The publication data currently available has been vetted by Vanderbilt faculty, staff, administrators and trainees. The data itself is retrieved directly from NCBI's PubMed and is automatically updated on a weekly basis to ensure accuracy and completeness.
If you have any questions or comments, please contact us.
Near the end of the Pleistocene epoch, populations of the woolly mammoth (Mammuthus primigenius) were distributed across parts of three continents, from western Europe and northern Asia through Beringia to the Atlantic seaboard of North America. Nonetheless, questions about the connectivity and temporal continuity of mammoth populations and species remain unanswered. We use a combination of targeted enrichment and high-throughput sequencing to assemble and interpret a data set of 143 mammoth mitochondrial genomes, sampled from fossils recovered from across their Holarctic range. Our dataset includes 54 previously unpublished mitochondrial genomes and significantly increases the coverage of the Eurasian range of the species. The resulting global phylogeny confirms that the Late Pleistocene mammoth population comprised three distinct mitochondrial lineages that began to diverge ~1.0-2.0 million years ago (Ma). We also find that mammoth mitochondrial lineages were strongly geographically partitioned throughout the Pleistocene. In combination, our genetic results and the pattern of morphological variation in time and space suggest that male-mediated gene flow, rather than large-scale dispersals, was important in the Pleistocene evolutionary history of mammoths.
The genus Acinetobacter is comprised of a diverse group of species, several of which have raised interest due to potential applications in bioremediation and agricultural purposes. In this work, we show that many species within the genus Acinetobacter possess the genetic requirements to assemble a functional type VI secretion system (T6SS). This secretion system is widespread among Gram negative bacteria, and can be used for toxicity against other bacteria and eukaryotic cells. The most studied species within this genus is A. baumannii, an emerging nosocomial pathogen that has become a significant threat to healthcare systems worldwide. The ability of A. baumannii to develop multidrug resistance has severely reduced treatment options, and strains resistant to most clinically useful antibiotics are frequently being isolated. Despite the widespread dissemination of A. baumannii, little is known about the virulence factors this bacterium utilizes to cause infection. We determined that the T6SS is conserved and syntenic among A. baumannii strains, although expression and secretion of the hallmark protein Hcp varies between strains, and is dependent on TssM, a known structural protein required for T6SS function. Unlike other bacteria, A. baumannii ATCC 17978 does not appear to use its T6SS to kill Escherichia coli or other Acinetobacter species. Deletion of tssM does not affect virulence in several infection models, including mice, and did not alter biofilm formation. These results suggest that the T6SS fulfils an important but as-yet-unidentified role in the various lifestyles of the Acinetobacter spp.
Functional analyses of candidate Heliothis virescens pheromone odorant receptors (HvORs) were conducted using heterologous expression in Xenopus oocytes. HvOR6 was found to be highly tuned to Z9-14:Ald, while HvOR13, HvOR14 and HvOR16 showed specificity for Z11-16:Ald, Z11-16:OAc and Z11-16:OH, respectively. HvOR15, which had been considered a candidate receptor for Z9-14:Ald did not respond to any of the pheromone compounds tested, nor to 50 other general odorants. Thus, while HvOR15 is specifically expressed in H. virescens male antennae, its role in pheromone reception remains unknown. Based on our results and previous research we can now assign pheromone receptors in H. virescens males to each of the critical H. virescens agonistic pheromone compounds and two antagonistic compounds produced by heterospecific females.
© 2010 The Authors. Insect Molecular Biology © 2010 The Royal Entomological Society.
Ferritin was purified from iron-fed Galleria mellonella hemolymph by ultra centrifugation and FPLC (Superose 6). SDS-PAGE revealed three bands of 26, 30, and 32 kDa. The ferritin 26 kDa subunit cDNA was obtained from RT-PCR using primer designed from N-terminal sequence analysis. 5'-RACE was used to obtain the complete protein coding sequence. The sequence encodes a 211 amino acid polypeptide including a 20 amino acid leader peptide. An IRE (iron-responsive element) sequence with a predicted stem-loop structure was present in the 5'-UTR of ferritin mRNA. Sequence alignment has a sequence identity with Calpodes ethlius (S)(74%), Drosophila melanogaster (50%), and Aedes aegypti (39%). Northern blot analysis indicated that there were 1.5- and 1.75-fold increases in the expression of ferritin mRNA after iron-fed fat body and midgut, respectively. Also, we confirmed that the ferritin mRNA is not expressed in adult ovary and testis. Arch.
Copyright 2001 Wiley-Liss, Inc.
A baculoviral expression vector consisting of a sequence encoding a six-histidine tag apposed to the human platelet 12-lipoxygenase cDNA, under control of the polyhedrin promoter, was constructed. Recombinant 12-lipoxygenase baculoviruses were used to infect Spodoptera frugiperda insect cells (Sf9). At 54 h post-infection, maximal 12-lipoxygenase activity and protein levels were achieved; the enzyme was purified to apparent homogeneity in a single step by nickel-ion-chelation chromatography in which the (His)6-tagged 12-lipoxygenase was eluted with 100 mM imidazole. The purified enzyme metabolized arachidonic acid almost exclusively to 12-hydroperoxyeicosatetraenoic acid with little, if any, epoxyalcohol or reduction products and had a Vmax of 2-4 mumol min-1 mg protein-1, Km of 10 microM and kcat of approximately 250 min-1. linoleic acid, on the other hand, was converted to (13S)-13-hydroperoxy-octadecadienoic acid at a rate which was about 2% of that obtained with arachidonic acid as substrate, but displayed the same Km. The enzyme was most active between pH 7.5-8 and activity was stimulated significantly in the presence of 0.006% Tween-20. A polyclonal antibody to the recombinant enzyme was generated and found to recognize a single 75-kDa band in platelets, human erythroleukemia cells and 12-lipoxygenase baculoviral-infected Sf9 cells by immunoblot and immunoprecipitation methods. 12-Lipoxygenase protein represented 0.1% of the total soluble protein in platelet preparations. In immunofluorescence experiments 12-lipoxygenase was observed in the cytoplasm of infected insect cells and in the human megakaryoblastic DAMI cell line. The isolation of large quantities of pure human platelet 12-lipoxygenase should facilitate detailed biochemical structure/function studies.
The beta and gamma subunits of heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) form tightly associated complexes. To examine functional differences among the large number of possible combinations of unique beta and gamma subunits, we have synthesized and characterized beta gamma complexes containing gamma 5 and gamma 7, two widely distributed gamma subunits. When either gamma 5 or gamma 7 is expressed concurrently with beta 1 or beta 2 subunits in a baculovirus/Sf9 cell system, all four subunit complexes support pertussis toxin-catalyzed ADP-ribosylation of rGi alpha 1 (where "r" indicates recombinant), indicating formation of functional complexes. Each of the complexes was purified by subunit exchange chromatography, using the G203A mutant of rGi alpha 1 as the immobilized ligand. The purified preparations were compared with other recombinant beta gamma subunits, including beta 1 gamma 1 and beta 1 gamma 2, for their ability to modulate type I and II adenylyl cyclase activities; stimulate phosphoinositide-specific phospholipase C beta; support pertussis toxin-catalyzed ADP-ribosylation of rGi alpha 1 and Go alpha; and inhibit steady-state GTP hydrolysis catalyzed by Gs alpha, Go alpha, and myristoylated rGi alpha 2. The results emphasize the unique properties of beta 1 gamma 1. The properties of the complexes containing gamma 5 or gamma 7 were similar to each other and to those of beta 1 gamma 2.
Arachidonate 12-lipoxygenase (12-LO) from porcine leukocytes was expressed in insect cells using a baculovirus expression vector. The recombinant 12-LO was expressed as an N-terminal fusion protein with a 31-amino acid polypeptide carrying a six-histidine tag and an enterokinase cleavage site. Maximal intracellular enzyme activity and protein levels were observed 48 h after infection of Spodoptera frugiperda cells with the recombinant virus. Cells were lysed and the recombinant protein was purified in a single step by Ni2+-nitrilotriacetate column chromatography. The purified enzyme migrated as a single band on sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. Recombinant enzyme catalyzed the formation of 12-hydroperoxy-5,8,10,14-eicosatetranoic acid and a small amount of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid. Chiral-phase HPLC analysis indicated that the 12-(S) enantiomer was the predominant product. The purified recombinant 12-lipoxygenase oxygenated linoleic acid to about 19% of the extent of oxygenation of arachidonic acid. Nordihydroguaiaretic acid and 5,8,11,14-eicosatetraynoic acid inhibited the recombinant enzyme with IC50's of 2.2 and 0.06 micM, respectively. Expression of cloned porcine leukocyte 12-LO in S. frugiperda cells and purification by Ni2+-nitrilotriacetate chromatography provides a straightforward method for isolation of milligram quantities of this form of 12-LO.
We have previously reported the purification of a palmitoyl-protein thioesterase (PPT) from bovine brain that removes palmitate from Ha-Ras (Camp, L. A., and Hofmann, S. L. (1993) J. Biol. Chem. 268, 22566-22574). In the current paper, we have isolated bovine and rat cDNA clones encoding PPT. The deduced amino acid sequence of PPT predicts a protein of 306 amino acids that contains amino acid motifs characteristic of thioesterases: "Gly-X-Ser-X-Gly" positioned near the NH2 terminus and "Gly-Asp-His" positioned near the COOH terminus of the protein. The identity of the PPT cDNA was further confirmed by expression in simian COS cells and insect Sf9 cells. Comparison of the DNA and protein sequence data suggests that a hydrophobic NH2-terminal sequence of 27 amino acid residues is removed from the primary translation product. Furthermore, the recombinant protein and the native protein purified from bovine brain contain complex asparagine-linked oligosaccharides and a large proportion of the expressed PPT is secreted from COS and Sf9 cells. Thus, while the palmitoyl-protein thioesterase will deacylate intracellular palmitoylated proteins such as Ha-Ras and the alpha subunits of heterotrimeric G proteins, the physiologic substrates are likely to be externally oriented or secreted proteins.
H-Ras, the protein product of the cellular homologue of the Harvey ras oncogene, undergoes a complex series of post-translational modifications that include C-terminal isoprenylation, proteolysis, methylation, and palmitoylation. Palmitoylation has been shown to enhance the transformation efficiency of H-Ras about 10-fold in vivo. A recent study (Magee, A. I., Gutierrez, L., McKay, I. A., Marshall, C. J., and Hall, A. (1987) EMBO J. 6, 3353-3357) has provided strong evidence that the palmitate undergoes a dynamic acylation-deacylation cycle, but details concerning the enzymology of this process and its regulation are lacking. To begin to dissect this event, we have developed an assay for the enzymatic removal of palmitate from [3H]palmitate-labeled H-Ras. This substrate was produced in a baculovirus expression system and was used to purify to homogeneity a novel 37-kDa enzyme from bovine brain cytosol that removes the radiolabeled palmitate. The purified enzyme is sensitive to diethyl pyrocarbonate and insensitive to phenylmethylsulfonyl fluoride and N-ethylmaleimide. Interestingly, the thioesterase recognizes H-Ras as a substrate only when H-Ras is in its native conformation (bound to Mg2+ and guanine nucleotide). The palmitoylated alpha subunits of the heterotrimeric G proteins are also substrates for the enzyme.
Discrete changes in silkmoth choriogenesis have occurred during evolution, as exemplified in the present report in Antheraea polyphemus and Hyalophora cecropia. At the level of morphology, the chorion of A. polyphemus has surface structures, called aeropyle crowns, that are absent from H. cecropia. Aeropyle crowns form during the very late period of choriogenesis and consist of two substructures--lamellae and filler. Filler is present in H. cecropia in greatly reduced amounts. At the level of protein synthesis, overall similarities in the two species are maintained until the very late period of choriogenesis, when synthesis of aeropyle crown components is maximal. In H. cecropia, very late period-specific proteins are reduced in number and abundance. Several of these minor proteins are candidates for E1 and E2, the components of filler. E1 and E2 RNAs are about 35 times more abundant in A. polyphemus, despite very similar gene copy numbers and times of expression in the two species. These results support the hypothesis that evolutionary changes in chorion morphology have resulted from regulatory changes in the expression of chorion genes, either at the level of transcription or mRNA decay. The hypothesis that evolutionary changes in chorion morphology are based on terminal addition onto a preexisting developmental program is discussed.