Showing papers by "Kettering University published in 2001"

Vascular Endothelial Growth Factor and Angiopoietin-1 Stimulate Postnatal Hematopoiesis by Recruitment of Vasculogenic and Hematopoietic Stem Cells

Abstract: Tyrosine kinase receptors for angiogenic factors vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) are expressed not only by endothelial cells but also by subsets of hematopoietic stem cells (HSCs). To further define their role in the regulation of postnatal hematopoiesis and vasculogenesis, VEGF and Ang-1 plasma levels were elevated by injecting recombinant protein or adenoviral vectors expressing soluble VEGF165, matrix-bound VEGF189, or Ang-1 into mice. VEGF165, but not VEGF189, induced a rapid mobilization of HSCs and VEGF receptor (VEGFR)2+ circulating endothelial precursor cells (CEPs). In contrast, Ang-1 induced delayed mobilization of CEPs and HSCs. Combined sustained elevation of Ang-1 and VEGF165 was associated with an induction of hematopoiesis and increased marrow cellularity followed by proliferation of capillaries and expansion of sinusoidal space. Concomitant to this vascular remodeling, there was a transient depletion of hematopoietic activity in the marrow, which was compensated by an increase in mobilization and recruitment of HSCs and CEPs to the spleen resulting in splenomegaly. Neutralizing monoclonal antibody to VEGFR2 completely inhibited VEGF165, but not Ang-1–induced mobilization and splenomegaly. These data suggest that temporal and regional activation of VEGF/VEGFR2 and Ang-1/Tie-2 signaling pathways are critical for mobilization and recruitment of HSCs and CEPs and may play a role in the physiology of postnatal angiogenesis and hematopoiesis.

Differentiation of Embryonic Stem Cell Lines Generated from Adult Somatic Cells by Nuclear Transfer

Abstract: Embryonic stem (ES) cells are fully pluripotent in that they can differentiate into all cell types, including gametes. We have derived 35 ES cell lines via nuclear transfer (ntES cell lines) from adult mouse somatic cells of inbred, hybrid, and mutant strains. ntES cells contributed to an extensive variety of cell types, including dopaminergic and serotonergic neurons in vitro and germ cells in vivo. Cloning by transfer of ntES cell nuclei could result in normal development of fertile adults. These studies demonstrate the full pluripotency of ntES cells.

Rab23 is an essential negative regulator of the mouse Sonic hedgehog signalling pathway

Abstract: The mouse open brain (opb) and Sonic hedgehog (Shh) genes have opposing roles in neural patterning: opb is required for dorsal cell types and Shh is required for ventral cell types in the spinal cord1,2,3. Here we show that opb acts downstream of Shh. Ventral cell types that are absent in Shh mutants, including the floor plate, are present in Shh opb double mutants. The organization of ventral cell types in Shh opb double mutants reveals that Shh-independent mechanisms can pattern the neural tube along its dorsal–ventral axis. We cloned opb by a map-based approach and found that it encodes Rab23, a member of the Rab family of vesicle transport proteins. The data indicate that dorsalizing signals activate transcription of Rab23 in order to silence the Shh pathway in dorsal neural cells.

Structure, mechanism, and evolution of the mRNA capping apparatus.

Abstract: Publisher Summary This chapter discusses the recent progress, concerning the mechanism of cap synthesis, by fungal and mammalian enzymes. Viral capping enzymes are discussed to the extent that their study illuminates the mechanistic features, shared by their cellular counterparts. The chapter discusses the structural features of the capping enzymes that are required for guanylyltransferase, triphosphatase, and methyltransferase activities. It also describes how these features are conserved in evolution. The essential structural elements illuminate the reaction mechanisms that are described briefly in this chapter. It emphasizes the importance of recent structure determinations in clarifying the mechanistic models of catalysis, opening new lines of biochemical investigation, and illuminating the surprising structural complexities for seemingly “simple” enzymatic steps. The chapter concludes with the following: (i) the cloning of genes and complementary DNA (cDNA) encoding the cap-forming enzymes from a wide variety of sources; (ii) the delineation of functional domains and catalytically essential amino acid side chains by mutagenesis; and (iii) the application of X-ray crystallography to determine the structure of the capping enzymes. The physical and functional organizations of the component activities diverged during evolution are also discussed in this chapter.

Active Disruption of an RNA-Protein Interaction by a DExH/D RNA Helicase

Abstract: All aspects of cellular RNA metabolism and the replication of many viruses require DExH/D proteins that manipulate RNA in a manner that requires nucleoside triphosphates. Although DExH/D proteins have been shown to unwind purified RNA duplexes, most RNA molecules in the cellular environment are complexed with proteins. It has therefore been speculated that DExH/D proteins may also affect RNA-protein interactions. We demonstrate that the DExH protein NPH-II from vaccinia virus can displace the protein U1A from RNA in an active adenosine triphosphate-dependent fashion. NPH-II increases the rate of U1A dissociation by more than three orders of magnitude while retaining helicase processivity. This indicates that DExH/D proteins can effectively catalyze protein displacement from RNA and thereby participate in the structural reorganization of ribonucleoprotein assemblies.

Defining the optimal surgeon experience for breast cancer sentinel lymph node biopsy: A model for implementation of new surgical techniques

Abstract: Objective To determine the optimal experience required to minimize the false-negative rate of sentinel lymph node (SLN) biopsy for breast cancer. Background Data Before abandoning routine axillary dissection in favor of SLN biopsy for breast cancer, each surgeon and institution must document acceptable SLN identification and false-negative rates. Although some studies have examined the impact of individual surgeon experience on the SLN identification rate, minimal data exist to determine the optimal experience required to minimize the more crucial false-negative rate. Methods Analysis was performed of a large prospective multiinstitutional study involving 226 surgeons. SLN biopsy was performed using blue dye, radioactive colloid, or both. SLN biopsy was performed with completion axillary LN dissection in all patients. The impact of surgeon experience on the SLN identification and false-negative rates was examined. Logistic regression analysis was performed to evaluate independent factors in addition to surgeon experience associated with these outcomes. Results A total of 2,148 patients were enrolled in the study. Improvement in the SLN identification and false-negative rates was found after 20 cases had been performed. Multivariate analysis revealed that patient age, nonpalpable tumors, and injection of blue dye alone for SLN biopsy were independently associated with decreased SLN identification rates, whereas upper outer quadrant tumor location was the only factor associated with an increased false-negative rate. Conclusions Surgeons should perform at least 20 SLN cases with acceptable results before abandoning routine axillary dissection. This study provides a model for surgeon training and experience that may be applicable to the implementation of other new surgical technologies.

The antibacterial arm of the Drosophila innate immune response requires an IκB kinase

Abstract: In both mammals and Drosophila, microbial infection activates Toll-like receptor (TLR) signaling pathways as a part of the innate host defense response (for review, see Anderson 2000). TLR-mediated signaling pathways are essential for appropriate responses to bacterial infection. In addition, mouse Tlr4 mediates septic shock associated with infection by gram-negative bacteria (Vogel 1992; Poltorak et al. 1998). The available data indicate that different microbial cell wall components activate different Toll-like receptor signaling pathways, which regulate distinct sets of target genes. In mammals, TLR4 is the prime mediator of responses to bacterial lipopolysaccharide, while TLR2 mediates responses to bacterial peptidoglycans (Poltorak et al. 1998; Takeuchi et al. 1999; for review, see Beutler 2000). The best-studied aspect of the Drosophila innate immune response is the rapid transcriptional induction of antimicrobial peptide genes in response to infection (Hultmark 1993; Hoffmann 1995). Infection by different classes of microorganisms leads to the preferential induction of particular subsets of antimicrobial peptides (Lemaitre et al. 1997), indicating that different microbial components activate different signaling pathways. At least two Toll-related signaling pathways are required for the activation of the Drosophila antimicrobial peptide genes. The Toll pathway itself, which was first identified because of its essential role in Drosophila embryonic patterning (Anderson et al. 1985), is essential for the induction of an antifungal peptide gene, drosomycin, although the antibacterial peptide genes are still induced in Toll pathway mutants (Lemaitre et al. 1996). Another Drosophila member of the Toll family, 18-wheeler, is required for the normal induction of attacin, an antibacterial peptide gene, but mutations in 18-wheeler do not prevent the induction of other antibacterial peptides (Williams et al. 1997). The imd gene is important for the induction of Diptericin and other antibacterial peptides (Lemaitre et al. 1995a; Corbo and Levine 1996) and, therefore, appears to be a component of a third signaling pathway activated by infection, but its biochemical function is not known. Each of the three Drosophila signaling pathways activated by infection leads to activation of NF-κB/Rel dimers, just as the mammalian TLRs activate NF-κB. All three Drosophila Rel proteins, Dorsal, Dif, and Relish, are expressed in the fat body cells that produce the antimicrobial peptides, and all three are activated within 30 min after infection by translocation from the cytoplasm to the nuclei (Ip at al. 1993; Lemaitre et al. 1995b; Stoven et al. 2000). Adults that lack Dif fail to induce Drosomycin, an antifungal peptide, and Defensin, which is active against gram-positive bacteria, but the other antimicrobial peptide genes are induced normally (Manfruelli et al. 1999; Meng et al. 1999; Rutschmann et al. 2000). Animals that lack Dorsal show normal induction of the antimicrobial peptide genes in response to infection (Lemaitre et al. 1995b), although Dorsal may act redundantly with Dif in larvae (Manfruelli et al. 1999; Rutschmann et al. 2000). Relish is a compound protein with an N-terminal Rel domain and a C-terminal IκB-like domain, similar to mammalian p100 and p105 (Dushay et al. 1996). Relish is activated by signal-dependent proteolysis, which liberates the N-terminal Rel domain, allowing it to translocate into nuclei (Stoven et al. 2000). Adults that lack Relish completely fail to induce the antibacterial peptides Diptericin and Cecropin and show reduced induction of the other antimicrobial peptides (Hedengren et al. 1999). The signaling pathway that activates Relish and controls induction of the antibacterial peptide genes has not been defined. We carried out a genetic screen to identify EMS-induced mutations on the Drosophila third chromosome that affect the antibacterial signaling pathway (Wu and Anderson 1998). A large number of mutants were identified and named ird (immune response deficient) mutants. This screen identified two alleles of the ird5 gene on the basis of the failure of homozygous mutant larvae to induce a diptericin-lacZ reporter gene in response to infection. Here we show that the ird5 gene is essential for antibacterial responses and encodes a Drosophila homolog of mammalian IκB kinases.

Concise Asymmetric Syntheses of Radicicol and Monocillin I

Abstract: Radicicol (1) exhibits potent anticancer properties in vitro, which are likely to be mediated through its high affinity (20 nM) for the molecular chaperone Hsp90. Recently, we reported the results of a synthetic program targeting radicicol (1) and monocillin I (2), highlighted by the application of ring-closing metathesis to macrolide formation. These efforts resulted in a highly convergent synthesis of radicicol dimethyl ether but failed in the removal of the two aryl methyl ethers. Simple exchange of these methyl ethers with more labile functionalities disabled a key esterification in the initial route. Through extended experimentation, a successful route to both natural products was secured, along with some intriguing results that emphasize the implications of this design on a broad range of fused benzoaliphatic targets, including analogues of these natural products.

Solid state microdosimetry.

Abstract: A review of solid state microdosimetry is presented with an emphasis on silicon-based devices. The historical foundations and basics of microdosimetry are briefly provided. Various methods of experimental regional microdosimetry are discussed to facilitate a comparison with the more recent development of silicon microdosimetry. In particular, the performance characteristics of a proportional gas counter and a silicon microdosimeter are compared. Recent improvements in silicon microdosimetry address the issues of requirement specification, non-spherical shape, tissue equivalence, sensitive volume definition (charge collection complexity) and low noise requirements which have previously impeded the implementation of silicon-based microdosimetry. A prototype based on silicon-on-insulator technology is described along with some example results from clinical high LET radiotherapy facilities. A brief summary of the applications of microdosimetry is included.

Pursuit of optimal carbohydrate-based anticancer vaccines: preparation of a multiantigenic unimolecular glycopeptide containing the Tn, MBr1, and Lewis(y) antigens.

Abstract: A novel preparation of nonnatural glycoamino acids starting from n-pentenyl glycosides is described. The approach involves a Horner-Emmons olefination with a suitably protected glycine-derived phosphonate, followed by catalytic asymmetric hydrogenation, which proceeds with excellent diastereomeric selectivity. The synthetic methodology was useful for the preparation of glycoamino acids containing the Tn antigen, the MBr1 antigen (Globo-H), the Le(y) antigen, and lactose. These glycoamino acids can also serve as units for peptide synthesis. The synthesis of polyvalent glycopeptides containing three different antitumor antigens is described (28 and 29), and these have been prepared for conjugation to carrier protein in order to access the immunogenicity for tumor immunotherapy applications.

Discovery through total synthesis: a retrospective on the himastatin problem.

Abstract: A total synthesis of a structure proposed for himastatin was accomplished. The non-identity of the fully synthetic material with himastatin necessitated a revision of the assigned structure. Confirmation of the revised stereostructure was subsequently confirmed through total synthesis. Among the achievements during this effort were i) stereospecific routes to both anti-cis and syn-cis pyrrolindoline substructures; ii) a practical synthesis to 5-hydroxypiperazic acid in enantiomerically pure form; iii) a Stille coupling leading to a complex bi-indole moiety, and iv) efficient protecting group management throughout the evolving depsipeptide domain. The outlines for a biological pharmacophore have been delineated. The alternating D- and L-substituents in the 6-mer as well as the biaryl linkage connecting the two identical subunits are critical for maintaining biological activity. This pattern is simulated in another antibiotic, and suggests a possible structural trend for future SAR investigations.

Structure and mechanism of the RNA triphosphatase component of mammalian mRNA capping enzyme

Abstract: The 5′ capping of mammalian pre-mRNAs is initiated by RNA triphosphatase, a member of the cysteine phosphatase superfamily. Here we report the 1.65 Å crystal structure of mouse RNA triphosphatase, which reveals a deep, positively charged active site pocket that can fit a 5′ triphosphate end. Structural, biochemical and mutational results show that despite sharing an HCxxxxxR(S/T) motif, a phosphoenzyme intermediate and a core α/β-fold with other cysteine phosphatases, the mechanism of phosphoanhydride cleavage by mammalian capping enzyme differs from that used by protein phosphatases to hydrolyze phosphomonoesters. The most significant difference is the absence of a carboxylate general acid catalyst in RNA triphosphatase. Residues conserved uniquely among the RNA phosphatase subfamily are important for function in cap formation and are likely to play a role in substrate recognition.

Accuracy of sentinel lymph node biopsy for patients with T2 and T3 breast cancers.

Abstract: Although numerous studies have demonstrated that sentinel lymph node (SLN) biopsy can accurately determine the axillary nodal status for early breast cancer some studies have suggested that SLN biopsy may be less reliable for tumors >2 cm in size This analysis was performed to determine whether tumor size affects the accuracy of SLN biopsy The University of Louisville Breast Cancer Sentinel Lymph Node Study is a prospective multi-institutional study involving 226 surgeons The study was approved by the Institutional Review Board of each institution, and informed consent was obtained from all patients Patients with clinical stage T1-2 N0 breast cancer were eligible for the study Some patients with T3 tumors were included because they were clinically staged as T2 but on final pathology were found to have tumors >5 cm This analysis includes 2148 patients who were enrolled from August 1997 through October 2000 All patients underwent SLN biopsy using a combination of radioactive colloid and blue dye injection followed by completion Level I/II axillary dissection Statistical comparison was performed by chi-square analysis The SLN identification rate, false negative rate, and overall accuracy of SLN biopsy were not significantly different among tumor stages T1, T2, and T3 We conclude that SLN biopsy is no less accurate for T2-3 breast cancers compared with T1 tumors

Strategies for Improving the Classroom Environment

Abstract: This paper describes some strategies for the educator seeking to better his/her classroom effectiveness. It was inspired by one of the technical sessions of the 29 th Annual IEEE/ASEE Frontiers in Education Conference in which over a dozen experienced college instructors engaged in a roundtable discussion of ways to improve a classroom environment. In this paper, those ideas are discussed and then supplemented with general advice and specific suggestions from the experience of the authors. The paper concludes with a bibliography of related reference material from a wide variety of educational sources.

Insights into long-range structural effects on the stereochemistry of aldol condensations: a practical total synthesis of desoxyepothilone F.

Abstract: A processable total synthesis of a potent antitumor agent, desoxyepothilone F (dEpoF, 21-hydroxy-12,13-desoxyepothilone B, 21-hydroxyepothilone D), has been accomplished. The route is highly convergent. The new technology has also been applied to a total synthesis of 12,13-desoxyepothilone (dEpoB). The crucial point of departure from previous syntheses of dEpoB and dEpoF involves presentation of the C1-C11 sector for Suzuki coupling with C3 in reduced form. Hitherto, the required S stereochemistry at C3 had been implemented via reduction of a keto function after Suzuki coupling. Whereas that chemistry worked quite well in a synthesis of dEpoB, it was not transferable to a high-yielding synthesis of dEpoF. The reduction of the keto group at C3 via a Noyori protocol after Suzuki coupling had proved to be very difficult. In our current approach, two consecutive aldol reactions are used to fashion the acyl sector. In the first aldol condensation, C6 becomes attached to C7. Following protection at C7, a two-carbon acetate equivalent is used to join C2 and C3 with very high asymmetric induction at C3. Only after this center has been implemented is the Suzuki reaction conducted. This major advance allowed us to synthesize dEpoF in a straightforward fashion. These findings found ready application in the total synthesis of dEpoB. Another part of the study involved analysis of the factors associated with aldol condensations joining C6 to C7. In the work described herein, the consequences of the status of C3 in promoting the C6-C7 aldol coupling are probed in detail. Dramatic stereochemical long-range effects uncovered during the study are described, and a working model to explain these effects has emerged.

Broad-complex, but not ecdysone receptor, is required for progression of the morphogenetic furrow in the Drosophila eye.

Abstract: The progression of the morphogenetic furrow in the developing Drosophila eye is an early metamorphic, ecdysteroid-dependent event. Although Ecdysone receptor-encoded nuclear receptor isoforms are the only known ecdysteroid receptors, we show that the Ecdysone receptor gene is not required for furrow function. DHR78, which encodes another candidate ecdysteroid receptor, is also not required. In contrast, zinc finger-containing isoforms encoded by the early ecdysone response gene Broad-complex regulate furrow progression and photoreceptor specification. br-encoded Broad-complex subfunctions are required for furrow progression and proper R8 specification, and are antagonized by other subfunctions of Broad-complex. There is a switch from Broad complex Z2 to Z1 zinc-finger isoform expression at the furrow which requires Z2 expression and responds to Hedgehog signals. These results suggest that a novel hormone transduction hierarchy involving an uncharacterized receptor operates in the eye disc.

Small molecule compositions for binding to hsp90

Abstract: Structural differences in binding pockets of members of the HSP90 family are exploited to achieve differential degradation of k nases and other signaling proteins by using small molecules which interact with the N-terminal binding pocket with an affinity greater than ADP and different from ansamycin antibiotics for at least one HSP90 family species (Figure 5). These small molecules are soluble in aqueous media, providing a further advantage over ansamycins. Pharmaceutical compositions contain a carrier and a molecule that includes a moiety which binds to the N-terminal pocket of at least one member of the HSP90 family. Such binding moieties have anti proliferative activity against tumor cells which depend on proteins requiring chaperones of the HSP90 family. Different chemical species have different activity, allowing the selection of Her2 degradation without degradation of Raf kinase. Thus, the binding moieties possess inherent targeting capacity. The small molecules can be linked to targeting moieties to target the activity to specific classes of cells. The invention includes treatment of diseases, including cancers. Dimeric forms of the binding moieties may be employed.

The epothilones, eleutherobins, and related types of molecules.

Abstract: Taxol is currently one of the most effective anticancer agents available. However, limitations due to multidrug-resistance (MDR) susceptibility and lack of aqueous solubility render it less than an ideal drug. These limitations, coupled with taxols unique mechanism of tumor inhibition, involving the stabilization of microtubule assembly, have spurred the search for more effective chemotherapeutic agents. This review will discuss the chemistry and biology of some of the most promising new molecules with taxol-like activity. The extended family of microtubule-stabilizing agents now includes the epothilones, eleutherobins, discodermolide, laulimalide and WS9885B. The epothilones have emerged as one of the most exciting new candidates for detailed structure-activity-related studies. A review of our efforts in the synthetic and biological aspects of this research is presented, as are the latest developments reported from other laboratories in academia and the pharmaceutical industry. The synthesis and str ucture-activity studies of eleutherobins, as well as recent progress with discodermolide, laulimalide and WS9885B are also reviewed. An abundance of exciting advances in chemistry and biology have emerged from these studies, and it is hoped that it will ultimately result in the development of new and more effective chemotherapeutic agents in the fight against cancer.

The current state of research on academic dishonesty among engineering students

Abstract: Academic dishonesty, or cheating, has become a serious problem at colleges and universities This is particularly true of engineering students who, are among the most likely to cheat in college The present paper reviews the literature as a basis of broadly understanding academic dishonesty This discussion focuses on three primary issues: (1) perceptions of and attitudes towards cheating, (2) reasons for student cheating, and (3) methods of promoting academic integrity A current research project being developed by the authors is also discussed The premise of this research is that students are constantly making ethical judgements between the pressure to cheat and their own moral beliefs and social norms The goal then is to uncover the reasons for and frequency of student cheating and to develop best practices for helping engineering students avoid this pressure Particular topics of discussion include a rationale for the research methodology, an outline of the questions we hope the survey will answer and a discussion of the ethical implications of conducting research of this type

Drosophila immunity: genes on the third chromosome required for the response to bacterial infection.

Abstract: We have screened the third chromosome of Drosophila melanogaster for mutations that prevent the normal immune response. We identified mutant lines on the basis of their failure to induce transcription of an antibacterial peptide gene in response to infection or their failure to form melanized clots at the site of wounding. These mutations define 14 genes [immune response deficient (ird) genes] that have distinct roles in the immune response. We have identified the molecular basis of several ird phenotypes. Two genes, scribble and kurtz/modulo, affect the cellular organization of the fat body, the tissue responsible for antimicrobial peptide production. Two ird genes encode components of the signaling pathways that mediate responses to bacterial infection, a Drosophila gene encoding a homolog of I kappa B kinase (DmIkk beta) and Relish, a Rel-family transcription factor. These genetic studies should provide a basis for a comprehensive understanding of the genetic control of immune responses in Drosophila.

Beam-steering of multi-chromatic light using acousto-optical deflectors and dispersion-compensatory optics

Abstract: Method and apparatus for steering a beam of light The method and apparatus are based on the discovery that a spectral dispersion of multi-chromatic light pulses by an acousto-optical deflector (11) can be significantly ameliorated by positioning a dispersive element, a prism (13) , along the path of the multi-chromatic light pulse in such a way that the dispersive element disperses the multi-chromatic light pulses in a direction opposite to the spectral dispersion caused by the acousto-optical deflector (11) The_method and apparatus are for use with ultrashort laser pulses in the visible and infrared ranges having a bandwith of up to about 40nm