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Application of Synthetic Peptides as Substrates for Reversible Phosphorylation

Description: Two highly homologous synthetic peptides MLC(3-13) (K-R-A-K-A-K-T-TK-K-R-G) and MLC(5-13) (A-K-A-K-T-T-K-K-R-G) corresponding to the amino terminal amino acid sequence of smooth muscle myosin light chain were utilized as substrates for protein kinase C purified from murine lymphosarcoma tumors to determine the role of the primary amino acid sequence of protein kinase C substrates in defining the lipid (phosphatidyl serine and diacylglycerol) requirements for the activation of the enzyme. Removal of the basic residues lysine and arginine from the amino terminus of MLC(3-13) did not have a significant effect on the Ka value of diacylglycerol. The binding of effector to calcium-protein kinase C appears to be random since binding of one effector did not block the binding of the other.
Date: August 1992
Creator: Abukhalaf, Imad Kazem
Partner: UNT Libraries

Genome sequencing reveals complex secondary metabolome in themarine actinomycete Salinispora tropica

Description: Recent fermentation studies have identified actinomycetes ofthe marine-dwelling genus Salinispora as prolific natural productproducers. To further evaluate their biosynthetic potential, we analyzedall identifiable secondary natural product gene clusters from therecently sequenced 5,184,724 bp S. tropica CNB-440 circular genome. Ouranalysis shows that biosynthetic potential meets or exceeds that shown byprevious Streptomyces genome sequences as well as other naturalproduct-producing actinomycetes. The S. tropica genome features ninepolyketide synthase systems of every known formally classified family,non-ribosomal peptide synthetases and several hybrid clusters. While afew clusters appear to encode molecules previously identified inStreptomyces species,the majority of the 15 biosynthetic loci are novel.Specific chemical information about putative and observed natural productmolecules is presented and discussed. In addition, our bioinformaticanalysis was critical for the structure elucidation of the novelpolyenemacrolactam salinilactam A. This study demonstrates the potentialfor genomic analysis to complement and strengthen traditional naturalproduct isolation studies and firmly establishes the genus Salinispora asa rich source of novel drug-like molecules.
Date: May 1, 2007
Creator: Udwary, Daniel W.; Zeigler, Lisa; Asolkar, Ratnakar; Singan,Vasanth; Lapidus, Alla; Fenical, William et al.
Partner: UNT Libraries Government Documents Department

Separation and Characterization of Variant Forms of Phosphoglucose Isomerase: Purification and Structural Analysis of Active Site Peptides from Human and Rabbit Phosphoglucose Isomerase

Description: A method has been developed for the rapid, quantitative separation of normal and abnormal phosphoglucose isoemrase allozymes from individuals heterozygous for genetic variant forms of the enzyme. The method utilizes a substrate gradient elution of the enzyme from carboxymethyl Biogel and is far superior in terms of resolution and recovery to methods based on electrophoresis and isoelectric focusing. Four different genetic variant forms of the enzyme were isolated and subjected to a systematic comparison of their physical, catalytic and stability properties. The physical and catalytic properties of the variants were similar; however, clear differences in the stability of the allozymes were apparent.
Date: May 1980
Creator: Gibson, David R.
Partner: UNT Libraries

Interplay between Bladder Microbiota and Urinary Antimicrobial Peptides: Mechanisms for Human Urinary Tract Infection Risk and Symptom Severity

Description: This article explores the interrelationship between the urinary microbiota and host antimicrobial peptides as mechanisms for urinary tract infection risk.
Date: December 8, 2014
Creator: Nienhouse, Vanessa; Gao, Xiang; Dong, Qunfeng; Nelson, David E.; Toh, Evelyn; McKinley, Kathleen et al.
Partner: UNT College of Arts and Sciences

The Use of Aryl Hydrazide Linkers for the Solid Phase Synthesis of Chemically Modified Peptides

Description: Since Merrifield introduced the concept of solid phase synthesis in 1963 for the rapid preparation of peptides, a large variety of different supports and resin-linkers have been developed that improve the efficiency of peptide assembly and expand the myriad of synthetically feasible peptides. The aryl hydrazide is one of the most useful resin-linkers for the synthesis of chemically modified peptides. This linker is completely stable during Boc- and Fmoc-based solid phase synthesis and yet it can be cleaved under very mild oxidative conditions. The present article reviews the use of this valuable linker for the rapid and efficient synthesis of C-terminal modified peptides, head-to-tail cyclic peptides and lipidated peptides.
Date: November 3, 2006
Creator: Woo, Y; Mitchell, A R & Camarero, J A
Partner: UNT Libraries Government Documents Department

An Appreciation of the Scientific Life and Acheivements of Bruce Merrifield

Description: Bruce Merrifield's scientific biography, 'Life During a Golden Age of Peptide Chemistry: The Concept and Development of Solid-Phase Peptide Synthesis', provides a history of solid phase-peptide synthesis (SPPS) from 1959 to 1993 [1]. While many readers will be familiar with SPPS literature after 1963, the inclusion of unpublished material from Merrifield's early laboratory notebooks opens a fascinating window on the development of SPPS from the formulation of concept in 1959 (p. 56, ref. 1) to the synthesis of a tetrapeptide four years later [2]. This early period was characterized by slow progress interrupted by numerous setbacks that led Bruce to later record (p. 90, ref. 1): 'At the end of the first two years the results were so poor, I wonder what made me think that this approach would ever succeed; but from the outset I had a strong conviction that this was a good idea, and I am glad that I stayed with it long enough'. Garland Marshall, Bruce's first graduate student (1963-1966), as well as later colleagues, were essentially unaware of the many highways, byways and dead ends that Bruce had explored in the early years [3].
Date: June 15, 2007
Creator: Mitchell, R
Partner: UNT Libraries Government Documents Department

R. Bruce Merrifield and Solid-Phase Peptide Synthesis: A Historical Assessment

Description: Bruce Merrifield, trained as a biochemist, had to address three major challenges related to the development and acceptance of solid-phase peptide synthesis (SPPS). The challenges were (1) to reduce the concept of peptide synthesis on a insoluble support to practice, (2) overcome the resistance of synthetic chemists to this novel approach, and (3) establish that a biochemist had the scientific credentials to effect the proposed revolutionary change in chemical synthesis. How these challenges were met is discussed in this article.
Date: December 4, 2007
Creator: Mitchell, A R
Partner: UNT Libraries Government Documents Department

SYNTHESIS OF PROTEINS BY NATIVE CHEMICAL LIGATION USING FMOC-BASED CHEMISTRY

Description: C-terminal peptide {alpha}-thioesters are valuable intermediates in the synthesis/semisynthesis of proteins by native chemical ligation. They are prepared either by solid-phase peptide synthesis (SPPS) or biosynthetically by protein splicing techniques. The present paper reviews the different methods available for the chemical synthesis of peptide {alpha}-thioesters using Fmoc-based SPPS.
Date: January 20, 2005
Creator: Camarero, J A & Mitchell, A R
Partner: UNT Libraries Government Documents Department

A Cell-Based Approach for the Biosynthesis/Screening of Cyclic Peptide Libraries against Bacterial Toxins

Description: Available methods for developing and screening small drug-like molecules able to knockout toxins or pathogenic microorganisms have some limitations. In order to be useful, these new methods must provide high-throughput analysis and identify specific binders in a short period of time. To meet this need, we are developing an approach that uses living cells to generate libraries of small biomolecules, which are then screened inside the cell for activity. Our group is using this new, combined approach to find highly specific ligands capable of disabling anthrax Lethal Factor (LF) as proof of principle. Key to our approach is the development of a method for the biosynthesis of libraries of cyclic peptides, and an efficient screening process that can be carried out inside the cell.
Date: October 24, 2007
Creator: Camarero, J. A.; Kimura, R.; Woo, Y.; Cantor, J. & Steenblock, E.
Partner: UNT Libraries Government Documents Department

Peptide Based Radiopharmaceuticals: Specific Construct Approach

Description: The objective of this project was to develop receptor based peptides for diagnostic imaging and therapy. A series of peptides related to cell adhesion molecules (CAM) and immune regulation were designed for radiolabeling with <sup>99m</sup>Tc and evaluated in animal models as potential diagnostic imaging agents for various disease conditions such as thrombus (clot), acute kidney failure, and inflection/inflammation imaging. The peptides for this project were designed by the industrial partner, Palatin Technologies, (formerly Rhomed, Inc.) using various peptide design approaches including a newly developed rational computer assisted drug design (CADD) approach termed MIDAS (Metal ion Induced Distinctive Array of Structures). In this approach, the biological function domain and the <sup>99m</sup>Tc complexing domain are fused together so that structurally these domains are indistinguishable. This approach allows construction of conformationally rigid metallo-peptide molecules (similar to cyclic peptides) that are metabolically stable in-vivo. All the newly designed peptides were screened in various in vitro receptor binding and functional assays to identify a lead compound. The lead compounds were formulated in a one-step <sup>99m</sup>Tc labeling kit form which were studied by BNL for detailed in-vivo imaging using various animals models of human disease. Two main peptides usingMIDAS approach evolved and were investigated: RGD peptide for acute renal failure and an immunomodulatory peptide derived from tuftsin (RMT-1) for infection/inflammation imaging. Various RGD based metallopeptides were designed, synthesized and assayed for their efficacy in inhibiting ADP-induced human platelet aggregation. Most of these peptides displayed biological activity in the 1-100 µM range. Based on previous work by others, RGD-I and RGD-II were evaluated in animal models of acute renal failure. These earlier studies showed that after acute ischemic injury the renal cortex displays RGD receptor with higher density. The results have indicated good diagnostic potential for their use in this clinical situation, as an imaging agent to ...
Date: October 21, 1997
Creator: Som, P; Rhodes, B A & Sharma, S S
Partner: UNT Libraries Government Documents Department

Synthesis and Screening of a Combinatorial Peptide Library for Ligands to Target Transferrin: Miniaturizing the Library

Description: Combinatorial libraries are used in the search for ligands that bind to target proteins. Fmoc solid-phase peptide synthesis is routinely used to generate such libraries. Microwave-assisted peptide synthesis was employed here to decrease reaction times by 80-90%. Two One-Bead-One-Compound combinatorial libraries were synthesized on 130μm beads (one containing 750 members and the other 16, 807). The use of smaller solid supports would have many important practical advantages including; increased library diversity per unit mass, smaller quantities of library needed to generate hits, and screening could be conducted by using a standard flow cytometer. To this end, a miniaturized peptide library was synthesized on 20 μm beads to demonstrate proof of principle. A small sample from the 16,807-member library was screened against transferrin-AlexaFluro 647, a protein responsible for iron transport in vivo. A number of hits were identified and sequenced using techniques coupling nanomanipulation with nanoelectrospray mass spectrometry.
Date: August 2010
Creator: Brown, Jennifer Marie
Partner: UNT Libraries

Breast Cancer Cells in Three-dimensional Culture Display an Enhanced Radioresponse after Coordinate Targeting of Integrin ?5?1 and Fibronectin

Description: Tactics to selectively enhance cancer radioresponse are of great interest. Cancer cells actively elaborate and remodel their extracellular matrix (ECM) to aid in survival and progression. Previous work has shown that {beta}1-integrin inhibitory antibodies can enhance the growth-inhibitory and apoptotic responses of human breast cancer cell lines to ionizing radiation, either when cells are cultured in three-dimensional laminin-rich ECM (3D lrECM) or grown as xenografts in mice. Here, we show that a specific {alpha} heterodimer of {beta}1-integrin preferentially mediates a prosurvival signal in human breast cancer cells that can be specifically targeted for therapy. 3D lrECM culture conditions were used to compare {alpha}-integrin heterodimer expression in malignant and nonmalignant cell lines. Under these conditions, we found that expression of {alpha}5{beta}1-integrin was upregulated in malignant cells compared with nonmalignant breast cells. Similarly, we found that normal and oncofetal splice variants of fibronectin, the primary ECM ligand of {alpha}5{beta}1-integrin, were also strikingly upregulated in malignant cell lines compared with nonmalignant acini. Cell treatment with a peptide that disrupts the interactions of {alpha}5{beta}1-integrin with fibronectin promoted apoptosis in malignant cells and further heightened the apoptotic effects of radiation. In support of these results, an analysis of gene expression array data from breast cancer patients revealed an association of high levels of {alpha}5-integrin expression with decreased survival. Our findings offer preclinical validation of fibronectin and {alpha}5{beta}1-integrin as targets for breast cancer therapy.
Date: April 7, 2010
Creator: Nam, Jin-Min; Onodera, Yasuhito; Bissell, Mina J & Park, Catherine C
Partner: UNT Libraries Government Documents Department

Laminin Mediates Tissue-specific Gene Expression in Mammary Epithelia

Description: Tissue-specific gene expression in mammary epithelium is dependent on the extracellular matrix as well as hormones. There is good evidence that the basement membrane provides signals for regulating beta-casein expression, and that integrins are involved in this process. Here, we demonstrate that in the presence of lactogenic hormones, laminin can direct expression of the beta-casein gene. Mouse mammary epithelial cells plated on gels of native laminin or laminin-entactin undergo functional differentiation. On tissue culture plastic, mammary cells respond to soluble basement membrane or purified laminin, but not other extracellular matrix components, by synthesizing beta-casein. In mammary cells transfected with chloramphenicol acetyl transferase reporter constructs, laminin activates transcription from the beta-casein promoter through a specific enhancer element. The inductive effect of laminin on casein expression was specifically blocked by the E3 fragment of the carboxy terminal region of the alpha 1 chain of laminin, by antisera raised against the E3 fragment, and by a peptide corresponding to a sequence within this region. Our results demonstrate that laminin can direct tissue-specific gene expression in epithelial cells through its globular domain.
Date: April 1, 1995
Creator: Streuli, Charles H; Schmidhauser, Christian; Bailey, Nina; Yurchenco, Peter; Skubitz, Amy P. N.; Roskelley, Calvin et al.
Partner: UNT Libraries Government Documents Department

New Tools for the site-specific attachment of proteins to surface

Description: Protein microarrays in which proteins are immobilized to a solid surface are ideal reagents for high-throughput experiments that require very small amounts of analyte. Such protein microarrays ('protein chips') can be used very efficiently to analyze all kind of protein interactions en masse. Although a variety of methods are available for attaching proteins on solid surfaces. Most of them rely on non-specific adsorption methods or on the reaction of chemical groups within proteins (mainly, amino and carboxylic acid groups) with complementary reactive groups. In both cases the protein is attached to the surface in random orientations. The use of recombinant affinity tags addresses the orientation issue, however in most of the cases the interaction of the tags are reversible (e.g., glutathione S-transferase, maltose binding protein and poly-His) and, hence, are not stable over the course of subsequent assays or require large mediator proteins (e.g., biotin-avidin and antigen antibody). The key for the covalent attachment of a protein to a solid support with a total control over the orientation is to introduce two unique and mutually reactive groups on both the protein and the surface. The reaction between these two groups should be highly selective thus behaving like a molecular 'Velcro'.
Date: June 17, 2005
Creator: Camarero, J A; Kwon, Y & Coleman, M A
Partner: UNT Libraries Government Documents Department

Programmed assembly of nanoscale structures using peptoids.

Description: Sequence-specific polymers are the basis of the most promising approaches to bottom-up programmed assembly of nanoscale materials. Examples include artificial peptides and nucleic acids. Another class is oligo(N-functional glycine)s, also known as peptoids, which permit greater sidegroup diversity and conformational control, and can be easier to synthesize and purify. We have developed a set of peptoids that can be used to make inorganic nanoparticles more compatible with biological sequence-specific polymers so that they can be incorporated into nucleic acid or other biologically based nanostructures. Peptoids offer degrees of modularity, versatility, and predictability that equal or exceed other sequence-specific polymers, allowing for rational design of oligomers for a specific purpose. This degree of control will be essential to the development of arbitrarily designed nanoscale structures.
Date: February 1, 2011
Creator: Ren, Jianhua (University of the Pacific, Stockton, CA); Russell, Scott (California State University, Stanislaus, Turlock, CA); Morishetti, Kiran (University of the Pacific, Stockton, CA); Robinson, David B.; Zuckermann, Ronald N. (Lawrence Berkeley National Laboratory, Berkeley, CA); Buffleben, George M. et al.
Partner: UNT Libraries Government Documents Department

Developing New Tools for the in vivo Generation/Screening of Cyclic Peptide Libraries. A New Combinatorial Approach for the Detection of Bacterial Toxin Inhibitors

Description: A new combinatorial approach for the biosynthesis and screening of small drug-like toxin inhibitors inside living cells is presented. This approach has been initially used as proof of principle for finding inhibitors against the LF factor from Bacillus anthracis. Key to our ''living combinatorial'' approach is the use of a living cell as a micro-chemical factory for both synthesis and screening of potential inhibitors for a given molecular recognition event (see Scheme 1). This powerful technique posses the advantage that both processes synthesis and screening happen inside the cell thus accelerating the whole screening/selection process.
Date: November 28, 2006
Creator: Camarero, J A
Partner: UNT Libraries Government Documents Department

Studies in Solid Phase Peptide Synthesis: A Personal Perspective

Description: By the early 1970s it had became apparent that the solid phase synthesis of ribonuclease A could not be generalized. Consequently, virtually every aspect of solid phase peptide synthesis (SPPS) was reexamined and improved during the decade of the 1970s. The sensitive detection and elimination of possible side reactions (amino acid insertion, N{sup {alpha}}-trifluoroacetylation, N{sup {alpha}{var_epsilon}}-alkylation) was examined. The quantitation of coupling efficiency in SPPS as a function of chain length was studied. A new and improved support for SPPS, the 'PAM-resin', was prepared and evaluated. These and many other studies from the Merrifield laboratory and elsewhere increased the general acceptance of SPPS leading to the 1984 Nobel Prize in Chemistry for Bruce Merrifield.
Date: June 1, 2007
Creator: Mitchell, A R
Partner: UNT Libraries Government Documents Department

Expanded breadth of the T-cell response to mosaic HIV-1 envelope DNA vaccination

Description: An effective AIDS vaccine must control highly diverse circulating strains of HIV-1. Among HIV -I gene products, the envelope (Env) protein contains variable as well as conserved regions. In this report, an informatic approach to the design of T-cell vaccines directed to HIV -I Env M group global sequences was tested. Synthetic Env antigens were designed to express mosaics that maximize the inclusion of common potential Tcell epitope (PTE) 9-mers and minimize the inclusion of rare epitopes likely to elicit strain-specific responses. DNA vaccines were evaluated using intracellular cytokine staining (ICS) in inbred mice with a standardized panel of highly conserved 15-mer PTE peptides. I, 2 and 3 mosaic sets were developed that increased theoretical epitope coverage. The breadth and magnitude ofT-cell immunity stimulated by these vaccines were compared to natural strain Env's; additional comparisons were performed on mutant Env's, including gpl60 or gpl45 with or without V regions and gp41 deletions. Among them, the 2 or 3 mosaic Env sets elicited the optimal CD4 and CD8 responses. These responses were most evident in CD8 T cells; the 3 mosaic set elicited responses to an average of 8 peptide pools compared to 2 pools for a set of3 natural Env's. Synthetic mosaic HIV -I antigens can therefore induce T-cell responses with expanded breadth and may facilitate the development of effective T -cell-based HIV -1 vaccines.
Date: January 1, 2009
Creator: Korber, Bette; Fischer, William & Wallstrom, Timothy
Partner: UNT Libraries Government Documents Department

Development of porous polymer monoliths for reverse-phase chromatography of proteins.

Description: The polymers developed in this project are intended for use as a stationary phase in reverse-phase chromatography of proteins, where the mobile phase is a solution of acetonitrile and a phosphate buffer, 6.6 pH. A full library of pore sizes have been developed ranging from 0.41{micro}m to 4.09 {micro}m; these pore sizes can be determined by the solvent ratio of tetrahydrofuran:methoxyethanol during polymerization. A column that can separate proteins in an isocratic mode would be a vast improvement from the common method of separating proteins through gradient chromatography using multiple solvents. In the stationary phase, the main monomers have hydrophobic tails, lauryl acrylate and steryl acrylate. Separations of small hydrophobic molecules and peptides (trial molecules) have efficiencies of 24,000-33,000 theoretical plates m{sup -1}. The combination of a highly non-polar stationary phase and a mobile phase where the polarity can be controlled provide for excellent separation.
Date: September 1, 2003
Creator: Shepodd, Timothy J. (Sandia National Laboratories, Albuquerque, NM) & Stephens, Christopher P. (Sandia National Laboratories, Albuquerque, NM)
Partner: UNT Libraries Government Documents Department

HIV evolution in early infection: selection pressures, patterns of insertion and deletion, and the impact of apobec

Description: The pattern of viral diversification in newly infected individuals provides information about the host environment and immune responses typically experienced by the newly transmitted virus. For example, sites that tend to evolve rapidly across multiple early-infection patients could be involved in enabling escape from common early immune responses, represent adaptation for rapid growth in a newly infected host, or reversion from less fit forms of the virus that were selected for immune escape in previous hosts. Here we investigated the diversification of HIV -I env coding sequences in 81 very early B SUbtype infections previously shown to have resulted from transmission or expansion of single viruses (n=78) or two closely related viruses (n=3). In these cases the sequence of the infecting virus can be estimated accurately, enabling inference of both the direction of substitutions as well as distinction between insertion and deletion events. By integrating information across multiple acutely infected hosts, we find evidence of adaptive evolution of HIV-1 envand identified a subset of codon sites that diversified more rapidly than can be explained by a model of neutral evolution. Of 24 such rapidly diversifying sites, 14 were either (i) clustered and embedded in CTL epitopes that were verified experimentally or predicted based on the individual's HLA or (ii) in a nucleotide context indicative of APOBEC mediated G-to-A substitutions, despite having excluded heavily hypermutated sequences prior to the analysis. In several cases, a rapidly evolving site was both embedded in an APOBEC motif and in a CTL epitope, suggesting that APOBEC may facilitate early immune escape. Ten rapidly diversifying sites could not be explained by CTL escape or APOBEC hypermutation, including the most frequently mutated site, in the fusion peptide of gp4l. We also examined the distribution, extent, and sequence context of insertions and deletions and provide evidence that the ...
Date: January 1, 2009
Creator: Korber, Bette; Bhattacharya, Tanmoy; Giorgi, Elena; Gaschen, B & Daniels, M
Partner: UNT Libraries Government Documents Department

Development of a biomarker for Geobacter activity and strain composition: Proteogenomic analysis of the citrate synthase protein during bioremediation of U(VI)

Description: Monitoring the activity of target microorganisms during stimulated bioremediation is a key problem for the development of effective remediation strategies. At the US Department of Energy's Integrated Field Research Challenge (IFRC) site in Rifle, CO, the stimulation of Geobacter growth and activity via subsurface acetate addition leads to precipitation of U(VI) from groundwater as U(IV). Citrate synthase (gltA) is a key enzyme in Geobacter central metabolism that controls flux into the TCA cycle. Here, we utilize shotgun proteomic methods to demonstrate that the measurement of gltA peptides can be used to track Geobacter activity and strain evolution during in situ biostimulation. Abundances of conserved gltA peptides tracked Fe(III) reduction and changes in U(VI) concentrations during biostimulation, whereas changing patterns of unique peptide abundances between samples suggested sample-specific strain shifts within the Geobacter population. Abundances of unique peptides indicated potential differences at the strain level between Fe(III)-reducing populations stimulated during in situ biostimulation experiments conducted a year apart at the Rifle IFRC. These results offer a novel technique for the rapid screening of large numbers of proteomic samples for Geobacter species and will aid monitoring of subsurface bioremediation efforts that rely on metal reduction for desired outcomes.
Date: February 15, 2010
Creator: Wilkins, M.J.; Callister, S.J.; Miletto, M.; Williams, K.H.; Nicora, C.D.; Lovley, D.R. et al.
Partner: UNT Libraries Government Documents Department