plasma-assisted-molecular-beam-epitaxy-n-polar-inaln-barrier-high

Plasma-assisted Molecular Beam Epitaxy of N-polar InAlN-barrier High-electron-mobility Transistors

Plasma-assisted molecular beam epitaxy is well suited for the epitaxial growth of III-nitride thin films and heterostructures with smooth, abrupt ...

Naval Research Laboratory

Trypanosoma cruzi glutathione-dependent peroxidase I (TcGPXI) can reduce fatty acid, phospholipid, and short chain organic hydroperoxides utilizing a novel redox cycle in which enzyme activity is linked to the reduction of trypanothione, a parasite-specific thiol, by glutathione. Here we show that TcGPXI activity can also be linked to trypanothione reduction by an alternative pathway involving the thioredoxin-like protein tryparedoxin. The presence of this new pathway was first detected using dialyzed soluble fractions of parasite extract. Tryparedoxin was identified as the intermediate molecule following purification, sequence analysis, antibody studies, and reconstitution of the redox cycle in vitro. The system can be readily saturated by trypanothione, the rate-limiting step being the interaction of trypanothione with the tryparedoxin. Both tryparedoxin and TcGPXI operate by a ping-pong mechanism. Overexpression of TcGPXI in transfected parasites confers increased resistance to exogenous hydroperoxides. TcGPXI contains a carboxyl-terminal tripeptide (ARI) that could act as a targeting signal for the glycosome, a kinetoplastid-specific organelle. Using immunofluorescence, tagged fluorescent proteins, and biochemical fractionation, we have demonstrated that TcGPXI is localized to both the glycosome and the cytosol. The ability of TcGPXI to use alternative electron donors may reflect their availability at the corresponding subcellular sites.

The Trypanosoma cruzi enzyme TcGPXI is a glycosomal peroxidase and can be linked to trypanothione reduction by glutathione or tryparedoxin.

Until recently, it had been thought that trypanosomes lack glutathione peroxidase activity. Here we report the subcellular localization and biochemical properties of a second glutathione-dependent peroxidase from Trypanosoma cruzi (TcGPXII). TcGPXII is a single-copy gene which encodes a 16 kDa protein that appears to be specifically dependent on glutathione as the source of reducing equivalents. Recombinant TcGPXII was purified and shown to have peroxidase activity towards a narrow substrate range, restricted to hydroperoxides of fatty acids and phospholipids. Analysis of the pathway revealed that TcGPXII activity could be readily saturated by glutathione and that the peroxidase functioned by a Ping Pong mechanism. Enzyme reduction was shown to be the rate-limiting step in this pathway. Using immunofluorescence, TcGPXII was shown to co-localize with a homologue of immunoglobulin heavy-chain binding protein (BiP), a protein restricted to the endoplasmic reticulum and Golgi. As the smooth endoplasmic reticulum is the site of phospholipid and fatty acid biosynthesis, this suggests that TcGPXII may play a specific role in the T. cruzi oxidative defence system by protecting newly synthesized lipids from peroxidation.

TcGPXII, a glutathione-dependent Trypanosoma cruzi peroxidase with substrate specificity restricted to fatty acid and phospholipid hydroperoxides, is localized to the endoplasmic reticulum.

Cytokines are the molecular messengers of the vertebrate immune system, coordinating the local and systemic immune responses to infective organisms. We report here functional and structural data on cytokine-like proteins from a eukaryotic pathogen. Two homologues of the human cytokine macrophage migration inhibitory factor (MIF) have been isolated from the parasitic nematode Brugia malayi. Both molecules (Bm-MIF-1 and Bm-MIF-2) show parallel functions to human MIF. They are chemotactic for human monocytes and activate them to produce IL-8, TNF-alpha, and endogenous MIF. The human and nematode MIF homologues share a tautomerase enzyme activity, which is in each case abolished by the mutation of the N-terminal proline residue. The crystal structure of Bm-MIF-2 at 1.8-A resolution has been determined, revealing a trimeric assembly with an inner pore created by beta-stranded sheets from each subunit. Both biological activity and crystal structure reveal remarkable conservation between a human cytokine and its parasite counterpart despite the considerable phylogenetic divide among these organisms. The strength of the similarity implies that MIF-mediated pathways play an important role in nematode immune evasion strategies.

Homologues of human macrophage migration inhibitory factor from a parasitic nematode. Gene cloning, protein activity, and crystal structure.

The Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) uniquely contains three cGMP binding sites, but also has a 'degenerate' fourth site. The role of each cGMP-binding site in PfPKG activation remains unknown. We have analysed the effect of mutation of each cGMP-binding site (individually and in combination) on PfPKG activation in vitro. The most striking result was that mutation of cGMP site 3 resulted in a 10-49-fold increase in the K (a((cGMP))) value and a 45-55% decrease in maximal activity compared with wild-type. Mutations involving only cGMP-binding sites 1 and 2 had less effect on both the K (a((cGMP))) values and the maximal activities. These results suggest that, although all three cGMP-binding sites are involved in PfPKG activation, cGMP-binding site 3 has the greatest influence on activation. A mutation in the fourth, degenerate cGMP-binding site decreased PfPKG maximal activity by 40%, but did not change the K (a((cGMP))) value for the PfPKG mutant, suggesting that this site does not bind cGMP, but is required for full activation of PfPKG. The distinct activation properties of PfPKG from mammalian isoforms may be exploitable in the design of a parasite-specific inhibitor and development of a novel anti-malarial drug.

The role of two novel regulatory sites in the activation of the cGMP-dependent protein kinase from Plasmodium falciparum.

The 8-amino and 9-hydroxy substituents of antimalarial cinchona alkaloids have the erythro orientation while their inactive 9-epimers are threo. From the X-ray structures a 90 degrees difference in torsion angle between the N1-H1 and C9-O12 bonds in the two series is believed to be important. In order to kill the malaria parasite, alkaloids must cross the erythrocyte and parasite membranes to accumulate in the acid digestive vacuole where they prevent detoxication of haematin produced during haemoglobin breakdown.

The relationship of physico-chemical properties and structure to the differential antiplasmodial activity of the cinchona alkaloids.

Serum amyloid A (SAA) is the major acute phase protein in man and most mammals. We observed SAA binding to a surprisingly large number of Gram-negative bacteria, including Escherichia coli, Salmonella typhimurium, Shigella flexneri, Klebsiella pneumoniae, Vibrio cholerae, and Pseudomonas aeruginosa. The binding was found to be high affinity and rapid. Importantly, this binding was not inhibited by high density lipoprotein with which SAA is normally complexed in serum. Binding was also observed when bacteria were offered serum containing SAA. Ligand blots following SDS-PAGE or two-dimensional gels revealed two major ligands of 29 and 35 kDa that bound SAA when probing with radiolabeled SAA or SAA and monoclonal anti-SAA. Following fractionation the ligand was found in the outer membrane fraction of E. coli and was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry to be outer membrane protein A (OmpA). OmpA-deficient E. coli did not bind SAA, and following purification of OmpA the protein retained binding activity. The ligands on other bacteria were likely to be homologues of OmpA because wild type, but not OprF-deficient, P. aeruginosa bound SAA.

Serum amyloid A protein binds to outer membrane protein A of gram-negative bacteria.

The disposal of misfolded proteins from the lumen of the endoplasmic reticulum (ER) is one of the quality control mechanisms present in the protein secretory pathway. Through ER-associated degradation, misfolded substrates are targeted to the cytosol where they are degraded by the proteasome. We have identified four maize (Zea mays) Der1-like genes (Zm Derlins) that encode homologs of Der1p, a yeast (Saccharomyces cerevisiae) protein implicated in ER-associated degradation. Zm Derlins are capable of functionally complementing a yeast Der1 deletion mutant. Such complementation indicates that the Der1p function is conserved among species. Zm Derlin genes are expressed at low levels throughout the plant, but appear prevalent in tissues with high activity of secretory protein accumulation, including developing endosperm cells. Expression of three of the four Zm Derlin genes increases during ER stress, with Zm Derlin1-1 showing the strongest induction. Subcellular fractionation experiments localized Zm Derlin proteins to the membrane fraction of microsomes. In maize endosperm, Zm Derlin proteins were found primarily associated with ER-derived protein bodies regardless of the presence of an ER stress response.

Identification and characterization of endoplasmic reticulum-associated degradation proteins differentially affected by endoplasmic reticulum stress.

We used whole genome scan association mapping to identify loci with major effect on oleic acid content in maize kernels. Single nucleotide polymorphism haplotypes at 8,590 loci were tested for association with oleic acid content in 553 maize inbreds. A single locus with major effect on oleic acid was mapped between 380 and 384 cM in the IBM2 neighbors genetic map on chromosome 4 and confirmed in a biparental population. A fatty acid desaturase, fad2, identified approximately 2 kb from the associated genetic marker, is the most likely candidate gene responsible for the differences in the phenotype. The fad2 alleles with high- and low-oleic acid content were sequenced and allelic differences in fad2 RNA level in developing embryos was investigated. We propose that a non-conservative amino acid polymorphism near the active site of fad2 contributes to the effect on oleic acid content. This is the first report of the use of a high resolution whole genome scan association mapping where a putative gene responsible for a quantitative trait was identified in plants.

Whole genome scan detects an allelic variant of fad2 associated with increased oleic acid levels in maize.

The Category Fluency Test (CFT) provides a sensitive measurement of cognitive capabilities in humans related to retrieval from semantic memory. In particular, it is widely used to assess progress of cognitive impairment in patients with dementia. Previous research shows that, in the first approximation, the intensity of tested individuals' responses within a standard 60-s test period decays exponentially with time, with faster decay rates for more cognitively impaired patients. Such decay rate can then be viewed as a global (macro) diagnostic parameter of each test. In the present paper we focus on the statistical properties of the properly de-trended time intervals between consecutive responses (inter-call times) in the Category Fluency Test. In a sense, those properties reflect the local (micro) structure of the response generation process. We find that a good approximation for the distribution of the de-trended inter-call times is provided by the Weibull Distribution, a probability distribution that appears naturally in this context as a distribution of a minimum of independent random quantities and is the standard tool in industrial reliability theory. This insight leads us to a new interpretation of the concept of "navigating a semantic space" via patient responses.

Random local temporal structure of category fluency responses.

Abstract We present a full-coordinate model of residues 1-319 of the polymerase domain of HIV-I reverse transcriptase. This model was constructed from the x-ray crystallographic structure of Jacobo-Molina et al. (Jacobo-Molina et al., P.N.A.S. USA 90, 6320-6324 (1993)) which is currently available to the degree of C- coordinates. The backbone and side-chain atoms were constructed using the MAXSPROUT suite of programs (L. Holm and C. Sander, Mol. Biol. 218, 183-194 (1991)) and refined through molecular modeling. A seven base pair A-form dsDNA was positioned in the nucleic acid binding cleft to represent the template-primer complex. The orientation of the template-primer complex in the nucleic acid binding cleft was guided by the positions of phosphorus atoms in the crystal structure. Two magnesium ions were placed in the active site in order to better understand the polymerization mechanism. The positions of metal ions in a number of structures guided the placement of ions in this study (L.S. Beese and T.A. Steitz, EMBO. J10, 25-33 (1991); T.A. Steitz and J.A. Steitz, P.N.A.S. USA 90, 6498-6502 (1993); D.L. Sloan et al., J. Biol. Chem. 250, 8913-8920 (1975); R.F. Setlik et al., J. Biomol. Str. Dyn. 10, 945-972 (1993)). The geometry of the active site allowed metal ions to be bound to Asp 110 and Asp 186 of the catalytic triad. However, due to spacial constraints, Asp 185 was found unable to bind to a metal ion. Due to its proximity to the attacking 3'OH group of the 3' terminal residue of the primer strand, it is proposed that this residue acts as a general base which abstracts a proton from the attacking group. Based on the locations of these metal ions with respect to the attacking group of the 3' end of the primer strand and to an incoming dTTP placed in the active site, we propose roles for the magnesium ions and discuss a mechanism through which chain elongation occurs. Also reported are the interactions between the polymerase domain and the template-primer complex observed in our model. These interactions are discussed in view of their possible roles in positioning the nucleic acid complex in the binding cleft and in regard to other structural and functional roles. The importance of these residues as observed in our model is compared to results from multiple sequence alignments and various mutational studies on HIV-I reverse transcriptase.

Article	Year
The Trypanosoma cruzi enzyme TcGPXI is a glycosomal peroxidase and can be linked to trypanothione reduction by glutathione or tryparedoxin. The Journal of biological chemistry\| PubMed ID: 11842085	2002
TcGPXII, a glutathione-dependent Trypanosoma cruzi peroxidase with substrate specificity restricted to fatty acid and phospholipid hydroperoxides, is localized to the endoplasmic reticulum. The Biochemical journal\| PubMed ID: 12049643	2002
Homologues of human macrophage migration inhibitory factor from a parasitic nematode. Gene cloning, protein activity, and crystal structure. The Journal of biological chemistry\| PubMed ID: 12221083	2002
The role of two novel regulatory sites in the activation of the cGMP-dependent protein kinase from Plasmodium falciparum. The Biochemical journal\| PubMed ID: 12817987	2003
The relationship of physico-chemical properties and structure to the differential antiplasmodial activity of the cinchona alkaloids. Malaria journal\| PubMed ID: 14505493	2003
Serum amyloid A protein binds to outer membrane protein A of gram-negative bacteria. The Journal of biological chemistry\| PubMed ID: 15705572	2005
Identification and characterization of endoplasmic reticulum-associated degradation proteins differentially affected by endoplasmic reticulum stress. Plant physiology\| PubMed ID: 15849299	2005
Whole genome scan detects an allelic variant of fad2 associated with increased oleic acid levels in maize. Molecular genetics and genomics : MGG\| PubMed ID: 17934760	2008
Random local temporal structure of category fluency responses. Journal of computational neuroscience\| PubMed ID: 21739237	2012
A Full-Coordinate Model of the Polymerase Domain of HIV-I Reverse Transcriptase and its Interaction With a Nucleic Acid Substrate. Journal of biomolecular structure & dynamics\| PubMed ID: 22671907	1994

David J. Meyer

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