Browsing by Author "Potts, Malcolm"

Now showing 1 - 20 of 44
  • Thumbnail Image
    Algorithms for Storytelling
    Kumar, Deept; Ramakrishnan, Naren; Helm, Richard F.; Potts, Malcolm (Department of Computer Science, Virginia Polytechnic Institute & State University, 2006)
    We formulate a new data mining problem called "storytelling" as a generalization of redescription mining. In traditional redescription mining, we are given a set of objects and a collection of subsets defined over these objects. The goal is to view the set system as a vocabulary and identify two expressions in this vocabulary that induce the same set of objects. Storytelling, on the other hand, aims to explicitly relate object sets that are disjoint (and hence, maximally dissimilar) by finding a chain of (approximate) redescriptions between the sets. This problem finds applications in bioinformatics, for instance, where the biologist is trying to relate a set of genes expressed in one experiment to another set, implicated in a different pathway. We outline an efficient storytelling implementation that embeds the CARTwheels redescription mining algorithm in an A* search procedure, using the former to supply next move operators on search branches to the latter. This approach is practical and effective for mining large datasets and, at the same time, exploits the structure of partitions imposed by the given vocabulary. Three application case studies are presented: a study of word overlaps in large English dictionaries, exploring connections between genesets in a bioinformatics dataset, and relating publications in the PubMed index of abstracts.
  • Thumbnail Image
    Baculovirus-directed expression of the phosphorylase kinase catalytic subunit: pseudosubstrate and calmodulin regulation
    Lanciotti, Robert Arthur (Virginia Tech, 1994-08-05)
    Phosphorylase kinase (EC 2.7.1.38) is a key enzyme involved in the regulation of the glycogenolysis pathway. It catalyzes the Ca²⁺-dependent phosphorylation and activation of the enzyme glycogen phosphorylase to make the active form glycogen phosphorylase. Phosphorylase kinase is composed of 4 subunits with a stoichiometry of (αβγδ)₄. The γ subunit is the catalytic subunit. The regulatory domain (residues 277-387) of γ contains a sequence resembling the sites phosphorylated in known γ substrates with the exception that a valine₃₃₂ occurs at the analogous position of the phosphorylated serine or threonine residue.
  • Thumbnail Image
    Candidate Gene Expression and SNP Analyses of Toxin-Induced Dilated Cardiomyopathy in the Turkey(Meleagris gallopavo)
    Lin, Kuan-chin (Virginia Tech, 2006-03-28)
    Dilated cardiomyopathy (DCM), a heart disease, affects many vertebrates including humans and poultry. The disease can be either idiopathic (IDCM) or toxin-induced. Idiopathic DCM often occurs without a consensus cause. Though genetic and other studies of IDCM are extensive, the specific etiology of toxin-induced is still unknown. Here, our objective was to compare the level of mRNA expression of two candidate genes including troponin T (cTnT) and phospholamban (PLN) using quantitative reverse transcription polymerase chain reaction (RT-PCR) in toxin-induced DCM affected and unaffected turkeys. Cardiac TnT and PLN were chosen because their spontaneous expression has been reported to be associated with IDCM. We also scanned these genes for single nucleotide polymorphisms (SNPs) that could be useful in evaluating their functions in the incidence and severity of toxin-induced DCM in turkeys. There were no significant differences between affected and unaffected birds in the expression of both cTnT and PLN. A total of 12 SNPs were detected in cTnT and PLN DNA sequences. One of the seven haplotypes detected in cTnT was the most frequent. Linkage analysis showed that cTnT gene was unlinked on the current turkey genetic map. Resources developed here, including SNPs, haplotypes, cDNA sequences, and the PCR-RFLP genotype procedure will be used for future investigations involving cTnT and PLN and toxin-induced DCM.
  • Thumbnail Image
    Carbohydrate-Interacting Proteins from Two Nostoc (Cyanobacteria) Species
    Jordan, Brian Robert (Virginia Tech, 2004-05-05)
    Cyanobacteria of the Nostoc genus are known for the thick, mucilaginous carbohydrate coatings that they produce. In this work, two examples of cyanobacterial glycobiology are considered, each of which involves a cyanobacterium of the Nostoc genus. The first portion of this work details attempts to obtain amino acid sequence information from the enzymes (glycosyltransferases) that are responsible for producing the extracellular polysaccharide (EPS) of Nostoc commune DRH1, ultimately to allow the transfer of this capacity to another organism. Two artificial substrates were synthesized for use in a capillary electrophoresis-based enzyme assay, which was used to look for glycosyltransferase activity in Nostoc commune DRH1 cell extracts. Glucuronosyltransferase activity was detected in association with Nostoc commune membrane material. The active enzyme displayed a divalent cation metal dependence (Mg+2) that is typical of glycosyltransferase enzymes purified from other organisms. Because the enzyme responsible for this activity held the potential to be EPS-related, its purification was attempted. The capillary electrophoresis-based enzyme assay and a 32P-labeled affinity tag were utilized to follow the glucuronosyltransferase enzyme through successive purification steps. The active enzyme was extracted from Nostoc commune membrane material using Triton X-100, and then purified by anion exchange chromatography. The active detergent extract was extremely unstable, and consequently, other purification techniques tested were unsuccessful in enriching activity. Affinity-labeling experiments indicated that the active enzyme was forming protein aggregates during these procedures, which were not amenable to in-gel protease digestion and peptide analysis by tandem mass spectrometry. The second portion of this work describes an investigation of an Anabaena (Nostoc) PCC 7120 soluble cell extract. Upon separation by sodium dodecyl sulfate ¡V polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent periodic acid-Schiff (PAS) staining of the resulting gel, the components of this cellular fraction produce a ladder-like pattern, which suggests that the extract may contain glycosylated protein. Analyses of several samples that were taken from within the PAS-staining region of such a gel revealed surface layer homology (SLH) domain-containing proteins, likely candidates to be covalently attached to or non-covalently interacting with carbohydrate. Various protein sequence analyses indicated that the detected SLH domain containing proteins belong to a family of (putative) cyanobacterial porins. Proteins in this family possess features that include a N-terminal signal sequence, a single SLH domain motif, followed by a coiled-coil region, and a C-terminal region that is homologous to the b-barrel-forming region of bacterial porins. All of these features were identified in the detected Anabaena (Nostoc) PCC 7120 SLH domain-containing proteins. Smith degradation was performed on a sample that was electroeluted from the PAS-staining region of a preparative-scale SDS-PAGE gel of the soluble cell extract. Subsequent analyses of the resulting sample by SDS-PAGE and mass spectrometry indicated that at least two SLH domain-containing proteins, encoded by all4499 and alr4550, were non-covalently interacting with the PAS-staining material. Following degradation, the PAS-staining material was still of sufficient size to detected by gel electrophoresis, and it continued to migrate in the absence of an interacting protein component. Protease digestion of a similarly prepared sample, and then subsequent analysis by SDS-PAGE and mass spectrometry, revealed that the region between amino acid residues #504 and #536, in the protein encoded by the alr4550 open reading frame, was interacting with the PAS-staining material. Monosaccharide composition analyses of this material revealed more carbohydrate constituents than are found in cyanobacterial primary (peptidoglycan) cell wall polymer alone, indicating that it contained a significant secondary cell wall polymer component as well.
  • Thumbnail Image
    Characterization of an operon containing a ribosomal protein gene and lipid biosynthetic genes in Escherichia coli K-12
    Oh, Won Shin (Virginia Tech, 1992)
    The plsX50 mutation is required together with plsX26 (encoding a Km- defective glycerol 3-phosphate acyltransferase) for the conferral of a glycerol 3- phosphate auxotrophic phenotype. A 4.9 kb segment of DNA complementing the plsX50 mutation have been cloned and sequenced. Six open reading frames (ORF’s) were found with five reading in the same direction and one in the opposite direction relative to the plsX gene. Each ORF encoded a protein, as demonstrated by radiolabeling in maxicells. ORF1 (orfY) encodes a protein of unknown function. ORF2 and ORF3 (rpmF) were sequenced prior to this study and encode a protein called G30k of unknown function and L32, a protein of the large ribosomal subunit, respectively. ORF4 complemented the p/sX50 mutation. ORFS was identified as fabH encoding 3-ketoacyl-ACP synthase III. ORF6 was identified as fabD encoding malonyl-CoA/ACP transacylase. The fabG gene encoding 3- ketoacyl-ACP reductase and the acpP gene encoding acy] carrier protein are located just downstream of the fabD gene. Northern and promoter activity analysis demonstrated that the rpmF-plsX-fabH-fabD-fabG-acpP genes comprise an operon suggesting a coordinate control of the synthesis of a ribosomal protein (L32), PlsX protein, and fatty acid biosynthetic enzymes. However, several features were identified that are likely to be important for differential expression of the individual genes. These include the presence of multiple promoters, an internal terminator (attenuator), differential degradation of transcripts, and differential efficiency of translation initiation. Portions of transcripts arising upstream of rpmF terminate at the attenuator located just downstream of the plsX initiation codon, and some of the transcripts continue into the plsX-jab genes. The fabH-fabD-fabG-acpP genes are also cotranscribed from a promoter located upstream of the fabH gene, within the pisX structural gene. There are additional cotranscripts responsible for the expression of the fabD-fabG-acpP genes. The acpP gene is encoded by several more transcripts. Transcription initiation sites upstream of rpmF were identified by primer extension analysis and the attenuator site was identified by S1 mapping analysis. N-terminal amino acid sequence analysis identified the translation initiation codons of orfY, plsX, fabH, fabD and fabG. Short intergenic distances (15 and 12 bp) found between fabH and fabD and between fabD and fabG implicate translational coupling as a mechanism for coordinate control of fabHDG expression. The p/sX50 mutation was identified as deletion of a single nucleotide from the 6th codon of plsX resulting in a frame shift nonsense mutation.
  • Thumbnail Image
    Characterization of IphP from Nostoc commune UTEX 584 and a Dual Specificity Protein Phosphatase from Anabaena PCC 7120
    Howell, Larry Daniel II (Virginia Tech, 1997-09-17)
    Protein phosphorylation is utilized universally as a mechanism of signal transduction. However, the use of tyrosine phosphorylation by bacteria has been a matter of dispute. Conventional wisdom dictated that "prokaryotic phosphorylation" was typified by phosphorylation of histidine and aspartate residues of proteins, while "eukaryotic phosphorylation" was characterized by modification of serine, threonine, or tyrosine residues. Increasing numbers of reports have emerged challenging the traditional view of "prokaryotic" and "eukaryotic" phosphorlyation. One of the strongest links unifying prokaryotic and eukaryotic protein phosphorylation to date is IphP, a genomically-encoded dual-specificity protein phosphatase from the cyanobacterium Nostoc commune UTEX 584 bearing the active-site signature sequence of eukaryotic tyrosine-specific and dual-specificity protein phosphatases. The catalytic properties and substrate specificity of IphP were examined in detail. The enzyme was able to discriminate among a variety of exogenous peptides and proteins. Kinetic studies revealed that IphP favors protein / peptide substrates over low molecular weight compounds. Heparin effected IphP activity in a substrate-dependent manner. Enzyme activity toward casein (P-Ser) and MAP kinase (P-Thr/P-Tyr) was stimulated in the presence of the polyanion, whereas activity was inhibited by heparin toward other protein substrates. Both stimulation and inhibition by heparin were dose-dependent. The ability to stimulate IphP activity toward select substrates was attributed to the ability of heparin to recruit the enzyme and substrate to the same microenvironment. To facilitate future genetic studies examining the role of tyrosine phosphorylation in cyanobacteria, we searched for evidence of protein tyrosine phosphorylation in Anabaena PCC 7120. In a collaborative effort with the laboratory of Dr. Potts, tyrosine phosphorylated proteins were identified in Anabaena utilizing several approaches, including comparative labelling with alpha- vs gamma-32P-ATP, phosphoamino acid analysis, and selective hydrolysis with a tyrosine specific protein phosphatase. Together, these data unequivocally demonstrate the presence of tyrosine-phosphorylated proteins in Anabaena PCC 7120. Extracts of Anabaena PCC 7120 were examined for protein tyrosine phosphatase activity. An apparent PTP activity was detected, partially purified, and characterized. The protein phosphatase was ~38kDa by SDS-PAGE and sucrose density gradient centrifugation and displayed dual-specificity protein phosphatase (DSP) activity in vitro. The enzyme was localized to the periplasm and was thus assigned the title PAD, for Periplasmic Anabaena DSP. Periplasmic phosphoproteins of ~120 and 55 kDa that had been radiolabelled in vitro were dephosphorylated by partially purified PAD. PAD activity varied in vivo ~5-fold in a rhthymic, seemingly diurnal manner. Periplasmic proteins, including the 55kDa protein, were labelled in vivo and the degree of radiolabel incorporated into these proteins varied inversely with PAD activity.
  • Thumbnail Image
    Characterization of structure, function and regulation of the speB gene in Escherichia coli
    Szumanski, Maria B. W. (Virginia Polytechnic Institute and State University, 1989)
    The speB gene of E. coli encodes agmatine ureohydrolase (AUH). AUH catalyses the hydrolysis of agmatine to urea and putrescine in a polyamine biosynthetic pathway. The plasmid pKA5, derived from an E. coli genomic library, was the source of a 2.97 kb restriction fragment containing the speB gene. Sequencing of this fragment revealed three intact open reading frames, ORF1 and ORF2 on one strand and ORF3 on the opposite strand, as well as a truncated open reading frame, ORF4, which terminated 92 kb upstream from ORF3. ORF2 and ORF3 were convergent, and overlapped by 85% of their sequence. ORF1 and ORF3 were separated by a sequence of two imperfect repeats containing four palindromes, three of which were overlapping. ORF3 represented the coding sequence of the speB gene. Two transcripts were detected from the speB gene: a shorter transcript, initiated 101 bp upstream from ORF3, and a polycistronic message, coding for ORF3 and ORF4. The short transcript was abundantly expressed when ORF4 sequences were deleted, but when ORF4 and its upstream sequences were present, the polycistronic message predominated and the amount of the monocistronic message was drastically reduced. The promoter producing the shorter transcript required only a -12 TATACT sequence for activity. Deletion of a 460 bp fragment comprising the 5'-region of ORF1 from a plasmid containing ORF1, ORF2 and speB reduced the activity of AUH by 83%. This fragment contained two divergently oriented promoters. The presence of ORF1 did not stimulate ß-galactosidase encoded by the speB promoter fused to lacΖ. Agmatine induced transcription from speB but not from the ORF4 nor the ORF1 promoters. cAMP caused an 88% reduction in the AUH activity of wild type E. coli K-12 but had no effect on the activity of plasmid encoded AUH. The activity of neither the speB nor the ORF4 promoters fused to lacΖ or phoA were influenced by cAMP; in contrast, the lacZ promoter fused to lacZ or phoA was stimulated by cAMP. Thus, the role of cAMP and CRP on speB expression is indirect and limited to a single copy state.
  • Thumbnail Image
    Characterization of the nifUHD cluster and a new myoglobin-like gene from Nostoc commune UTEX 584
    Angeloni, Stephen V. (Virginia Tech, 1992-03-04)
    Sequence analysis of the entire 3.5 kb HindIII genomic DNA fragment previously isolated from Nostoc commune UTEX 584 (Defrancesco and Potts 1988), determined the exact locations of the nifU, nifH, and nifD genes and identified two potential stem loop structures, a direct repeat, and an ORF that codes for a protein with a predicted amino acid sequence similar to that of myoglobin from Paramecium caudatum. The N. commune UTEX 584 myoglobin-like protein has a predicted length of 118 amino acids and molecular mass of 12,906 Da. A PCR copy of the gene (glbN) was cloned for overexpression of the protein. The recombinant protein was purified and used for spectral analysis and for the production of polyclonal antisera. Treatment of the recombinant protein with dithionite and CO resulted in spectral shifts characteristic of hemoproteins that bind oxygen. While some of the spectral characteristics are unique to the protein, in general the spectra were more like those of globins than cytochromes. Based on these characteristics and the sequence similarity to the P. caudatum mnyoglobin, we proposed the name cyanoglobin, with the gene designation glbN and the protein designation GlbN. Western analysis of GlbN expression was performed on N. commune UTEX 584 and two species of Anabaena (Anabaena sp. PCC 7120 and Anabaena variabilis). In N. commune UTEX 584 a protein with a molecular mass similar to that predicted for GlbN was detected. This protein was produced in increased amounts under the same growth conditions that resulted in increased production of nitrogenase reductase (the nifH gene product). No proteins of similar size to GlbN were detected in Anabaena sp. PCC 7120 or A. variabilis. A possible function of GlbN may be for oxygen storage, transport, or protection of the nitrogenase system. These functions as well as those of the direct repeat and the potential stem loop structures and their relationship to nitrogen fixation or other physiological processes in N. commune UTEX 584 require further analysis.
  • Thumbnail Image
    Connecting the Dots between PubMed Abstracts
    Hossain, M. Shahriar; Gresock, Joseph; Edmonds, Yvette M.; Helm, Richard F.; Potts, Malcolm; Ramakrishnan, Naren (PLOS, 2012-01-03)
    Background There are now a multitude of articles published in a diversity of journals providing information about genes, proteins, pathways, and diseases. Each article investigates subsets of a biological process, but to gain insight into the functioning of a system as a whole, we must integrate information from multiple publications. Particularly, unraveling relationships between extra-cellular inputs and downstream molecular response mechanisms requires integrating conclusions from diverse publications. Methodology We present an automated approach to biological knowledge discovery from PubMed abstracts, suitable for “connecting the dots” across the literature. We describe a storytelling algorithm that, given a start and end publication, typically with little or no overlap in content, identifies a chain of intermediate publications from one to the other, such that neighboring publications have significant content similarity. The quality of discovered stories is measured using local criteria such as the size of supporting neighborhoods for each link and the strength of individual links connecting publications, as well as global metrics of dispersion. To ensure that the story stays coherent as it meanders from one publication to another, we demonstrate the design of novel coherence and overlap filters for use as post-processing steps. Conclusions We demonstrate the application of our storytelling algorithm to three case studies: i) a many-one study exploring relationships between multiple cellular inputs and a molecule responsible for cell-fate decisions, ii) a many-many study exploring the relationships between multiple cytokines and multiple downstream transcription factors, and iii) a one-to-one study to showcase the ability to recover a cancer related association, viz. the Warburg effect, from past literature. The storytelling pipeline helps narrow down a scientist's focus from several hundreds of thousands of relevant documents to only around a hundred stories. We argue that our approach can serve as a valuable discovery aid for hypothesis generation and connection exploration in large unstructured biological knowledge bases.
  • Thumbnail Image
    Effect of dietary zinc or pyridoxine deficiency upon estrogen directed gene expression in the rat uterus
    Gunesekera, Bhadra Manel (Virginia Tech, 1990-06-05)
    In the present study the effect of diets restricted in either zinc or pyridoxine upon estrogen directed gene expression in the mature rat uterus was tested. Sexually mature female rats were maintained on zinc-adequate (40 mg/kg diet) ad libitum or restricted-fed, pyridoxine-deficient, or zinc-deficient ( < 1 mg/kg diet or 3mg/kg diet) ad libitum-fed diets for 35 days. All animals were bilaterally ovariectomized and used for experimentation at 14 days post ovariectomy. On day 35 each rat was injected intraperitoneally with estrogen. They were killed at different times post injection and thymidine kinase (TK, EC 2.7.1.21) or creatine kinase (CK, EC 2.7.3.2) activity was assayed in uteri cytosol fractions. In addition the steady state level of ckb mRNA in uteri cytosol fractions was measured following estrogen administration. The weight gain of the rats fed the low zinc and low pyridoxine diets was significantly lower than those fed the zinc-adequate diet ad libitum. The consumption of the zinc-deficient diet resulted in a significant decrease in plasma zinc while a pyridoxine deficient diet produced a significant reduction in plasma pyridoxine. Vehicle-injected uterine TK activity was 2-3 pmoles of d-TMP/min/mg protein. The TK activity was significantly increased (p < 0.05) 42 h post estrogen injection on the zinc-adequate diet ad libitum and pair-wt fed rats. This activity was sustained at 48 h post injection prior to declining to control values within 60 h. The maximum (4-fold) increase occurred at 36 h post estrogen injection in pyridoxine-deficient rats which was sustained at 42 & 48 h. The increase in uterine TK activity was 3-fold at 42 hand 48 h post injection. However this increase was not significantly different from the peak value seen in zinc-adequate and pyridoxine-deficient diet fed rats. No measurable effect of estrogen on CK activity was observed on the zinc adequate or zinc-deficient diet fed rats using a coupled enzyme assay. However the time course of ckb mRNA induction on the zinc-adequate pair-wt fed rats following estrogen administration paralleled the time course of estrogen induced protein (IP) synthesis previously observed by Gorski et al. (1970). IP is now known to be the brain type isoenzyme of creatine kinase. An induction of ckb mRNA between 0-3 h post estrogen injection was not observed on the zinc-deficient diet fed rats. However in a subsequent experiment an induction of uterine ckb mRNA 2 h following estrogen administration was observed in zinc-deficient rats. The possible reasons for this discrepancy are discussed. Zinc ions are known to be required to enable the estrogen receptor complex to bind to DNA and initiate transcription. It has been hypothesized that inadequate provision of dietary zinc may therefore reduce compliance to estrogen directed gene expression by limiting the efficiency of recruitment of zinc ions for stabilization of the zinc finger of the steroid receptor. The results of the present study failed to support this hypothesis at this moderate level of zinc depletion.
  • Thumbnail Image
    The effect of insulin-like growth factor-I (IGF-I), and an IGF-I-like factor secreted by human lung fibroblasts, on the growth of human lung carcinoma cells in vitro
    Ankrapp, David P. (Virginia Tech, 1993-05-05)
    The concentration of insulin-like growth factor I (IGF-I) in tissue taken from human non-small cell lung carcinoma (NSCLC) is 1.4- to 7-fold higher than the concentration of IGF-I in the surrounding normal lung tissue and therefore IGF-I may be involved in the growth of NSCLC. In this study it was determined that NSCLC cell lines (A549, A427, SK-LU-1) expressed the type I IGF-I receptor protein and IGF-I stimulated the proliferation of low density plated (2000 cells/cm² growth area) carcinoma cells by 1.6- to 3- fold above control after a four day inCUbation period under serum-free conditions (A549, A427) or in the presence of 0.25% serum (SK-LU-1). In addition, when added to detergent-solubilized type I IGF receptors from A549 cells, IGF-I stimulated [1] a dose-dependent increase in the autophosphorylation of the type I IGF receptor, and [2] a dose-dependent increase (1.5- to 4-fold) in the phosphorylation of a tyrosine kinase-specific substrate. These results suggest that the growth promoting activity of IGF-I for the lung carcinoma cells was mediated through the activation of the type I IGF receptor.
  • Thumbnail Image
    Endogenous Stress Signaling within Human Multicellular Aggregates (Spheroids)
    Jack, Graham Dillon (Virginia Tech, 2006-07-10)
    A wide variety of adherent mammalian cells can be induced into a reversible state of metabolic arrest (quiescence) by conversion to non-adherent multicellular aggregates. These "spheroids" can be maintained at room temperature under oxygen- and nutrient-deprived conditions for extended periods of time (weeks) as well as converted back to viable proliferating monolayers. Herein it is shown that HEK293 spheroid arrest and recovery requires the co-activation of both NF-kB and JNK, and chemical inhibition of either NF-κB nuclear translocation or JNK phosphorylation leads to cell death. Cytokine profiling within the aggregates during the arrest and recovery process is suggestive that a cyclical cascade was in operation, leading to endogenous cytokine production of TNF-Alpha, IL-1Beta, and IL-8, thereby propagating the cellular stress signal within cells as well as throughout the aggregate. Cytokines exist in vivo as mixtures, yet tissue culture studies delineating how cells respond to these molecules are often performed using individual effectors added exogenously. Are the results obtained in these studies true representations of physiological responses? As HEK293 multicellular aggregates (spheroids) survive long term arrest by endogenous cytokine (TNF-α and IL-1β) and chemokine (IL-8) signaling, adherent monolayer cells were evaluated for their ability to provide a "spheroid signal response" when exposed to TNF-α, IL-1β and IL-8 individually, and in combination, at concentrations observed in the aggregates. The spheroid signal transduction response was only observed when all three cytokines were present, demonstrating that signal transduction cascade mechanisms are cytokine-profile dependent. To determine if similar processes were involved in the arrest and recovery of multicellular aggregates derived from other cell types, the responses of primary human foreskin fibroblasts (HFF-2) and a glioblastoma cell line (T98G) were characterized, utilizing the procedures developed in the HEK293 study. Both the T98G and HFF-2 cell lines entered and exited from the long term arrest utilizing an autocrine response. However, while the carcinoma cell line was dependent upon NF-κB for survival, its signaling partner was Gadd45α and signaling occurred through the p38 pathway. Primary fibroblast arrest and recovery proceeded through the p38 pathway as well, but was independent of NF-κB. Thus, three different cell types and transformation states (HEK293, HFF-2, and T98G) provided three different routes to survival, all with cyclical cytokine production and signaling. These routes cannot be measured or modulated effectively in adherent monolayers. Multicellular aggregates provide higher ordered systems that can be used to describe signaling pathways within a cell, highlighting the role of autocrine responses and the synergistic relationships between cytokines and neighboring cells.
  • Thumbnail Image
    Erwinia carotovora extracellular proteases: characterization and role in soft rot
    Kyöstiö, Sirkka R. M. (Virginia Tech, 1990-12-03)
    Erwinia carotovora subsp. carotovora (Ecc) strain EC14, a Gram-negative bacterium, causes soft rot on several crops, including potato. Maceration of potato tuber tissue is caused by secreted pectolytic enzymes. Other cell-degrading enzymes may also have roles in pathogenesis, including cellulases, phospholipases, and protease(s). The objectives of this research were to (1) characterize Ecc extracellular protease (Prt) and (2) elucidate its role in potato soft rot. A gene encoding a Prt, prt1, was cloned from cosmid pCA7 containing Ecc genomic DNA into plasmid pSK1. Escherichia coli transformed with pSK1 or pSK23, a subclone of pSK1, produced intracellularly a 38 kDa Prt with the same pI (4.8) as the secreted Ecc Prt. Prt1 activity produced by E.coli/pSK23 was inhibited by phenanthroline, which inhibits Zn-metalloproteases, but not by Ecc intracellular proteins. Analysis of deletion mutants indicated a 1.2 kb region necessary for Prt1 production. Sequencing of the pSK1 insert revealed a 1,041 bp open reading frame (ORF1) corresponding to the prtl region. ORF1 encodes a putative polypeptide of 347 amino acids with a total molecular mass of 38.8 kDa. The location of the prt1 promoter was determined to be 173 to 1,173 bp upstream from ORF1 by constructing transcriptional fusions to lacZ in plasmid pCD267. Primer extension revealed the start of prt1 mRNA 205 bp upstream of ORF1. The deduced amino acid sequence of the prt1 was compared to other proteases; it is similar to several bacterial Zn-metalloproteases. Prt1 production by Ecc was not observed during growth in rich broth; however, Northern analysis showed prt] mRNA accumulation in Ecc grown in planta. The role of prt1 in soft rot was determined by constructing a Prt1-deficient Ecc; prt1 insertionally inactivated by a kanamycin resistance gene was used to replace wildtype prt11 in the Ecc genome by homologous recombination. This mutant (L-957) had approximately 60 to 80% reduced Prt activity suggesting the presence of a second Prt (Prt2). Prt2 was purified from Ecc culture supernatant. This protease, also a metalloprotease, has a molecular mass of 45 kDa and pI of 4.8. Its amino terminal sequence had Significant sequence identity to metalloproteases from Erwinia chrysanthemi and Serratia marcescens, but not to Prt1. Further, unlike Prt1i, Prt2 was inhibited by Ecc intracellular proteins. The effect of proteases in potato tuber maceration was measured using L-957 and L-763, a Tn5 transposon mutant constructed previously. L-763 had no extracellular protease activity and may have been mutated in a regulatory region. Both mutants macerated significantly less tuber tissue than the wildtype Ecc. Reduced maceration of L-957 and L-763 was correlated with slower in planta growth. This suggests Prt1 production provides a nutritional advantage for Ecc growth on potato.
  • Thumbnail Image
    Evalutating Biological Data Using Rank Correlation Methods
    Slotta, Douglas J. (Virginia Tech, 2005-05-05)
    Analyses based upon rank correlation methods, such as Spearman's Rho and Kendall's Tau, can provide quick insights into large biological data sets. Comparing expression levels between different technologies and models is problematic due to the different units of measure. Here again, rank correlation provides an effective means of comparison between the two techniques. Massively Parallel Signature Sequencing (MPSS) transcript abundance levels to microarray signal intensities for Arabidopsis thaliana are compared. Rank correlations can be applied to subsets as well as the entire set. Results of subset comparisons can be used to improve the capabilities of predictive models, such as Predicted Highly Expressed (PHX). This is done for Escherichia coli. Methods are given to combine predictive models based upon feedback from experimental data. The problem of feature selection in supervised learning situations is also considered, where all features are drawn from a common domain and are best interpreted via ordinal comparisons with other features, rather than as numerical values. This is done for synthetic data as well as for microarray experiments examining the life cycle of Drosophila melanogaster and human leukemia cells. Two novel methods are presented based upon Rho and Tau, and their efficacy is tested with synthetic and real world data. The method based upon Spearman's Rho is shown to be more effective.
  • Thumbnail Image
    Exploring Siderophore-Mineral Interaction Using Force Microscopy and Computational Chemistry
    Kendall, Treavor Allen (Virginia Tech, 2003-04-11)
    The forces of interaction were measured between the siderophore azotobactin and the minerals goethite (FeOOH) and diaspore (AlOOH) in solution using force microscopy. Azotobactin was covalently linked to a hydrazide terminated atomic force microscope tip using a standard protein coupling technique. Upon contact with each mineral surface, the adhesion force between azotobactin and goethite was two to three times the value observed for the isostructural Al-equivalent diaspore. The affinity for the solid iron oxide surface reflected in the force measurements correlates with the specificity of azotobactin for aqueous ferric iron. Further, the adhesion force between azotobactin and goethite significantly decreases when small amounts of soluble iron are added to the system suggesting a significant specific interaction between the azotobactin and the mineral surface. Changes in the force signature with pH and ionic strength were fairly predictable when considering mineral solubility, the charge character of the mineral surfaces, the molecular structure of azotobactin, and the intervening solution. Molecular and quantum mechanical calculations which were completed to further investigate the interaction between azotobactin and iron/aluminum oxide surfaces, and to more fully understand the force measurements, also showed an increased force affinity for Fe over Al. Ab initio calculations on siderophore fragment analogs suggest the iron affinity can be attributed to increased electron density associated with the Fe-O bond compared to the Al-O bond; an observation that correlates with iron's larger electronegativity compared to aluminum. Attachment of the ligand to each surface was directed by steric forces within the molecule and coulombic interactions between the siderophore oxygens and the metals in the mineral. Chelating ligand pairs coordinated with neighboring metal atoms in a bidentate, binuclear geometry. Upon simulated retraction of azotobactin from each surface, the Fe-O(siderophore) bonds persisted into a higher force regime than Al-O(siderophore) bonds, and surface metals were removed from both minerals. Extrapolation of the model to more realistic hydrated conditions using a PCM model in the quantum mechanical calculations and water clusters in the molecular mechanical model demonstrated that the presence of water energetically favors and enhances metal extraction, making this a real possibility in a natural system.
  • Thumbnail Image
    Extracellular Matrix (ECM)
    Helm, Richard F.; Potts, Malcolm (Springer, 2012)
    The region of space at the periphery of cyanobacterial cells is the interface between the environment and intracellular processes. This metaspace may include a structure appressed to the outer wall and membrane, such as an extracellular polysaccharide (EPS), a structural and/or physiological discontinuity modulating metabolite fl ow, as well as a temporal fl ux that accompanies stress or cell division. The functional framework within this region is designed to recognize environmental perturbations and relay physical and biochemical information to the cell interior, and perhaps to the cell community, for the appropriate physiological response. Communication between the environment and the cells is thus initiated within this extracellular milieu, which is therefore an important spatial domain in cyanobacteria. The ECM of cyanobacterial cells is multifaceted. It is not only a complex and dynamic mixture of polysaccharides, proteins, cell remnants and lower molecular weight secondary metabolites, but a hyperspace that tunes seasonal as well as shortterm stochastic modulations in environmental conditions. Such stresses result in changes in both the composition and organization of the matrix as cyanobacterial cells adjust to the environmental perturbations. This chapter provides a critical appraisal of the ecology and evolution of the cyanobacterial ECM compared with other prokaryotes. Emphasis is placed on how little is understood about this “occupied space” and several hypotheses and examples are presented in an effort to promote additional investigations of this oft-ignored interface.
  • Thumbnail Image
    Fatty acid synthase is a major polypeptide constituent of cytosolic lipoprotein and is associated with components of the milk lipid secretory pathway
    Keon, Brigitte H. (Virginia Tech, 1993-05-15)
    Most of the lipid present in lactating mammary gland cytosol was associated with a high molecular weight aggregate isolated from cytosol by gel exclusion chromatography or by density gradient centrifugation. The major polypeptide constituent of this lipoprotein aggregate was the monomer of fatty acid synthase (FAS). The major milk lipid globule proteins, butyrophilin (8u) and xanthine oxidase (XO), as well as the small GTP-binding protein ARF, also were present. This lipoprotein complex was abundant in cytosol from lactating but not from involuting mammary glands. HPTLC analysis of lipids extracted from the low density FAS (LDFAS) complex demonstrated the presence of the five major milk phospholipids as well as triacylglycerols, cholesterol, unesterified fatty acids, and diacylglycerols. ³²P-labeled phospholipids present in cytosol could be transferred to microlipid droplets (MLD) and endoplasmic reticulum (ER), in vitro, and could be precipitated along with FAS, and other polypeptide constituents of the LDFAS complex. Complexed FAS could be separated from noncorrlplexed FAS by density gradient centrifugation, native PAGE, and gel exclusion chromatography. A large amount of phospholipid consistently was retained with the complexed form of FAS. These results suggest that FAS migrates to a low density fraction by virtue of its association to other proteins and lipids. FAS was found to be associated with ER, intracellular lipid droplets, and the milk lipid globule membrane (MLGM). A similar complex to LDFAS was isolated from ER from liver and mammary gland homogenates following incubation in buffer containing ATP. Polypeptide constituents of this complex had similar electrophoretic patterns to LDFAS, but behaved differently from LDFAS constituents when fractionated with the detergent TX-114. While most of the polypeptides in LDFAS partitioned equally into the detergent and aqueous phases, a constituent with an approximate molecular weight of 70 kDa was enriched in the detergent phase. For the ER-derived FAS complex, most of the polypeptides remained in the aqueous phase but the detergent phase also was enriched with a polypeptide similar in size to the LDFAS detergent enriched constituent. Western blot analysis failed to detect Bu in the ER-derived complex. However, protein disulfide isomerase (PDI) was detected in this complex as well as a polypeptide with approximate molecular weight 50 kDa that cross-reacted with PDI antibody. Extraction of lipids from this ER-derived complex demonstrated the presence of large quantities of unesterified fatty acids, with relatively low amounts of complex lipids. In studies using ¹²⁵I labeled LDFAS, labeled polypeptides were shown to associate with ER and intracellular lipid droplets and their dissociation was stimulated by ATP. Immunocytochemistry using antibody to rat liver FAS revealed distribution of FAS at localized regions of the cytoplasmic surface of rough endoplasmic reticulum and on surfaces of intracellular lipid droplets. Electron micrographs of the LDFAS complex showed a homogeneous morphology of granular, symmetrical particles ranging in size from 40 nm to 170 nm in diameter. These particles resembled low density lipoprotein (LDL) in morphology. From the available data, the following model was proposed for a possible involvement of FAS in lipid droplet secretion in the mammary gland. During active lipogenesis, FAS is targeted to ER membrane by association with a signal or targeting peptide(s) in the cytosol. The signal peptide then binds to selected regions of ER where signal receptors reside. Binding of FAS may initiate synthesis and accumulation of triacylglycerol between ER membrane bilayers. Upon the achievement of a "critical mass", the lipid core may be released into the cytoplasm in an ATP-dependent manner, surrounded by the membrane components that provided the hydrophobic pocket for lipid accumulation. Butyrophilin and the 70 kDa detergent-extractable constituent released from the ER and present in LDFAS are possible sources of such a function. Polypeptides from the cytosolic leaflet of the ER, and proteins peripherally associated with the leaflet then would comprise the polypeptide constituents of the lipid particle.
  • Thumbnail Image
    Genomics-Based Analysis of Antibody Response to Sheep Red Blood Cells in Chickens
    Geng, Tuoyu (Virginia Tech, 2007-05-18)
    Immune response provides vertebrates an important mechanism to fight pathogens and to reduce the incidence of diseases. Defining the molecular basis of antibody response may facilitate genetic improvement in the immune response of animals to pathogens. For almost 4 decades, antibody titers in response to challenge by sheep red blood cells (anti-SRBC) have provided an investigative tool in the efforts to define molecular mechanisms that underlie vertebrate immune response. The overall objective of this dissertation research was to identify DNA markers associated with anti-SRBC response in chickens. Specific objectives were: to develop a resource population for QTL analysis for anti-SRBC, to identify DNA markers and genes associated with primary anti-SRBC, and to evaluate the allelic frequencies in non-selected chicken populations of candidate markers associated with either high or low anti-SRBC response. These objectives tested the hypothesis that genetic control of a chicken's response to SRBC is polygenic. The resource population developed consisted of F1, backcross, and F2 derived from reciprocal crosses of birds from parental lines in the 28th generation of divergent selection for low (L) and high (H) anti-SRBC. The mean anti-SRBC titers of the parental lines were significantly different, with 11.5 for H and 2.6 for L (P<0.05). That for the 4 groups of F2 progeny ranged from 6.3 to 7.5, while those of the 8 groups of backcross progeny ranged from 3.9 to 13.3. Four of 555 random primers used to screen the parental H and L anti-SRBC lines were informative by amplifying seven line-specific fragments (P<0.0025). Each of the 7 line-specific fragments was converted to a sequence characterized amplified region (SCAR) within which single nucleotide polymorphisms (SNPs) were identified and tested for association with anti-SRBC. Only two of the seven SCARs in the parental lines were associated (P<0.05) with anti-SRBC level in the backcross resource population. Additionally, from analysis of the parental L and H anti-SRBC lines using microarrays, a total of 57 line-specific SNPs were also identified. Twenty of the line-specific SNPs were in and/or near genes previously reported to have immunity-related function. Microarray-based gene expression profiling of pooled RNA samples from L and H anti-SRBC birds identified three differentially expressed genes. In summary, this dissertation describes resources that include candidate SCARs and SNPs as well as differentially expressed genes that may be useful for the identification of genes that underlie antibody response.
  • Thumbnail Image
    Glycolipids in mouse F9 teratocarcinoma cells: some changes associated with retinoic acid-induced differentiation
    Gorbea, Carlos M. (Virginia Tech, 1991-01-15)
    To investigate the changes in glycolipid biosynthesis during early embryogenesis mouse F9 teratocarcinoma cells were induced to differentiate in vitro in the presence of retinoic acid. Control embryonal carcinoma cells and their differentiated derivatives, RNF9 cells, were metabolically-radiolabeled with [6-3H]galactose or [6-3H]glucosamine, and their glycolipids were compared. The neutral and acidic glycolipid fractions from both cell lines were subjected to ozonolysis and alkali fragmentation or endoglycoceramidase digestion to release the glycolipid-derived oligosaccharides. The neutral oligosaccharides were separated according to size by gel filtration and high performance liquid chromatography. These analyses indicated that differentiated F9 cells synthesized less high molecular weight oligosaccharides (containing more than 5 sugar residues) relative to controls. Serial lectin affinity chromatography on columns of immobilized Helix pomatia. Wisteria f1oribunda. Griffonia simplicifolia-I and Ricinus communis-' agglutinins followed by reduction and permethylation revealed that globoside (GaINAcβ1, 3Galα1, 4Galβ1, 4Glc) and lactose (Galβ1,4Glc) are the principal glycolipid-derived oligosaccharides synthesized by F9 and RNF9 cells. An increased biosynthesis of these components was observed in RNF9 cells relative to controls. These changes paralleled the reduced biosynthesis of Forssman pentasaccharide (GaiNAcα 1 ,3GaINAcβ1 ,3Galα1 ,4Galβ1 ,4Glc) reported previously. Normalization of the incorporation of 3H-monosaccharides in glycolipid-derived oJigosaccharides to the number of cells indicated a 2-6 fold increase in the incorporation of radioactive precursors in RAlF9 cells relative to F9 controls, suggesting that an enhancement in glycosphingolipid biosynthesis accompanies the differentiation of F9 cells. The monosialylganglioside-derived oligosaccharides obtained from F9 and RAlF9 cells were separated by anion exchange chromatography. A reduced biosynthesis of high molecular weight components was observed in RAlF9 cells when compared with undifferentiated F9. Lectin affinity chromatography on immobilized Maackia amurensis agglutinin followed by reduction and permethylation indicated a dramatic increase in the synthesis of GM1 (Galβ1,3Ga1NAcβ1,4[NeuAcα2,3] Galβ1,4Glc) and GM3 (NeuAcα2,3Galβ1,4Glc) in RAlF9 cells relative to controls. These changes were accompanied by a decrease in the synthesis of sialyltetrasaccharide a (NeuAcα2,3Galβ1 ,3GlcNAcβ1 ,3Galβ1,4Glc) and sialylparagloboside (NeuAcα2,3Galβ1 ,4GlcNAβ1 ,3Galβ1 ,4Glc) in the differentiated cells. These observations are in agreement with previous reports in leukemic and human embryonal carcinoma cell lines and may be related to the growth arrest and antigenic changes associated with F9 differentiation. In the work reported herein, serial lectin affinity chromatography in concert with permethylation analysis prove to be powerful methods for the isolation and characterization of glycolipid-derived oligosaccharides. The application of these methods has allowed the unequivocal identification of main glycosphingolipid components as well as of some representing less than 1 % of the total glycolipids synthesized by two cell lines. This information should provide the basis for further studies involving glycosyltransferas.
  • Thumbnail Image
    Identification of a Low Molecular Weight Protein Tyrosine Phosphatase and Its Potential Physiological Substrates in Synechocystis sp. PCC 6803
    Mukhopadhyay, Archana (Virginia Tech, 2006-02-10)
    The predicted protein product of open reading frame slr0328 from Synechocystis sp. PCC 6803, SynPTP, possesses significant amino acid sequence similarity with known low molecular weight protein tyrosine phosphatases (PTPs). To determine the gross functional properties of this hypothetical protein, open reading frame slr0328 was cloned, and its predicted protein product was expressed in E. coli. The recombinant protein, SynPTP, was purified by metal ion column chromatography. The catalytic activity of SynPTP was examined toward several exogenous protein substrates that had been phosphorylated on either tyrosine residues or serine residues. SynPTP exhibited phosphatase activity toward tyrosine phosphorylated protein substrates (Vmax toward phosphotyrosyl 32P-casein was 1.5 nmol/min/mg). However, no phosphatase activity was detected toward serine phosphorylated protein substrates. SynPTP displayed phosphohydrolase activity toward several organophosphoesters including para-nitrophenyl phosphate (p-NPP), beta-napthyl phosphate and phosphotyrosine but not toward alpha-napthyl phosphate, phosphoserine, or phosphothreonine. Kinetic analysis indicated that the Km (0.6 mM) and Vmax (3.2 mmole/min/mg) values for SynPTP toward pNPP are similar to those of other known bacterial low molecular weight PTPs. The protein phosphatase activity of SynPTP was inhibited by sodium orthovanadate, a known inhibitor for tyrosine phosphatases, but not by okadaic acid, an inhibitor for many serine/threonine phosphatases. Mutagenic alteration of the predicted catalytic cysteine, Cys7, to serine abolished enzyme activity. Several phosphotyrosine containing proteins were detected from the whole cell extracts of Synechocystis sp. PCC 6803 through immunoreactions using anti-phosphotyrosine antibody. SynPTP was observed to dephosphorylate three of these proteins in vitro. Two of these proteins were identified by peptide-mass fingerprinting analysis, as PsaD (photosystem I subunit II) and CpcD (phycocyanin rod linker protein). In addition, several phosphotyrosine proteins were detected from the soluble and membrane fractions of Synechocystis sp. PCC 6803 cell extracts by in vitro substrate trapping as potential endogenous substrates of SynPTP. Two of these proteins were identified as the alpha and beta subunits of phycocyanin. We therefore speculate that SynPTP might be involved in the regulation of photosynthesis in Synechocystis sp. PCC 6803.