of a high-energy vinyl anionic intermediate. To investigate the presence of an anionic intermediate, 13C isotope effect studies have been performed using the alternate substrate 5-fluoro-OMP (OMP = orotidine 5'-monophosphate). Isotope effects obtained for the wild-type enzyme with OMP and 5-fluoro-OMP are 1.0255 and 1.0106, respectively, corresponding to a decrease of approximately 1.5% for 5-fluoro-OMP. With the K59A enzyme, the intrinisic isotope effects show a similar decrease of approximately 1.9% from 1.0543 with OMP to 1.0356 with 5-fluoro-OMP. This decrease results from the inductive effect of the fluorine, which stabilizes the carbanion intermediate by electron withdrawal and produces a reaction with an earlier transition state. The isotope effect for the decarboxylation of the slow substrate 2'-deoxy-OMP produced a intrinsic isotope effect of nearly 1.0461.     

The Orf18 gene product from conjugative transposon Tn916 is an ArdA antirestriction protein that inhibits type I DNA restriction-modification systems

Gene orf18, which is situated within the intercellular transposition region of the conjugative transposon Tn916 from the bacterial pathogen Enterococcus faecalis, encodes a putative ArdA (alleviation of restriction of DNA A) protein. Conjugative transposons are generally resistant to DNA restriction upon transfer to a new host. ArdA from Tn916 may be responsible for the apparent immunity of the transposon to DNA restriction and modification (R/M) systems and for ensuring that the transposon has a broad host range. The orf18 gene was engineered for overexpression in Escherichia coli, and the recombinant ArdA protein was purified to homogeneity. The protein appears to exist as a dimer at nanomolar concentrations but can form larger assemblies at micromolar concentrations. R/M assays revealed that ArdA can efficiently inhibit R/M by all four major classes of Type I R/M enzymes both in vivo and in vitro. These R/M systems are present in over 50% of sequenced prokaryotic genomes. Our results suggest that ArdA can overcome the restriction barrier following conjugation and so helps increase the spread of antibiotic resistance genes by horizontal gene transfer.     

Caveolae as potential mediators of MCH-signaling pathways

The melanin-concentrating hormone receptor (MCHR) 1 is a G protein-coupled receptor involved in the regulation of appetite and energy expenditure in mammals. Here, we show that MCHR1 partitions to lipid rafts in stably expressing Chinese hamster ovary cells. In addition to co-fractionating with lipid rafts containing caveolin-1 on sucrose gradients, caveolin-1 was present in MCHR1 immunoprecipitates, suggesting that MCHR1 complexes with caveolae. The cholesterol-depleting drug methyl-β-cyclodextrin impaired MCH-mediated ERK signaling. These data suggest that a functional interaction between MCHR1 and caveolin-1 in lipid rafts exists and provide a basis for further biochemical studies to understand the significance on MCH-mediated signal transduction events.