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Very solid evidence suggests that the core of full length PrPSc is a 4-rung β-solenoid, and that individual PrPSc subunits stack to form amyloid fibers. We recently used limited proteolysis to map the β-strands and connecting loops that... more
Very solid evidence suggests that the core of full length PrPSc is a 4-rung β-solenoid, and that individual PrPSc subunits stack to form amyloid fibers. We recently used limited proteolysis to map the β-strands and connecting loops that make up the PrPSc solenoid. Using high resolution SDS-PAGE followed by epitope analysis, and mass spectrometry, we identified positions ~116/118, 133-134, 141, 152-153, 162, 169 and 179 (murine numbering) as Proteinase K (PK) cleavage sites in PrPSc. Such sites likely define loops and/or borders of β-strands, helping us to predict the threading of the β-solenoid. We have now extended this approach to recombinant PrPSc (recPrPSc). The term recPrPSc refers to bona fide recombinant prions prepared by PMCA, exhibiting infectivity with attack rates of ~100%. Limited proteolysis of mouse and bank vole recPrPSc species yielded N-terminally truncated PK-resistant fragments similar to those seen in brain-derived PrPSc, albeit with varying relative yields. Alo...
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A relational database, the Available Pathways Database (APD), has been constructed of microbial natural products, their producing strains, and their biosynthetic pathways. The database allows the ready selection of donor strains for... more
A relational database, the Available Pathways Database (APD), has been constructed of microbial natural products, their producing strains, and their biosynthetic pathways. The database allows the ready selection of donor strains for combinatorial biology experiments. It provides the same type of resource for combinatorial biology as the Available Chemicals Directory (ACD) does for combinatorial chemical library generation. Its cataloging ability can also provide insight into novel aspects of biosynthetic routes. In particular, no 10-unit Type I polyketides were found in the compilation of this edition of the APD (Version I).
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Prions (PrP(Sc)) are molecular pathogens that are able to convert the isosequential normal cellular prion protein (PrP(C)) into a prion. The only demonstrated difference between PrP(C) and PrP(Sc) is conformational: they are isoforms. A... more
Prions (PrP(Sc)) are molecular pathogens that are able to convert the isosequential normal cellular prion protein (PrP(C)) into a prion. The only demonstrated difference between PrP(C) and PrP(Sc) is conformational: they are isoforms. A given host can be infected by more than one kind or strain of prion. Five strains of hamster-adapted scrapie [Sc237 (=263K), drowsy, 139H, 22AH, and 22CH] and recombinant PrP were reacted with five different concentrations (0, 1, 5, 10, and 20 mM) of reagent (N-hydroxysuccinimide ester of acetic acid) that acetylates lysines. The extent of lysine acetylation was quantitated by mass spectrometry. The lysines in rPrP react similarly. The lysines in the strains react differently from one another in a given strain and react differently when strains are compared. Lysines in the C-terminal region of prions have different strain-dependent reactivity. The results are consistent with a recently proposed model for the structure of a prion. This model proposes that prions are composed of a four-rung β-solenoid structure comprised of four β-sheets that are joined by loops and turns of amino acids. Variation in the amino acid composition of the loops and β-sheet structures is thought to result in different strains of prions.
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Infectious proteins (prions) are, ironically, defined by their resistance to proteolytic digestion. A defining characteristic of the transmissible isoform of the prion protein (PrP(Sc)) is its partial resistance to proteinase K (PK)... more
Infectious proteins (prions) are, ironically, defined by their resistance to proteolytic digestion. A defining characteristic of the transmissible isoform of the prion protein (PrP(Sc)) is its partial resistance to proteinase K (PK) digestion. Diagnosis of prion disease typically relies upon immunodetection of PK-digested PrP(Sc) by Western blot, ELISA or immunohistochemical detection. PK digestion has also been used to detect differences in prion strains. Thus, PK has been a crucial tool to detect and, thereby, control the spread of prions. PK has also been used as a tool to probe the structure of PrP(Sc). Mass spectrometry and antibodies have been used to identify PK cleavage sites in PrP(Sc). These results have been used to identify the more accessible, flexible stretches connecting the β-strand components in PrP(Sc). These data, combined with physical constraints imposed by spectroscopic results, were used to propose a qualitative model for the structure of PrP(Sc). Assuming tha...
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Prions are molecular pathogens, able to convert a normal cellular prion protein (PrP(C)) into a prion (PrP(Sc)). The information necessary for this conversion is contained in the conformation of PrP(Sc). Mass spectrometry (MS) and... more
Prions are molecular pathogens, able to convert a normal cellular prion protein (PrP(C)) into a prion (PrP(Sc)). The information necessary for this conversion is contained in the conformation of PrP(Sc). Mass spectrometry (MS) and small-molecule covalent reactions have been used to study prions. Mass spectrometry has been used to detect and quantitate prions in the attomole range (10⁻¹⁸ mole). MS-based analysis showed that both possess identical amino acid sequences, one disulfide bond, a GPI anchor, asparagine-linked sugar antennae, and unoxidized methionines. Mass spectrometry has been used to define elements of the secondary and tertiary structure of wild-type PrP(Sc) and GPI-anchorless PrP(Sc). It has also been used to study the quaternary structure of the PrP(Sc) multimer. Small molecule reagents react differently with the same lysine in the PrP(C) conformation than in the PrP(Sc) conformation. Such differences can be detected by Western blot using mAbs with lysine-containing e...
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PrP(Sc) is an infectious protein. The only experimentally verified difference between PrP(Sc) and its normal cellular isoform (PrP(C)) is conformational. This work describes an approach to determining the presence of surface exposed or... more
PrP(Sc) is an infectious protein. The only experimentally verified difference between PrP(Sc) and its normal cellular isoform (PrP(C)) is conformational. This work describes an approach to determining the presence of surface exposed or sequestered amino acids present in the PrP(Sc) isoform. The N-hydroxysuccinimide esters of acetic acid and 4-trimethylammoniumbutyric acid were synthesized and reacted with detergent-solubilized brain extracts from Me7-infected mice, uninfected mice, 263K-infected hamsters or uninfected hamsters. These reaction mixtures were analyzed by western blots probed with the antibodies 3F4, 6D11, 7D9, AG4, AH6, GE8 or MAB5424. The 3F4, 6D11, AH6, and GE8 antibodies recognize an epitope that is encrypted in the PrP(Sc) isoform, but exposed in the PrP(C) isoform. These reagents permit the detection of prion infected brain extracts without the need for proteinase K digestion. In addition they can be used, with an appropriate antibody, to determine which amino aci...
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1. [3-3H]-Squalene was fed to 11 marine sponges containing a mixture of "common" sterol side chains. All of these sponges possess significant quantities of cholesterol, but their ability to biosynthesize it differs widely. 2.... more
1. [3-3H]-Squalene was fed to 11 marine sponges containing a mixture of "common" sterol side chains. All of these sponges possess significant quantities of cholesterol, but their ability to biosynthesize it differs widely. 2. All the sponges possess significant quantities of delta 22 sterols, yet none of them was able to introduce the delta 22 double bond. 22-Dehydro-24-norcholesterol and 24-methyl-22-dehydro-27-norcholesterol side chains also originate from the diet. 3. These sponges biosynthesized between 40 and 80% of their sterols, a typical value being 70%. The remainder is derived from the diet or by modification of dietary sterols.
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12 Screening of Combinatorial Biology Libraries for Natural Products Discovery Christopher J. Silva and Paul Brian Cubist Pharmaceuticals, Vancouver, British Columbia, Canada Todd Peterson Genicon Sciences Corporation, San Diego,... more
12 Screening of Combinatorial Biology Libraries for Natural Products Discovery Christopher J. Silva and Paul Brian Cubist Pharmaceuticals, Vancouver, British Columbia, Canada Todd Peterson Genicon Sciences Corporation, San Diego, California I. INTRODUCTION The ...
The cellular prion protein (PrP(C)) is a highly conserved protein, which is anchored to the outer surface of the plasma membrane. Even though its physiological function has already been investigated in different cell or mouse models where... more
The cellular prion protein (PrP(C)) is a highly conserved protein, which is anchored to the outer surface of the plasma membrane. Even though its physiological function has already been investigated in different cell or mouse models where PrP(C) expression is either upregulated or depleted, its exact physiological role in a mammalian organism remains elusive. Recent studies indicate that PrP(C) has multiple functions and is involved in cognition, learning, anxiety, locomotion, depression, offensive aggression and nest building behavior. While young animals (3 months of age) show only marginal abnormalities, most of the deficits become apparent as the animals age, which might indicate its role in neurodegeneration or neuroprotection. However, the exact biochemical mechanism and signal transduction pathways involving PrP(C) are only gradually becoming clearer. We report the observations made in different studies using different Prnp0/0 mouse models and propose that PrP(C) plays an imp...
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Chapter 22 Overview Immobilization for High-Throughput Screening Nicole M. Nasby, Todd. C. Peterson, and Christopher I. Silva Introduction Immobilization is an important and emerging approach in biotechnology for investigation into... more
Chapter 22 Overview Immobilization for High-Throughput Screening Nicole M. Nasby, Todd. C. Peterson, and Christopher I. Silva Introduction Immobilization is an important and emerging approach in biotechnology for investigation into microbial and cellular processes. ...
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ABSTRACT Calysterol (1) was shown not to be biosynthesized by direct dehydrogenation of its cyclopropane analog, dihydrocalysterol (5). Loss of H-28 from 5 during the biosynthesis of 1 was demonstrated, which implies that either... more
ABSTRACT Calysterol (1) was shown not to be biosynthesized by direct dehydrogenation of its cyclopropane analog, dihydrocalysterol (5). Loss of H-28 from 5 during the biosynthesis of 1 was demonstrated, which implies that either 24-H-isocalysterol (2) or (23S)-23H-isocalysterol (4) is the direct dehydrogenation product of 5. A novel synthesis of 5 is described which allowed facile introduction of tritium at C-28.
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The cellular prion protein (PrPC) is a highly conserved protein whose exact physiological role remains elusive. In the present study, we investigated age-dependent behavioral abnormalities in PrPC-knockout (Prnp0/0) mice and wild-type... more
The cellular prion protein (PrPC) is a highly conserved protein whose exact physiological role remains elusive. In the present study, we investigated age-dependent behavioral abnormalities in PrPC-knockout (Prnp0/0) mice and wild-type (WT) controls. Prnp0/0 mice showed age-dependent behavioral deficits in memory performance, associative learning, basal anxiety, and nest building behavior. Using a hypothesis-free quantitative proteomic investigation, we found that loss of PrPC affected the levels of neurofilament proteins in an age-dependent manner. In order to understand the biochemical basis of these observations, we analyzed the phosphorylation status of neurofilament heavy chain (NF-H). We found a reduction in NF-H phosphorylation in both Prnp0/0 mice and in PrPC-deficient cells. The expression of Fyn and phospho-Fyn, a potential regulator for NF phosphorylation, was associated with PrPC ablation. The number of β-tubulin III-positive neurons in the hippocampus was diminished in Prnp0/0 mice relative to WT mice. These data indicate that PrPC plays an important role in cytoskeletal organization, brain function, and age-related neuroprotection. Our work represents the first direct biochemical link between these proteins and the observed behavioral phenotypes.
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Daptomycin is a 13 amino acid, cyclic lipopeptide produced by a non-ribosomal peptide synthetase (NRPS) mechanism inStreptomyces roseosporus. A 128 kb region ofS. roseosporusDNA was cloned and verified by heterologous expression... more
Daptomycin is a 13 amino acid, cyclic lipopeptide produced by a non-ribosomal peptide synthetase (NRPS) mechanism inStreptomyces roseosporus. A 128 kb region ofS. roseosporusDNA was cloned and verified by heterologous expression inStreptomyces lividansto contain the daptomycin biosynthetic gene cluster (dpt). The cloned region was completely sequenced and three genes (dptA,dptBC,dptD) encoding the three subunits of an NRPS were identified. The catalytic domains in the subunits, predicted to couple five, six or two amino acids, respectively, included a novel activation domain and amino-acid-binding pocket for incorporating the unusual amino acidl-kynurenine (Kyn), three types of condensation domains and an extra epimerase domain (E-domain) in the second module. Novel genes (dptE,dptF) whose products likely work in conjunction with a unique condensation domain to acylate the first amino acid, as well as other genes (dptI,dptJ) probably involved in supply of the non-proteinogenic amino...
Research Interests: Microbiology, Gene expression, Multidisciplinary, Streptomyces, Sequencing, and 14 moreNatural Product, Bacteria, Cyclic peptides, Daptomycin, Molecular cloning, Anti-Bacterial Agents, Phylogenetic Tree, Amino Acid Sequence, Base Sequence, Heterologous Expression, Molecular Sequence Data, conserved sequence , HPLC Chromatography, and stereoisomerism
... by isomerization of the tertiary carbonium ion (9) to the requisite secondary carbonium ion at (2-23 (lo), might be the enzymatic step leading to the cyclopropyl sterols ( 1-4).7*8 However, when this was tested experimentally with... more
... by isomerization of the tertiary carbonium ion (9) to the requisite secondary carbonium ion at (2-23 (lo), might be the enzymatic step leading to the cyclopropyl sterols ( 1-4).7*8 However, when this was tested experimentally with [3H]SAM in a cell ... (6) (a) Proudfoot, JR; Djerassi, C ...
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Daptomycin and the A21978C antibiotic complex are lipopeptides produced by Streptomyces roseosporus and also in recombinant Streptomyces lividans TK23 and TK64 strains, when a 128 kbp region of cloned S. roseosporus DNA containing the... more
Daptomycin and the A21978C antibiotic complex are lipopeptides produced by Streptomyces roseosporus and also in recombinant Streptomyces lividans TK23 and TK64 strains, when a 128 kbp region of cloned S. roseosporus DNA containing the daptomycin gene cluster is inserted site-specifically in the phiC31 attB site. A21978C fermentation yields were initially much lower in S. lividans than in S. roseosporus, and detection was complicated by the production of host metabolites. However A21978C production in S. lividans was improved by deletion of genes encoding the production of actinorhodin and by medium optimization to control the chemical form of the calcium dependent antibiotic (CDA). This latter compound has not previously been chemically characterized as a S. lividans product. Adding phosphate to a defined fermentation medium resulted in formation of only the phosphorylated forms of CDA, which were well separated from A21978C on chromatographic analysis. Adjusting the level of phosphate in the medium led to an improvement in A21978C yield from 20 to 55 mg/l.
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Šormosterol ((24R)-24,25-methylenecholesterol, XVIII, a product of the novo sterol biosynthesis in the marine sponge Lissodendoryx topsenti, was shown, trough the use of suitably labeled precursors, to be a new alternative product of the... more
Šormosterol ((24R)-24,25-methylenecholesterol, XVIII, a product of the novo sterol biosynthesis in the marine sponge Lissodendoryx topsenti, was shown, trough the use of suitably labeled precursors, to be a new alternative product of the initial S-adenosylmethionine (SAM) methylation of the 24,25 double bond in the sterol side chain. Additional labeling experiments demonstrated that the cyclopropane ring is not further modifies in vivo by the sponge.
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We developed a sensitive mass spectrometry-based method of quantitating the prions present in a variety of mammalian species. Calibration curves relating the area ratios of the integrated MRM signals from selected analyte peptides and... more
We developed a sensitive mass spectrometry-based method of quantitating the prions present in a variety of mammalian species. Calibration curves relating the area ratios of the integrated MRM signals from selected analyte peptides and their oxidized analogues to their homologous stable isotope labeled internal standards were prepared. The limit of detection (LOD) and limit of quantitation (LOQ) for the synthetic peptides from human, sheep, deer, cow, and mouse PrP were determined to be below 100 amol. Nonanalyte peptides that were characteristic of prions were included in the multiple reaction monitoring method, thereby allowing for both the quantitation and confirmation of the presence of prions in the attomole range. This method was used to quantitate the prions present in brains of hamsters or mice 5 weeks after inoculation (ic) with either four hamster-adapted prion strains (139H, drowsy, 22AH, and 22CH) or four mouse-adapted prion strains (Me7, Me7-298, RML, and 79A). The prions from different brain regions of a sheep naturally infected with scrapie were quantitated. All of the rodent-adapted prion strains were detectable in the asymptomatic animals. In sheep, prions were detectable in the obex, anterior portion of the cerebrum, and the nonobex/nonanterior portion of the cerebrum. This mass spectrometry-based approach can be used to quantitate and confirm the presence of prions before detectable pathology.
Research Interests: Chemical Engineering, Analytical Chemistry, Mass Spectrometry, Ion Chromatography, Quantitative analysis, and 14 morePrion Diseases, Humans, Mice, Sheep, Animals, Method, Analytical, Signal, Quantitative Analysis, Amino Acid Sequence, Standard Curve, Reference standards, Cricetinae, and limit of detection
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Shiga-like toxins (verotoxins) are a class of AB5 holotoxins that are primarily responsible for the virulence associated with Shiga-like toxin producing Escherichia coli (STEC) infections. The holotoxins are composed of a pentamer of... more
Shiga-like toxins (verotoxins) are a class of AB5 holotoxins that are primarily responsible for the virulence associated with Shiga-like toxin producing Escherichia coli (STEC) infections. The holotoxins are composed of a pentamer of identical subunits (B subunit) responsible for delivering the catalytic subunit (A subunit) to a host cell and facilitating endocytosis of the toxin into the cell. The B subunits are not associated with toxicity. We developed a multiple reaction monitoring method based on analyzing conserved peptides, derived from the tryptic digestion of the B subunits. Stable-isotope-labeled analogues were prepared and used as internal standards to identify and quantify these characteristic peptides. We were able to detect and quantify Shiga toxins (Stx), Shiga-like toxin type 1 (Stx1) and type 2 (Stx2) subtypes, and to distinguish among most of the known subtypes. The limit of detection for digested pure standards was in the low attomole range/injection (~10 attomole...
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Research Interests: Analytical Chemistry, Nanotechnology, Food supply, Animals, High Pressure Liquid Chromatography, and 10 moreLiquid Chromatography / Electrospray Ionization Mass Spectrometry, Prions, Liquid Chromatography, Neurodegenerative Disease, Reproducibility of Results, Microchemistry, Sensitivity and Specificity, Brain Chemistry, Mass Spectrometer, and The American
A relational database, the Available Pathways Database (APD), has been constructed of microbial natural products, their producing strains, and their biosynthetic pathways. The database allows the ready selection of donor strains for... more
A relational database, the Available Pathways Database (APD), has been constructed of microbial natural products, their producing strains, and their biosynthetic pathways. The database allows the ready selection of donor strains for combinatorial biology experiments. It provides the same type of resource for combinatorial biology as the Available Chemicals Directory (ACD) does for combinatorial chemical library generation. Its cataloging ability can also provide insight into novel aspects of biosynthetic routes. In particular, no 10-unit Type I polyketides were found in the compilation of this edition of the APD (Version I).
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We employed a sensitive mass spectrometry-based method to deconstruct, confirm, and quantitate the prions present in elk naturally infected with chronic wasting disease and sheep naturally infected with scrapie. We used this approach to... more
We employed a sensitive mass spectrometry-based method to deconstruct, confirm, and quantitate the prions present in elk naturally infected with chronic wasting disease and sheep naturally infected with scrapie. We used this approach to study the oxidation of a methionine at position 216 (Met216), because this oxidation (MetSO216) has been implicated in prion formation. Three polymorphisms (Ile218, Val218, and Thr218) of sheep recombinant prion protein were prepared. Our analysis showed the novel result that the proportion of MetSO216 was highly dependent upon the amino acid residue at position 218 (I > V > T), indicating that Ile218 in sheep and elk prion protein (PrP) renders the Met216 intrinsically more susceptible to oxidation than the Val218 or Thr218 analogue. We were able to quantitate the prions in the attomole range. The presence of prions was verified by the detection of two confirmatory peptides: GENFTETDIK (sheep and elk) and ESQAYYQR (sheep) or ESEAYYQR (elk). This approach required much smaller amounts of tissue (600 μg) than traditional methods of detection (enzyme-linked immunosorbent assay, Western blot, and immunohistochemical analysis) (60 mg). In sheep and elk, a normal cellular prion protein containing MetSO216 is not actively recruited and converted to prions, although we observed that this Met216 is intrinsically more susceptible to oxidation.