• The cloning and identification of a streptonigrin resistance gene from Escherichia coli

      Heller, Phillip Ferdinand; Speedie, Marilyn K. (1996)
      Streptonigrin is a antineoplastic antibiotic whose mode of action is dependent on formation of the hydroxyl radical. A streptonigrin resistance gene was cloned from a partial Sau3A digest of E. coli DNA. One to 10 Kilobase (Kb) fragments were ligated into the vector pUC18 and transformed into E. coli (DH5{dollar}\alpha{dollar}). Once prepared, the library was replica plated and then screened with differing streptonigrin concentrations in a top agar overlay. Clones exhibiting growth into the overlay at the highest STN concentrations (pH191, pH192) were rescued from the overlay and grown for plasmid isolation and for determination of theminimum inhibitory concentration (MIC) for streptonigrin in LB broth, 3 and 1 {dollar}\mu{dollar}g/ml respectively. The isolated plasmids were mapped by restriction endonuclease digestion followed by agarose gel electrophoresis. Restriction sites were assigned based on known location in the multicloning site of the vector, leading to the production of an approximate physical map of the insert in the plasmid. The resistance element (StnR) was localized by deletion studies on pH191. Sequencing and analysis of the deoxynucleotide sequence of pH191 led to the identification of an open reading frame (StnR) with a high homology (97.6%) to the first 185 residues of riboflavin synthase (RibC). The resistance gene was expressed in S. lividans on pIJ702 where it yielded streptonigrin resistance in excess of that found in the wild type producing species, S. flocculus (40 {dollar}\mu{dollar}g/ml). Expression of StnR and RibC in E. coli via the vector pET3C resulted in strains with elevated streptonigrin resistance as compared to the pET3C control in both solid and liquid media. A gene imparting resistance to the hydroxyl radical producing drug streptonigrin has been identified as the riboflavin synthase from E. coli.
    • Contributions of dissolution and intestinal permeation to oral drug product quality

      Ginski, Mark Joseph; Polli, James E. (1999)
      Dissolution and intestinal permeation are the primary biopharmaceutical factors controlling oral drug product performance. The objective of this research was to develop in vitro dissolution and Caco-2 cell monolayers, both separately and in combination, as tools to characterize the biopharmaceutical performance of oral drug products. Firstly, in vitro dissolution methods were developed to elucidate the degree of drug solubility-controlled or formulation-controlled drug dissolution from immediate release dosage forms. Results indicate dissolution from formulations may be controlled by disintegration or diffusion through the aqueous boundary layer. Drug solubility and formulation design dictated the degree of disintegration or diffusion control. Secondly, Caco-2 monolayer permeability was quantitatively related to human drug absorption kinetics. Results suggest a rank-order relationship between Caco-2 permeability and in vivo intestinal permeation rate, providing a kinetic interpretation of Caco-2 monolayer permeability. Caco-2 monolayer methods were also used to develop a simple kinetic model to elucidate P-glycoprotein effects on intestinal drug transport. Results suggest P-glycoprotein substantially reduced net fexofenadine transport, which was well characterized by the simple kinetic model. Thirdly, "static" and "dynamic" dissolution/Caco-2 systems were developed. Each system integrated in vitro dissolution and Caco-2 monolayers to predict the relative contributions of dissolution and intestinal permeation to overall drug absorption kinetics. Results indicate the "static" system accurately predicted the relative contributions of dissolution and intestinal permeation to overall drug absorption and the "dynamic" system simultaneously assessed the effect of formulation changes on dissolution and intestinal permeation. Hence, in vitro dissolution and Caco-2 cell monolayer methods were successfully developed to characterize the biopharmaceutical performance of oral drug products. Separately, these methods identified factors controlling drug product dissolution, estimated the absorption potential of oral drug candidates and elucidated P-glycoprotein effects on intestinal drug transport. In combination, these methods elucidated the degree to which dissolution and intestinal permeation limits overall drug product performance and characterized excipient effects on dissolution and intestinal permeation.
    • Identification of proteins that interact with Escherichia coli DNA topoisomerase III in vivo

      Kumar, Uplaksh S.; DiGate, Russell J. (2000)
      Topoisomerase III (Topo III) belongs to the type I DNA Topoisomerase family and is encoded by the topB gene. This enzyme is capable of relaxing and decatenating DNA. Although structure-activity relationships within the protein have been extensively studied, there is very limited information about the function of the enzyme in vivo. In this dissertation we have been able to identify proteins that interact with Topo III based on phage display library and biochemical purification. A region of Penicillin Binding Protein 1B (PBP1B) of E. coli was identified by phage display. This enzyme catalyzes the polymerization and insertion of peptidoglycan precursors into the bacterial cell wall during cell elongation. Using a modified Elisa assay we have confirmed that PBP1B and Topo III interact in vitro. To study the in vivo interaction, a gfp-topB (green fluorescence protein) fusion protein was constructed and the localization of Topo III in a PBP1B deletion mutant and its isogenic parent strain was observed under fluorescence microscopy. Topo III is located at the septum and the poles in the wild type cells, however no localization of Topo III was found in PBP1B mutant cells. In addition, a lethal effect due to the overexpression of Topo III can be rescued in a PBP1B mutant. All these results indicate that the two proteins interact in vivo. The localization of a mutant Topo III has also been examined. The overexpression Topo III containing a G432D mutation is not lethal to the cells. No localization of this mutant was observed regardless of the presence or absence of PBP1B. This suggests that the mutation may identify the region of Topo III that interacts with PBP1B. A biochemical approach has been designed to identify additional proteins that interact with Topo III. A partially purified protein has been shown to inhibit the relaxation activity of Topo III. This protein may be a useful candidate in the design of specific inhibitors of Topo III. In conclusion, the function of Topo III is not only restricted to DNA metabolism but also plays a role in cell division. The identification of the inhibitor of Topo III could also yield a new antibiotic.
    • Investigation of sigma and dextromethorphan-like neuroprotection using glutamate-induced LDH release, cellular morphology and dynamic calcium signaling

      Klette, Kevin Louis; Moreton, J. Edward (1995)
      The role of the putative sigma receptor in mediating neuroprotection against glutamate induced neuronal injury was examined in mature cultured rat cortical neurons. With the exception of the sigma1, selective ligand (+)-3-PPP, all of the sigma receptor ligands tested were neuroprotective, preventing glutamate induced morphological changes and increases in LDH release. When corrected for relative sigma versus PCP binding site affinity, it appears that a positive correlation exists between neuroprotective potency and sigma1, site affinity. None of the sigma ligands were neurotoxic when tested alone at concentrations at least 5-30 times their respective neuroprotective EC{dollar}\sb{lcub}50{rcub}{dollar} values. The effect of neuroprotective sigma ligands on the unique calcium responses evoked by glutamate, NMDA, potassium chloride (KCl) and trans-ACPD were investigated to elucidate the mechanism of sigma-mediated neuroprotection. In general, except for (+)-3-PPP all of the sigma ligands studied interfered with glutamate and NMDA induced (Ca{dollar}\sp{lcub}2+{rcub}\rbrack\sb{lcub}\rm i{rcub}{dollar} signaling, but, highly sigma{dollar}\sb1{dollar} selective ligands also lacking substantial PCP binding site affinity (i.e. carbetapentane, DTG and haloperidol) were much less effective in altering calcium influx induced by 80 {dollar}\mu{dollar}M glutamate. In contrast to glutamate, KCl (50 mM) produced changes in (Ca{dollar}\sp{lcub}2+{rcub}\rbrack\sb{lcub}\rm i{rcub}{dollar} which were not neurotoxic to the neurons as measured by LDH release. Sigma ligands which lack substantial PCP site afflinity were very effective in altering KCl induced calcium signaling while the sigma/PCP site ligand (+)-cyclazocine was ineffective or, in the case of (+)-SKF 10047, much less effective. Similar to the effects of sigma ligands on KCl induced calcium dynamics, the sigma selective ligands DTG, haloperidol, (+)-pentazocine, and carbetapentane were very effective in altering intracellular calcium dynamics evoked by trans-ACPD while the sigma/PCP ligand (+)-SKF 10047 was ineffective or, in the case of (+)-cyclazocine, much less effective. Importantly, (+)-3-PPP, a non-neuroprotective sigma selective ligand, actually potentiated the calcium response elicited by trans-ACPD. The ability of sigma ligands applied at maximal neuroprotective concentrations to attenuate receptor and/or voltage-gated changes in calcium dynamics suggests that modulation of neurotoxic (Ca{dollar}\sp{lcub}2+{rcub}\rbrack\sb{lcub}\rm i{rcub}{dollar} plays a significant role in sigma-mediated neuroprotection. The unique modulatory effects of sigma ligands on the buffering of neuronal (Ca{dollar}\sp{lcub}2+{rcub}\rbrack\sb{lcub}\rm i{rcub}{dollar} will likely have numerous therapeutic applications in the treatment of CNS injury and other neurodegenerative disorders.
    • Mechanism of action of 1-beta-D arabinofuranosyl cytosine (Ara-C): The biological consequences of the incorporation of Ara-C into a eukaryotic DNA replication origin

      Yao, Lei; Hickey, Robert J., Ph.D. (1997)
      1-beta-D arabinofuranosyl cytosine (Ara-C) is a highly effective drug used to treat acute leukemia. Yet, despite its high efficacy, the precise mechanism of action of ara-C mediating the cell killing and inhibition of DNA synthesis remains unclear. Ara-C is converted to the active form, ara-CTP, by cellular kinases. Ara-CTP inhibits the DNA synthesis by inhibiting DNA polymerase and blocks the elongation of the DNA strand. However, the studies using intact cells and viral system indicate that ara-C is mainly incorporated into DNA at internucleotide linkages and ara-C slows but does not terminate the DNA synthesis, suggesting the alternative mechanism of action of ara-C. We have investigated the biological effects of the incorporation of ara-Cinto DNA using the SV40 DNA replication system. Ara-C incorporation into SV40 origin caused a site specific inhibition of T-Ag (an initiator protein of DNA replication) and RP-A (human single stranded DNA binding protein) binding to the origin DNA, and reduction in the stability of DNA. Ara-C substitution at T-Ag binding site I domain inhibited more effectively the binding of T-Ag and promoted greater reduction in the stability of DNA duplexes compared to the ara-C substitution at T-Ag binding site II and early mRNA domains, and contributes to the ara-C substitutions at other sites in inhibiting T-Ag binding and reducing the stability of the DNA duplex. The ara-C substitutions at both site I and early mRNA domains also disrupted RP-A binding. Furthermore ara-C substituted origin DNA has lower affinity for T-Ag binding than unsubstituted DNA. These site specific effects of ara-C incorporation may be sequence dependent. Moreover, ara-C incorporation within the SV40 origin appears to have essentially no effect on the enzymatic unwinding of origin DNA by T-Ag helicase and the initiation and subsequent elongation of the origin DNA by the human multiprotein DNA replication complex (MRC). The data suggest that the ara-C incorporation changes the structure of DNA, which alters the binding affinity of the origin DNA for T-Ag and RP-A binding, but does not disrupt the enzymatic processes involved in replication of the DNA. Our results support the findings that: (1) ara-C is a weak competitive inhibitor of DNA polymerase; and, (2) ara-C incorporation slows, but does not terminate DNA synthesis.
    • Structural and electrochemical properties of wild-type and mutant cytochromes b-(5)

      Sarma, Siddhartha P.; Guiles, Ronald (1996)
      The main objective of this thesis project was to gain a better understanding of the protein structural factors that modulate heme reduction potentials in b-type cytochromes. Rat liver microsomal cytochrome {dollar}b\sb5{dollar} was chosen as a model system because the availability of a synthetic gene coding for the protein permits mutagenic experiments to be performed. The synthetic gene encoding for cytochrome {dollar}b\sb5{dollar} was subcloned into a high yield bacterial overexpression system that enabled isotopic enrichment of proteins. Site directed mutants of cytochrome {dollar}b\sb5{dollar} were prepared by subcloning the synthetic gene into bacteriophage DNA m 13mp18. Mutants of cytochrome {dollar}b\sb5{dollar} were designed to achieve reorientation of individual axial imidazole ligands. The orientation of the axial ligand planes is thought to modulate the reduction potential of bis(imidazole) axially ligated heme proteins. The A67V (alanine to valine) mutation resulted in a reorientation of the H63 imidazole ring and a shift in reduction potential by {dollar}-20{dollar} mV. Structural characterization of the A67V mutant protein was achieved using homonuclear and heteronuclear NMR methods. Calculation of the orientation of the components of paramagnetic susceptibility tensor showed that the wild type and mutant proteins differed only in the orientation of the z-component. The rotation of the z-component of the susceptibility tensor is in the same direction as the rotation of the H63 imidazole ring. EPR and Near-IR data suggest that the stability of iron d-orbital energy levels in the reduced proteins may be significant in determining the reduction potential of b-type heme proteins. Multidimensional double and triple resonance NMR methods have been applied to assign the backbone and side-chain {dollar}\sp{lcub}13{rcub}{dollar}C resonances for both equilibrium conformers of ferricytochrome {dollar}b\sb5{dollar} On the basis observed NOEs and backbone {dollar}\sp{lcub}13{rcub}{dollar}C chemical shifts, the solution secondary structure of cytochrome {dollar}b\sb5{dollar} has been determined. The {dollar}\sp{lcub}13{rcub}{dollar}C chemical shifts of backbone and side-chain atoms are relatively insensitive to paramagnetic effects. The reliability of such methods in anisotropic paramagnetic systems, where large pseudocontact shifts are observed, is evaluated through calculations of the magnitude of such shifts.
    • Structural and functional characterization of the human apical sodium-dependent bile acid transporter

      Banerjee, Antara; Swaan, Peter W. (2005)
      The human apical sodium-dependent bile acid transporter (hASBT), an essential component of the enterohepatic circulation (EHC), is responsible for bile acid reabsorption from the lumen of the distal ileum and plays a critical role in bile acid and cholesterol homeostasis. Lack of a crystal structure for ASBT, limits our understanding of the structural and functional determinants of transport. The work in this dissertation was carried out to characterize the structural components and determine their overall role in ASBT function. In particular, the work described here was aimed to (1) elucidate and further understand the topological framework of hASBT by epitope insertion, (2) evaluate the role of N-glycosylation and the N-terminal domain in ASBT function by alanine scanning mutagenesis, (3) probe the role of the endogenous cysteines using thiol modifiers and various bile acid conjugates, (4) and to determine the sodium, and bile acid translocation pathway comprising residues of transmembrane (TM) domain seven and extracellular loop (EL) three using substituted cysteine accessibility method. Due to conflicting experimental evidence, the membrane topography of ASBT, predicted to comprise 7 to 9 putative TM domains, remains unresolved. Our results from epitope insertion clearly, support a 7TM model. The work that followed was aimed at characterizing some of the functionally critical domains of this transporter. Alanine scanning mutagenesis showed that the N-terminal region was vital for function and mutation of the N-glycosylation site was responsible for reduced uptake activity but did not impact trafficking of the protein to the plasma membrane. Evaluation of the endogenous cysteine residues revealed that multiple Cys residues are essential for hASBT function and C270A in combination with methanethiosulfonate (NITS) reagents and bile acid-NITS conjugates can aid in defining the putative ligand binding region(s). Cysteine mutants of EL3 and TM7 were generated using C270A, and in conjunction with thiol-modification it was shown that EL3 forms the primary sodium interaction site while TM7 lines the substrate translocation pathway. Furthermore, critical residues involved in the process were also identified. Overall, the work carried out in this dissertation will aid in the advancement of drug design and improve our understanding of the features that are essential for recognition of effective inhibitors for this transporter.
    • Structural basis of Escherichia coli DNA topoisomerase III-catalyzed decatenation

      Li, Zhiyu; DiGate, Russell J. (1999)
      Escherichia coli topoisomerase III (Topo III) consists of four domains folding into a torus with a central cavity of approximately 25 A in diameter. In order to assess the DNA binding function of this hole, a pair of cysteine residues were introduced into Topo III at I299 and A494 in two adjacent domains near the hole. In the absence of reducing reagent, the C299 and C494 form a disulfide bond. By regulating the oxidation and reduction of this disulfide bond, a double stranded or a single stranded DNA has been trapped within the Topo III central hole. Topo III possesses a single stranded DNA binding region in addition to the central hole. A single stranded DNA gel shift assay has been applied to the Topo III/double stranded DNA complex, a slowly migrating complex consisting of the Topo III, double stranded DNA, and single stranded DNA has been observed. Based on these observations and other evidence, a 'strand passage' model of Topo III-catalyzed decatenation has been developed. A seventeen amino acid residue domain has been also identified in Topo III that is essential for Topo III-mediated resolution of DNA replication intermediates in vitro. Deletion of this domain completely abrogates Topo III-catalyzed resolution of DNA replication intermediates and decatenation of multiply linked plasmid DNA dimers. The modified polypeptide, however, was capable of relaxing negatively supercoiled DNA substrates. The presence of this domain has been detected in multiple plasmid encoded topoisomerases raising the possibility that these enzymes may also be decatenases. The polypeptide encoded by the plasmid RN traE gene shows extensive protein sequence homology with E. coli topB, the gene encoding Topo III. The traE gene product has been cloned, expressed and purified. The TraE protein exhibits topoisomerase activity similar to that of Topo III. Similar to E. coli Topo III, the TraE protein is a potent decatenase and can substitute for Topo III activity in vivo. The biochemical properties of the TraE protein in vitro suggest that the protein may be involved in the resolution of plasmid DNA replication intermediates during either vegetative replication, or in conjugative DNA transfer. Putative homologues of Topo III have been found to be encoded by other broad host range, conjugative plasmids isolated from both gram negative and gram positive organisms suggesting that Topo III-like polypeptides may have an essential role in the propagation of many promiscuous plasmids.
    • Structure/function analysis of Escherichia coli DNA topoisomerase III

      Zhang, HongLiang; DiGate, Russell J. (1994)
      The generalized nucleic acid-binding domain of Escherichia coli DNA topoisomerase III (Topo III) has been identified using a selection procedure designated to isolate inactive Topo III polypeptides. Deletion of this binding domain, contained within the carboxyl terminal 50 amino acid residues of Topo III, results in a drastic reduction in the ability of the enzyme to bind to single-stranded DNA and RNA substrates. Successive truncation of the enzyme within this region results in the gradual loss of nucleic binding activity and in a gradual change in the mechanism of Topo III-catalyzed relaxation of negatively supercoiled DNA. Truncation of the enzyme to 605 amino acids (Topo III contains 653 amino acids) reduced DNA relaxation activity and binding activity to 1-2% the activity of the full length polypeptide. The reduction of nucleic acid binding activity of the truncated polypeptides does not result in a loss of cleavage site specificity for the enzyme, suggesting other amino acids are involved in the positioning of the nucleic acid within the nicking/closing site of the topoisomerase. Topo III binds asymmetrically to its substrate relative to its cleavage site. Topo III protects 14 nucleotides (12 nucleotides upstream of the cleavage site and 2 nucleotides downstream) from nuclease P1 digestion. The minimum substrate requirement for Topo III-catalyzed cleavage of DNA is only 7 nucleotides; however, this minimum substrate exhibits the same asymmetric sequence preference (6 nucleotides upstream of the cleavage site and 1 nucleotide downstream). In addition, a substrate that contained a Topo III cleavage/binding site formed a more stable complex with the enzyme than a substrate that did not contain a Topo III cleavage/binding site. An oligonucleotide with a Topo III cleavage/binding site was a better competitor for Topo III than an oligonucleotide without the site. Based on these results, a model is proposed in which Topo III binds to DNA and diffuses along the substrate until a cleavage site is located. When a cleavage site is found, Topo III halts and stably binds to the cleavage site. A hybrid of molecule consisting of Topo III cleavage domain and the Topo I binding domain has been used to demonstrate that the domains for cleavage and binding could be separated. The hybrid was created by fusing the first 604 amino acids of Topo III with the carboxyl-terminal 313 amino acids of Topo I. Addition of the C-terminal amino acids of Topo I restored both DNA relaxation activity and DNA binding activity to the enzyme; however, the ability of the enzyme to segregate newly synthesized replication molecules was not drastically enhanced. This suggests that C-terminal domain of Topo III may specifically contribute to its ability to decatenate newly replicated chromosomes.
    • Synthesis and cytotoxic mechanism study of aziridinyl spermidine analogues

      Yuan, Zhi-Min; Callery, Patrick S. (1993)
      In this study, the three-membered alkylating moiety, aziridine, was chosen to replace a terminal amino group of the polyamine, spermidine, to generate N{dollar}\sp1{dollar}- and N{dollar}\sp8{dollar}-aziridinyl spermidine analogues. Cytotoxicity was tested in vitro against L1210 murine lymphoblastic leukemic and HL60 human leukemic cell lines. Aminoguanidine, a serum amine oxidase inhibitor, reduced the cell growth inhibitory effect of spermidine approximately 6- or 20-fold in HL60 or L1210 cells, respectively, but did not show any effect on the cytotoxicity induced by aziridinyl spermidines. N{dollar}\sp1{dollar}-Aziridinyl spermidine was slightly more potent than the N{dollar}\sp8{dollar}-isomer (IC{dollar}\sb{lcub}50{rcub}{dollar} values 0.15 {dollar}\mu{dollar}M vs 0.3 {dollar}\mu{dollar}M for L1210 cells and 0.14 {dollar}\mu{dollar}M vs 0.28 {dollar}\mu{dollar}M for HL60 cells). Assessment of cell viability with a flow cytometric technique using fluorescein diacetate and propidium revealed that both N{dollar}\sp{lcub}1{rcub}{dollar}- and N{dollar}\sp8{dollar}-aziridinyl spermidines were cytotoxic. Both aziridinyl spermidine analogues inhibited (H{dollar}\sp3{dollar}) -thymidine and (H{dollar}\sp3{dollar}) -uridine incorporation into L1210 cells in a dose-dependent fashion. This effect was observed after an exposure time as short as one hour. The fact that the cytotoxicity induced by these two aziridinyl derivatives of spermidine was potentiated by 24 hour pretreatment of L1210 cells with 100 {dollar}\mu{dollar}M {dollar}\alpha{dollar}-diflouromethylornithine (DFMO), and were prevented by coincubation with 3.7 {dollar}\mu{dollar}M exogenous spermidine suggests a mechanistic relationship of the aziridinyl spermidine analogues with the polyamine system. Under the same conditions, pretreatment with DFMO and coincubation with spermidine had no effect on the cytotoxicity induced by thiotepa. The cell growth inhibitory effect induced by DFMO (100 {dollar}\mu{dollar}M for 24 hours) was overcome by washing away the test compound and replenishment with 3.7 {dollar}\mu{dollar}M spermidine. However, the cytotoxicity induced by aziridinyl spermidines was not affected at all. These observations suggest that the cytotoxic effect of aziridinyl compounds is irreversible. The accumulation of both N{dollar}\sp1{dollar}- and N{dollar}\sp8{dollar}-aziridinyl spermidine increased proportionally with increasing extracellular concentrations. Enhancement of cellular accumulation of both aziridinyl compounds by DFMO pretreatment provided evidence to support the argument that the aziridinyl spermidine analogues might utilize the polyamine transport system to enter cells. Other evidence which strengthens this argument is the fact that both N- and N{dollar}\sp8{dollar}-aziridinyl spermidines inhibited the uptake of natural spermidine in a dose-dependent manner. The perturbation of polyamine biochemistry by the test compounds was characterized by their ability to deplete cellular putrescine, as well as spermidine and spermine. Finally, results from DNA alkylation studies showed the formation of aziridinyl spermidine-DNA adducts, suggesting that DNA might be one target of the test compounds. (Abstract shortened by UMI.)