Showing papers by "Kettering University published in 2000"

From the Laboratory to the Clinic: A Retrospective on Fully Synthetic Carbohydrate‐Based Anticancer Vaccines

Abstract: This review provides an account of our explorations into oligosaccharide and glycoconjugate construction for the creation and evaluation of vaccines based on carbohydrate-centered tumor antigens. Our starting point was the known tendency of transformed cells to express selective carbohydrate motifs in the form of glycoproteins or glycolipids. Anticancer vaccines derived from carbohydrate-based antigens could be effective targets for immune recognition and attack. Obtaining significant quantities of such structures from natural sources is, however, extremely difficult. With the total synthesis of tumor-associated carbohydrate antigens accomplished, we began to evaluate at the clinical level whether the human immune system can respond to such fully synthetic antigens in a focused and useful way. Toward this goal, we have merged the resources of chemistry and immunology in an attack on the problem. The synthesis and immunoconjugation of various tumor-associated carbohydrate antigens and the results of such constructs in mice vaccinations will be described. For fashioning an effective vaccine, conjugation to a suitable immunogenic carrier was necessary and conjugates of KLH (keyhole limpet cyanin) have consistently demonstrated the relevant immunogenicity. Preclinical and clinical studies with synthetic conjugate carbohydrate vaccines show induction of IgM- and IgG-antibody responses. Another approach to anticancer vaccines involves the use of clustered glycopeptides as targets for immune attack. Initial attention has been directed to mucin related O-linked glycopeptides. Synthetic trimeric clusters of glycoepitopes derived from the Tn-, TF- and Lewis(y)-antigens, appropriately bioconjugated, have been demonstrated to be immunogenic. The hope is that patients immunized in an adjuvant manner with synthetic carbohydrate vaccines would produce antibodies reactive with cancer cells and that the production of such antibodies would mitigate against tumor spread, thereby enabling a more favorable survival and "quality of life" prognosis.

Chemotaxis of primitive hematopoietic cells in response to stromal cell–derived factor-1

Abstract: Stromal cell-derived factor-1 (SDF-1) provides a potent chemotactic stimulus for CD34(+) hematopoietic cells. We cultured mobilized peripheral blood (PB) and umbilical cord blood (CB) for up to 5 weeks and examined the migratory activity of cobblestone area-forming cells (CAFCs) and long-term culture-initiating cells (LTC-ICs) in a transwell assay. In this system, SDF-1 or MS-5 marrow stromal cells placed in the lower chamber induced transmembrane and transendothelial migration by 2- and 5-week-old CAFCs and LTC-ICs in 3 hours. Transmigration was blocked by preincubation of input CD34(+) cells with antibody to CXCR4. Transendothelial migration of CB CAFCs and LTC-ICs was higher than that of PB. We expanded CD34(+) cells from CB in serum-free medium with thrombopoietin, flk-2 ligand, and c-kit ligand, with or without IL-3 and found that CAFCs cultured in the absence of IL-3 had a chemotactic response equivalent to noncultured cells, even after 5 weeks. However, addition of IL-3 to the culture reduced this response by 20-50%. These data indicate that SDF-1 induces chemotaxis of primitive hematopoietic cells signaling through CXCR4 and that the chemoattraction could be downmodulated by culture ex vivo.

The DExH protein NPH-II is a processive and directional motor for unwinding RNA.

Abstract: All aspects of cellular RNA metabolism and processing involve DExH/D proteins, which are a family of enzymes that unwind or manipulate RNA in an ATP-dependent fashion1 DExH/D proteins are also essential for the replication of many viruses, and therefore provide targets for the development of therapeutics2 All DExH/D proteins characterized to date hydrolyse nucleoside triphosphates and, in most cases, this activity is stimulated by the addition of RNA or DNA1 Several members of the family unwind RNA duplexes in an NTP-dependent fashion in vitro1,3; therefore it has been proposed that DExH/D proteins couple NTP hydrolysis to RNA conformational change in complex macromolecular assemblies4 Despite the central role of DExH/D proteins, their mechanism of RNA helicase activity remains unknown Here we show that the DExH protein NPH-II unwinds RNA duplexes in a processive, unidirectional fashion with a step size of roughly one-half helix turn We show that there is a quantitative connection between ATP utilization and helicase processivity, thereby providing direct evidence that DExH/D proteins can function as molecular motors on RNA

Novel class of cytodifferentiating agents and histone deacetylase inhibitors, and methods of use thereof

Abstract: The present invention provides the compound having formula (I), wherein each of R1 and R2 is, substituted or unsubstituted, aryl, cycloalkyl, cycloalkylamino, naphtha, pyridineamino, piperidino, t-butyl, aryloxy, arylalkyloxy, or pyridine group; wherein A is an amido moiety, -O-, -S-, -NH-, or -CH2-; and wherein n is an integer from 3 to 8. The present invention also provides a method of selectively inducing growth arrest, terminal differentiation and/or apoptosis of neoplastic cells and thereby inhibiting proliferation of such cells. Moreover, the present invention provides a method of treating a patient having a tumor characterized by proliferation of neoplastic cells. Lastly, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically acceptable amount of the compound above.

Immunization of ovarian cancer patients with a synthetic Lewisy-protein conjugate vaccine : A phase 1 trial

Abstract: As the initial step in developing carbohydrate-based vaccines for the treatment of ovarian cancer patients in an adjuvant setting, 25 patients were immunized with a Lewisy pentasaccharide (Ley)-keyhole limpet hemocyanin (KLH)-conjugate vaccine together with the immunological adjuvant QS-21. Four different doses of the vaccine, containing 3, 10, 30, and 60 μg of carbohydrate were administered s.c. at 0, 1, 2, 3, 7, and 19 weeks to groups of 6 patients. Sera taken from the patients at regular intervals were assayed by ELISA for reactivity with naturally occurring forms of Ley (Ley-ceramide and Ley mucin) and by flow cytometry and a complement-dependent cytoxicity assay for reactivity with Ley-expressing tumor cells. The majority of the patients (16/24) produced anti-Ley antibodies as assessed by ELISA, and a proportion of these had strong anti-tumor cell reactivity as assessed by flow cytometry and complement-dependent cytotoxicity. One serum, analyzed in detail, was shown to react with glycolipids but not with glycoproteins or mucins expressed by ovarian cancer cell line OVCAR-3. The vaccine was well tolerated and no gastrointestinal, hematologic, renal, or hepatic toxicity related to the vaccine was observed. On the basis of this study, Ley-KLH should be a suitable component for a polyvalent vaccine under consideration for the therapy of epithelial cancers. Int. J. Cancer 87:79–85, 2000. © 2000 Wiley-Liss, Inc.

Accelerating identification of single nucleotide polymorphisms and alignment of clones in genomic sequencing

Abstract: The present invention is directed to a method of assembling genomic maps of an organism's DNA or portions thereof. A library of an organism's DNA is provided where the individual genomic segments or sequences are found on more than one clone in the library. Representations of the genome are created, and nucleic acid sequence information is generated from the representations. The sequence information is analyzed to determine clone overlap from a representation. The clone overlap and sequence information from different representations is combined to assemble a genomic map of the organism. Once the genomic map is obtained, genomic sequence information from multiple individuals can be applied to the map and compared with one another to identify single nucleotide polymorphisms. These single nucleotide polymorphisms can be detected, and alleles quantified, by conducting (1) a global PCR amplification which creates a genome representation, and (2) a ligation detection reaction process whose ligation products are captured by hybridization to a support.

Iron speciation in the Arabian Sea

Abstract: Fe(III) speciation was measured in seawater collected as part of the United States Joint Global Ocean Flux (US JGOFS) Arabian Sea Process Study, Cruise TN045, March 14–April 10, 1995. The Fe-binding capacity of organic seawater ligands was measured in filtered seawater (<0.4 μm) collected from surface depths and throughout the oxygen minimum zone (OMZ). Seawaters from three stations on the southern line (S2, S9, and S11) were examined. Total Fe concentrations measured at the three sites ranged from: 1.25±0.21 nM to 1.30±0.01 nM (S2); 1.67±0.50 nM to 2.63±0.54 nM (S9); and 1.40±0.11 nM to 1.70±0.29 nM (S11). Cathodic stripping voltammetry (CSV) with 1-nitroso-2-napthol (1N2N) as the competitive ligand (pH 6.9) was used to determine conditional stability constants and Fe-binding ligand concentrations in seawater. Conditional stability constants for FeL complexes ranged from log KFeL=21.6±0.1 to 22.5±0.9 at the three sites. Total ligand concentrations ranged from 1.47±0.06 nM to 6.33±1.16 nM over all sites, but increased by a factor of 2–3 from the surface to the oxygen minimum zone (OMZ), suggesting that Fe-binding ligands may be produced during organic matter degradation. Ligand concentrations were consistently higher than total iron concentrations at every site measured, with an average “excess” ligand concentration of 2.15±1.50 (n=10). “Excess” ligand concentrations in the OMZ were 2 to 20 times higher than surface waters (upper 100 m). Formation-rate constants (kf) and dissociation-rate constants (kd) between added Fe3+ and seawater ligands were measured using a kinetic approach at ambient seawater pH, allowing independent calculation of the conditional stability constant, since K=kf/kd. Using the kinetic approach, conditional stability constants ranged from log KFeL=20.5±0.1 to 22.9±0.1. Although log K values are comparable in magnitude to those reported in the Pacific and Northwestern Atlantic Oceans, measured total ligand concentrations in the Arabian Sea are higher. This suggests that in areas that receive high Fe inputs through upwelling and/or atmospheric deposition, marine organisms may produce `excess’ ligands to keep Fe soluble in seawater for extended intervals.

Processes controlling the distribution and cycling of manganese in the oxygen minimum zone of the Arabian Sea

Abstract: Vertical and horizontal distributions of dissolved and particulate manganese were investigated in the Arabian Sea (Northwestern Indian Ocean) during the 1995 Spring Intermonsoon period (March–April; US JGOFS Process Cruise 2; TN045). The region is characterized by an intense, basin-wide oxygen minimum zone (OMZ) that strongly influences the manganese distribution. In the OMZ, two distinct dissolved Mn (d-Mn) maxima were observed, at depths of 200–300 m and 600 m, respectively. The latter peak displayed concentration maxima of approximately 6 nanomolar and was largely confined to stations north of 19°N latitude (Stations N2–N7). This mid-depth maximum was associated with the low oxygen core of the OMZ ([O2] Saager et al. (1989, Geochimica et Cosmochimica Acta, 53, 2259–2267). This signal was largely absent at stations along the southern transect, likely due to oxidative scavenging of d-Mn to the suspended particulate phase. Mid-depth particulate Mn maxima at some southern stations (Stations S4–S11) appear to be remnants of this feature. The upper d-Mn maximum (200–300 m depth) was more widely distributed than the 600 m peak, with d-Mn concentrations of ∼3 to as high as 8 nm at most stations east of about 62°E longitude. The signal was everywhere correlated with the secondary nitrite maximum, at stations within the main denitrification zone delineated by Naqvi (1991) . Nepheloid layers, presumably bacterial, also were associated with this depth interval. Particulate Mn profiles displayed corresponding concentration minima and low Mn/Al and reactive/refractory Mn ratios for this same depth interval, suggesting reductive dissolution of Mn-oxyhydroxides. These observations imply that in situ microbially mediated processes may be the predominant source of d-Mn in the upper OMZ, while horizontal advection is more important deeper in the water column.

A second generation synthesis of the MBr1 (globo-H) breast tumor antigen: new application of the n-pentenyl glycoside method for achieving complex carbohydrate protein linkages.

Abstract: A new synthesis of the hexasaccharide MBr1 antigen (globo-H) is reported. A revised construction with improved efficiency was necessary because an anti-cancer vaccine containing this antigen is entering phase II and phase III clinical trials for prostate cancer. The key feature of this second generation synthesis is the preparation of globo-H as its n-pentenyl glycoside. This group serves as an anomeric protecting group and as a linker for bioconjugation to carrier protein. The resultant synthesis allows for the production of suitable quantities of globo-H for clinical trials.

Characterization of an ATP-dependent DNA ligase from the thermophilic archaeon Methanobacterium thermoautotrophicum

Abstract: We report the production, purification and characterization of a DNA ligase encoded by the thermophilic archaeon Methanobacterium thermoautotrophicum. The 561 amino acid MTH: ligase catalyzed strand-joining on a singly nicked DNA in the presence of a divalent cation (magnesium, manganese or cobalt) and ATP (K(m) 1.1 microM). dATP can substitute for ATP, but CTP, GTP, UTP and NAD(+) cannot. MTH: ligase activity is thermophilic in vitro, with optimal nick-joining at 60 degrees C. Mutational analysis of the conserved active site motif I (KxDG) illuminated essential roles for Lys251 and Asp253 at different steps of the ligation reaction. Mutant K251A is unable to form the covalent ligase-adenylate intermediate (step 1) and hence cannot seal a 3'-OH/5'-PO(4) nick. Yet, K251A catalyzes phosphodiester bond formation at a pre-adenylated nick (step 3). Mutant D253A is active in ligase-adenylate formation, but defective in activating the nick via formation of the DNA-adenylate intermediate (step 2). D253A is also impaired in phosphodiester bond formation at a pre-adenylated nick. A profound step 3 arrest, with accumulation of high levels of DNA-adenylate, could be elicited for the wild-type MTH: ligase by inclusion of calcium as the divalent cation cofactor. MTH: ligase sediments as a monomer in a glycerol gradient. Structure probing by limited proteolysis suggested that MTH: ligase is a tightly folded protein punctuated by a surface-accessible loop between nucleotidyl transferase motifs III and IIIa.

Vom Labor zur Klinik: vollsynthetische Antitumor‐Impfstoffe auf Kohlenhydratbasis

Abstract: Dieser Aufsatz gibt einen Uberblick uber die Untersuchungen unserer Arbeitsgruppe zur Synthese von Oligosacchariden und Glycokonjugaten mit dem Ziel der Herstellung und Erprobung von Impfstoffen auf der Basis kohlenhydrathaltiger Tumorantigene. Unser Ausgangspunkt war die bekannte Tendenz transformierter Zellen, auf der Zelloberflache vergleichsweise selektiv Kohlenhydratmotive in Form von Glycoproteinen oder Glycolipiden zu exprimieren. Angeregt wurden diese Untersuchungen dadurch, dass derartige Antitumor-Impfstoffe effektive Targets fur Immunerkennung und -angriff sein konnten; es ist jedoch schwierig, ausreichende Substanzmengen aus naturlichen Quellen zu gewinnen. Nachdem die Totalsynthese tumorassoziierter Kohlenhydratantigene gelungen war, war im klinischen Umfeld zu klaren, ob das menschliche Immunsystem auf solche vollsynthetischen Antigene zielgerichtet und sinnvoll antworten kann. Dafur wurden die Ressourcen von Chemie und Immunologie zu einem gemeinsamen Angriff zusammengefuhrt. Die Synthese von tumorassoziierten Kohlenhydratantigenen sowie die Immunkonjugation und die Ergebnisse der Impfung von Mausen mit solchen Konstrukten werden beschrieben. Hierfur war die Konjugation an einen immunogenen Trager notwendig, wofur sich KLH-Konjugate als geeignet erwiesen. In praklinischen und klinischen Studien mit synthetischen Kohlenhydratkonjugaten als Impfstoffen lies sich die Induktion einer IgM- und IgG-Antikorper-Reaktion zeigen. Eine andere Strategie bestand in der Verwendung von Glycopeptidclustern als Targets fur den Immunangriff. Anfanglich richtete sich unsere Aufmerksamkeit auf mucinahnliche, O-glycosidisch gebundene Glycopeptide. Synthetische trimere Cluster von Glycoepitopen, die sich von den Tn-, TF- und Ley-Antigenen ableiteten, erwiesen sich nach geeigneter Biokonjugation als immunogen. Die Hoffnung ist, dass Patienten, die mit den synthetischen Kohlenhydrat-Impfstoffen in Kombination mit einem Adjuvans immunisiert werden, Antikorper gegen Tumorzellen produzieren und dass die Produktion solcher Antikorper der Ausbreitung des Tumors entgegenwirkt, wodurch die Uberlebenschancen und die Prognose fur die „Lebensqualitat” verbessert werden.

Human mutations in glucose 6-phosphate dehydrogenase reflect evolutionary history

Abstract: Glucose 6-phosphate dehydrogenase (G6PD) is a cytosolic enzyme encoded by a housekeeping X-linked gene whose main function is to produce NADPH, a key electron donor in the defense against oxidizing agents and in reductive biosynthetic reactions. Inherited G6PD deficiency is associated with either episodic hemolytic anemia (triggered by fava beans or other agents) or life-long hemolytic anemia. We show here that an evolutionary analysis is a key to understanding the biology of a housekeeping gene. From the alignment of the amino acid (aa) sequence of 52 glucose 6-phosphate dehydrogenase (G6PD) species from 42 different organisms, we found a striking correlation between the aa replacements that cause G6PD deficiency in humans and the sequence conservation of G6PD: two-thirds of such replacements are in highly and moderately conserved (50-99%) aa; relatively few are in fully conserved aa (where they might be lethal) or in poorly conserved aa, where presumably they simply would not cause G6PD deficiency. This is consistent with the notion that all human mutants have residual enzyme activity and that null mutations are lethal at some stage of development. Comparing the distribution of mutations in a human housekeeping gene with evolutionary conservation is a useful tool for pinpointing amino acid residues important for the stability or the function of the corresponding protein. In view of the current explosive increase in full genome sequencing projects, this tool will become rapidly available for numerous other genes.

Structure-Function Analysis of Yeast mRNA Cap Methyltransferase and High-Copy Suppression of Conditional Mutants by AdoMet Synthase and the Ubiquitin Conjugating Enzyme Cdc34p

Abstract: Here we present a genetic analysis of the yeast cap-methylating enzyme Abd1p. To identify individual amino acids required for Abd1p function, we introduced alanine mutations at 35 positions of the 436-amino acid yeast protein. Two new recessive lethal mutations, F256A and Y330A, were identified. Alleles F256L and Y256L were viable, suggesting that hydrophobic residues at these positions sufficed for Abd1p function. Conservative mutations of Asp-178 established that an acidic moiety is essential at this position ( i.e. , D178E was viable whereas D178N was not). Phe-256, Tyr-330, and Asp-178 are conserved in all known cellular cap methyltransferases. We isolated temperature-sensitive abd1 alleles and found that abd1-ts cells display a rapid shut-off of protein synthesis upon shift to the restrictive temperature, without wholesale reduction in steady-state mRNA levels. These in vivo results are consistent with classical biochemical studies showing a requirement for the cap methyl group in cap-dependent translation. We explored the issue of how cap methylation might be regulated in vivo by conducting a genetic screen for high-copy suppressors of the ts growth defect of abd1 mutants. The identification of the yeast genes SAM2 and SAM1 , which encode AdoMet synthase, as abd1 suppressors suggests that Abd1p function can be modulated by changes in the concentration of its substrate AdoMet. We also identified the ubiquitin conjugating enzyme Cdc34p as a high-copy abd1 suppressor. We show that mutations of Cdc34p that affect its ubiquitin conjugation activity or its capacity to interact with the E3-SCF complex abrogate its abd1 suppressor function. Moreover, the growth defect of abd1 mutants is exacerbated by cdc34-2. These findings suggest a novel role for Cdc34p in gene expression and engender a model whereby cap methylation or cap utilization is negatively regulated by a factor that is degraded when Cdc34p is overexpressed.

The chemistry and immunochemistry of blood group A, B, H, and Lewis antigens: past, present and future.

Abstract: This article traces reseach on the chemistry and immunochemistry of blood group A, B, H, and Lewis antigens from early work on the identification of soluble sources of these antigens, through the elucidation of the structures of the carbohydrate epitopes responsible for these specificities, to recent work on exploring their possible use as cancer vaccines. The various approaches used in the isolation of oligosaccharides from mucins for use in structural studies are discussed, as are recent efforts in the chemical systhesis of blood group-active oligosaccharides.

Recombinogenic flap ligation pathway for intrinsic repair of topoisomerase IB-induced double-strand breaks.

Abstract: Topoisomerase IB catalyzes recombinogenic DNA strand transfer reactions in vitro and in vivo. Here we characterize a new pathway of topoisomerase-mediated DNA ligation in vitro (flap ligation) in which vaccinia virus topoisomerase bound to a blunt-end DNA joins the covalently held strand to a 5' resected end of a duplex DNA containing a 3' tail. The joining reaction occurs with high efficiency when the sequence of the 3' tail is complementary to that of the scissile strand immediately 5' of the cleavage site. A 6-nucleotide segment of complementarity suffices for efficient flap ligation. Invasion of the flap into the duplex apparently occurs while topoisomerase remains bound to DNA, thereby implying a conformational flexibility of the topoisomerase clamp around the DNA target site. The 3' flap acceptor DNA mimics a processed end in the double-strand-break-repair recombination pathway. Our findings suggest that topoisomerase-induced breaks may be rectified by flap ligation, with ensuing genomic deletions or translocations.

Total synthesis and antitumor activity of 12,13-desoxyepothilone F: an unexpected solvolysis problem at C15, mediated by remote substitution at C21.

Abstract: A new epothilone analogue, 12,13-desoxyepothilone F (dEpoF, 21-hydroxy-12,13-desoxyepothilone B, 21-hydroxyepothilone D), was synthesized and evaluated for antitumor potential. A convergent strategy employed for the semipractical synthesis of 12,13-desoxyepothilone B (dEpoB) has been utilized to yield an amount of dEpoF sufficient for relevant biological studies. The results from an in vitro assay reveal that this new analogue is highly active against various tumor cell lines with a potency comparable to that of dEpoB. In particular, the growth of resistant tumor cells is inhibited by dEpoF at concentrations where paclitaxel (Taxol) is basically ineffective. A preliminary assessment of its in vivo activity is also promising. The new analogue, containing an additional hydroxyl group at C21, exhibits advantages over other epothilones in terms of water solubility, and can serve as a readily functionalizable handle to produce other useful compounds for pertinent biological studies.