Showing papers by "Kettering University published in 1999"

The internal workings of a DNA polymerase clamp-loading machine

Abstract: Replicative DNA polymerases are multiprotein machines that are tethered to DNA during chain extension by sliding clamp proteins. The clamps are designed to encircle DNA completely, and they are manipulated rapidly onto DNA by the ATP-dependent activity of a clamp loader. We outline the detailed mechanism of gamma complex, a five-protein clamp loader that is part of the Escherichia coli replicase, DNA polymerase III holoenzyme. The gamma complex uses ATP to open the beta clamp and assemble it onto DNA. Surprisingly, ATP is not needed for gamma complex to crack open the beta clamp. The function of ATP is to regulate the activity of one subunit, delta, which opens the clamp simply by binding to it. The delta' subunit acts as a modulator of the interaction between delta and beta. On binding ATP, the gamma complex is activated such that the delta' subunit permits delta to bind beta and crack open the ring at one interface. The clamp loader-open clamp protein complex is now ready for an encounter with primed DNA to complete assembly of the clamp around DNA. Interaction with DNA stimulates ATP hydrolysis which ejects the gamma complex from DNA, leaving the ring to close around the duplex.

Transcription therapy for cancers

Abstract: The present invention provides a method of treating a neoplastic condition in an individual, comprising the step of administering to said individual a pharmacologically effective dose of a retinoic acid and/or an inhibitor of histone deacetylase. Also provided is a pharmaceutical composition, comprising a retinoic acid, an inhibitor of histone deacetylase and a pharmaceutically acceptable carrier. Further provided is a method of inducing terminal differentiation of tumor cells in a tumor in an individual in need of such treatment, comprising the step of administering to said individual a pharmacologically effective dose of a retinoic acid and/or an inhibitor of histone deacetylase.

Nomenclature for factors of the HLA system, 1998.

Abstract: 1. The Naming of New Genes Within the HLA Region The Committee agreed in 1996 to defer the naming of new genes within the HLA region. However, in view of the great interest and work being carried out on the MIC (MHC Class I chain related) genes, already named by the Human Genome Nomenclature Committee as MICA, MICB, MICC, MICD and MICE in April and September 1994 [15, 16], it was agreed that they should be included within the official HLA nomenclature for the purpose of naming alleles of these genes. In addition the change of name of the gene HLADNA to HLA-DOA, as published in the monthly updates, was confirmed, following further results showing that the gene codes for the alpha chain associated with HLA-DOB. The updated list of genes in the HLA region is given in Table 1.

Fusion receptors specific for prostate-specific membrane antigen and uses thereof

Abstract: A fusion receptor composition which is effective to promote a cellular immune response to prostate-specific membrane antigen (PSMA) in vivo when the fusion receptors is expressed by T lymphocytes has the structure: PSMA-scFv: connector: cytoplasmic domain. The PSMA-scFv in this structure is a single chain antibody cloned from the V region genes of a hybridoma specific for PSMA. The connector region is provided to give a spacing between the OSMA-scFv and the cytoplasmic domain, such that both can retain substantial function. A suitable connector is the CD8 hinge, although other connectors of greater or lesser length might be used. The cytoplasmic domain is included to direct the function of the fusion receptor. One exemplary cytoplasmic domain which can be used in the fusion receptor of the invention is a T cell receptor z-chain cytoplasmic domain. An expression vector encoding the fusion receptor is transduced into primary T lymphocytes obtained from an individual to be treated. The transduced lymphocytes are returned to the patient where cells expressing the fusion receptor secrete interleukin 2 and proliferate in response to PSMA-positive cells. The resulting cytotoxic lymphocytes specifically lyse cells expressing PSMA and thus can be used to target PSMA-positive tumor cells and neovasculature.

Final report of Intergroup Trial 0122 (ECOG PE-289, RTOG 90-12): Phase II trial of neoadjuvant chemotherapy plus concurrent chemotherapy and high-dose radiation for squamous cell carcinoma of the esophagus.

Abstract: Purpose: To determine the outcome of neoadjuvant chemotherapy followed by concurrent chemotherapy plus high-dose radiation therapy in patients with local/regional squamous cell carcinoma of the esophagus. Methods and Materials: Forty-five patients with clinical Stage T1-4N0-1M0 squamous cell carcinoma were entered on a prospective single-arm study, of which 38 were eligible. Patients received 3 monthly cycles of 5-FU (1000 mg/m 2 /24 h × 5 days) and cisplatin (100 mg/m 2 day 1; neoadjuvant segment) followed by 2 additional monthly cycles of 5-FU (1000 mg/m 2 /24 h × 5 days) and cisplatin (75 mg/m 2 day 1) plus concurrent 6480 cGy (combined modality segment). The median follow-up in surviving patients was 59 months. Results: For the 38 eligible patients, the primary tumor response rate was 47% complete, 8% partial, and 3% stable disease. The first site of clinical failure was 39% local/regional and 24% distant. For the total patient group, there were 6 deaths during treatment, of which 9% (4/45) were treatment related. The median survival was 20 months. Actuarial survival at 3 years was 30%, and at 5 years, 20%. Conclusion: This intensive neoadjuvant approach does not appear to offer a benefit compared with conventional doses and techniques of combined modality therapy. However, high dose radiation (6480 cGy) appears to be tolerable, and is being tested further in Intergroup Trial INT 0123.

Acoustic monopoles, dipoles, and quadrupoles: An experiment revisited

Abstract: A simple and inexpensive demonstration of acoustic monopole, dipole, and quadrupole sources utilizes four 4-in. boxed loudspeakers and a homemade switch box. The switch box allows the speakers to be driven in any combination of phase relationships. Placing the speakers on a rotating stool allows students to measure directivity patterns for monopole, dipole, and quadrupole speaker combinations. Stacking the speakers in a square, all facing the same direction, allows students to aurally compare the frequency and amplitude dependence of sound radiation from monopoles, dipoles, and quadrupoles.

A fully synthetic globo h carbohydrate vaccine induces a focused humoral response in prostate cancer patients : a proof of principle

Abstract: Human trials on the globo H carbohydrate vaccine (see picture, KLH=the carrier protein keyhole limpet hemocyanin) show that it produces strong IgM, and in some cases IgG, responses in patients with progressive and recurrent prostate cancer. Furthermore, these antibodies not only recognize synthetic antigens, but also globo H-positive tumors in biopsy extracts and tumor tissues.

Total Synthesis of (+)‐Halichlorine: An Inhibitor of VCAM‐1 Expression

Abstract: The diastereoselective addition of the highly functionalized organozinc compound 1 to the aldehyde 2 in the presence of the chiral amino alcohol 3 (-->4) is a key step in the first total synthesis of (+)-halichlorine. A series of protections/deprotections and a macrolaconization complete the synthesis. Halichlorine selectively inhibits the expression of the cell adhesion molecule VCAM-1. TBS=tert-butyldimethylsilyl.

Regulation of transendothelial migration of hematopoietic progenitor cells.

Abstract: Transendothelial migration of hematopoietic progenitor cells occurs in the bone marrow during mobilization and homing, and may therefore play a key role in the trafficking of hematopoietic stem cells. We hypothesize that adhesion molecules, chemokines, and paracrine cytokines are involved in this multifactorial process. As suggested in several studies, downregulation of adhesion molecules (e.g., integrins) may contribute to mobilization of progenitors due to a decreased avidity to bone marrow stromal and endothelial cells, which express the corresponding ligands. Using an in vitro model of transendothelial migration, we have shown that only a small number of more mature, committed progenitors migrates spontaneously under the control of adhesion molecules of the beta-2-integrin family and their corresponding endothelial/stromal ligands. However, transendothelial migration of progenitors in vitro is substantially enhanced by the chemokine stromal cell-derived factor-1 (SDF-1), which is constitutively produced by bone marrow stromal cells. More primitive progenitors also respond to this chemokine. In addition, the ligand for SDF-1, the chemokine receptor CXCR-4, is expressed in greater levels on bone marrow CD34+ cells as compared to mobilized progenitors, suggesting that downregulation of chemokine receptors occurs during progenitor mobilization. Indeed, bone marrow CD34+ cells migrate more avidly in response to SDF-1 than mobilized progenitors. Paracrine cytokines may also play a role in hematopoietic stem cell trafficking, since growth factor-stimulated hematopoietic cells produce cytokines that act on endothelial cells (e.g., vascular endothelial growth factor, VEGF), modifying their proliferation, motility, permeability, and fenestration. We conclude that transendothelial migration of hematopoietic progenitor cells is regulated by adhesion molecules, paracrine cytokines, and chemokines. Cytotoxic therapy as well as exogenously administered hematopoietic growth factors may affect adhesion molecule expression, the local cytokine and chemokine milieu, and chemokine receptor expression, which indirectly results in mobilization of hematopoietic stem cells.

Inhibition of interleukin 2 signaling and signal transducer and activator of transcription (STAT)5 activation during T cell receptor-mediated feedback inhibition of T cell expansion.

Abstract: Limitation of clonal expansion of activated T cells is necessary for immune homeostasis, and is achieved by growth arrest and apoptosis. Growth arrest and apoptosis can occur passively secondary to cytokine withdrawal, or can be actively induced by religation of the T cell receptor (TCR) in previously activated proliferating T cells. TCR-induced apoptosis appears to require prior growth arrest, and is mediated by death receptors such as Fas. We tested whether TCR religation affects T cell responses to interleukin (IL)-2, a major T cell growth and survival factor. TCR ligation in activated primary human T cells blocked IL-2 induction of signal transducer and activator of transcription (STAT)5 DNA binding, phosphorylation of STAT5, Janus kinase (Jak)1, Jak3, and Akt, and kinase activity of Jak1 and Jak3. Inhibition was mediated by the mitogen-activated protein kinase kinase (MEK)–extracellular stimulus–regulated kinase (ERK) signaling pathway, similar to the mechanism of inhibition of IL-6 signaling we have described previously. TCR ligation blocked IL-2 activation of genes and cell cycle regulatory proteins, and suppressed cell proliferation and expansion. These results identify TCR-induced inhibition of IL-2 signaling as a novel mechanism that underlies antigen-mediated feedback limitation of T cell expansion, and suggest that modulation of cytokine activity by antigen receptor signals plays an important role in the regulation of lymphocyte function.

Purified populations of stem cells

Abstract: The invention is directed to a purified population of mammalian endothelial, muscle, or neural stem cells. The invention further provides methods for isolating such populations of cells; methods for using such populations of cells for treating mammals; methods for making vectors for gene therapy; and methods for carrying out gene therapy with such vectors.

Mutational analysis of Escherichia coli DNA ligase identifies amino acids required for nick-ligation in vitro and for in vivo complementation of the growth of yeast cells deleted for CDC9 and LIG4

Abstract: We report that the NAD-dependent Escherichia coli DNA ligase can support the growth of Saccharomyces cerevisiae strains deleted singly for CDC9 or doubly for CDC9 plus LIG4. Alanine-scanning mutagenesis of E.coli DNA ligase led to the identification of seven amino acids (Lys115, Asp117, Asp285, Lys314, Cys408, Cys411 and Cys432) that are essential for nick-joining in vitro and for in vivo complementation in yeast. The K314A mutation uniquely resulted in accumulation of the DNA-adenylate intermediate. Alanine substitutions at five other positions (Glu113, Tyr225, Gln318, Glu319 and Cys426) did not affect in vivo complementation and had either no effect or only a modest effect on nick-joining in vitro. The E113A and Y225A mutations increased the apparent K (m)for NAD (to 45 and 76 microM, respectively) over that of the wild-type E. coli ligase (3 microM). These results are discussed in light of available structural data on the adenylylation domains of ATP- and NAD-dependent ligases. We observed that yeast cells containing only the 298-amino acid Chlorella virus DNA ligase (a 'minimal' eukaryotic ATP-dependent ligase consisting only of the catalytic core domain) are relatively proficient in the repair of DNA damage induced by UV irradiation or treatment with MMS, whereas cells containing only E.coli ligase are defective in DNA repair. This suggests that the structural domains unique to yeast Cdc9p are not essential for mitotic growth, but may facilitate DNA repair.

The t(8;21) fusion protein, AML1/ETO, transforms NIH3T3 cells and activates AP-1

Abstract: The 8;21 translocation is the most common cytogenetic abnormality in human acute myelogenous leukemia, joining the AML1 gene on chromosome 21, to the ETO gene on chromosome 8, forming the AML1/ETO fusion gene. The AML1/ETO fusion protein has been shown to function mainly as a transcriptional repressor of AML1 target genes and to block AML1 function in vitro and in vivo. However, AML1/ETO can also activate the BCL-2 promoter and cause enhanced hematopoietic progenitor self-renewal in vitro, suggesting gain-of-functions unique to the fusion protein. We used NIH3T3 cells to determine the transforming capacity of AML1/ETO, and to further characterize its mechanism of action. Expression of AML1/ETO in NIH3T3 cells caused cell-type specific cell death, and cellular transformation, characterized by phenotypic changes, anchorage-independent growth, and tumor formation in nude mice. In contrast, neither expression of AML1A, AML1B or ETO altered the normal growth pattern of the cells. To investigate the mechanism of transformation by AML1/ETO, we analysed the levels of activated, phosphorylated c-Jun (ser63) and other constituents of the AP-1 complex, in the presence of various AML1/ETO related proteins. Expression of AML1/ETO increased the level of c-Jun-P (ser63), and activated AP-1 dependent transcription, which was inhibited by expression of a dominant-negative c-Jun protein. Mutational analysis revealed that the runt homology domain (RHD) and a C-terminal transcriptional repression domain in AML1/ETO are required for transformation, activation of c-Jun and increased AP-1 activity. These results establish the transforming potential of the t(8;21) fusion protein and link this gain-of-function property to modulation of AP-1 activity.

Trimeric antigenic o-linked glycopeptide conjugates, methods of preparation and uses thereof

Abstract: The present invention provides novel α-O-linked glycoconjugates such as α-O-linked glycopeptides, as well as convergent methods for synthesis thereof. The general preparative approach is exemplified by the synthesis of the mucin motif commonly found on epithelial tumor cell surfaces. The present invention further provides compositions and methods of treating cancer using the α-O-linked glycoconjugates.

Transcriptional activation by artificial recruitment in yeast is influenced by promoter architecture and downstream sequences

Abstract: The idea that recruitment of the transcriptional machinery to a promoter suffices for gene activation is based partly on the results of “artificial recruitment” experiments performed in vivo. Artificial recruitment can be effected by a “nonclassical” activator comprising a DNA-binding domain fused to a component of the transcriptional machinery. Here we show that activation by artificial recruitment in yeast can be sensitive to any of three factors: position of the activator-binding elements, sequence of the promoter, and coding sequences downstream of the promoter. In contrast, classical activators worked efficiently at all promoters tested. In all cases the “artificial recruitment” fusions synergized well with classical activators. A classical activator evidently differs from a nonclassical activator in that the former can touch multiple sites on the transcriptional machinery, and we propose that that difference accounts for the broader spectrum of activity of the typical classical activator. A similar conclusion is reached from studies in mammalian cells in the accompanying paper [Nevado, J., Gaudreau, L., Adam, M. & Ptashne, M. (1999) Proc. Natl. Acad. Sci. USA 96, 2674–2677].

Discovery Through Total Synthesis-Epimerization at C7 in the CP Compounds: Is (7S)-CP-263,114 a Fermentation Product?

Abstract: Sometimes a surprise can occur in a total synthesis. The preparative investigation of the title compound CP-263,114 and of CP-225,917 (1)—both natural products that were first isolated in 1997 by Pfizer—has shown that CP compounds of the 7R series can be converted into the 7S series (for example, 2); (7S)-CP-263,114 is itself apparently a fermentation product.

IL-4 Selectively Inhibits IL-2-Triggered Stat5 Activation, But Not Proliferation, in Human T Cells

Abstract: IL-2 activates several distinct signaling pathways that are important for T cell activation, proliferation, and differentiation into both Th1 and Th2 phenotypes. IL-4, the major cytokine that promotes differentiation of Th2 cells, has been shown to block signaling of the Th1-promoting cytokine IL-12. As IL-2 synergizes with IL-12 in promoting Th1 differentiation, the effects of IL-4 on IL-2 signal transduction were investigated. IL-4 suppressed activation of DNA binding and tyrosine phosphorylation of the transcription factor Stat5 by IL-2, and suppressed the expression of the IL-2-inducible genes CD25, CIS, the PGE2 receptor, and cytokine responsive (CR) genes CR1 and CR8. Activation of Stat5 by cytokines that share a common gamma receptor subunit, IL-2, IL-7, and IL-15, was suppressed by preculture in IL-4. Activation of the Jak1 and Jak3 kinases that are proximal to Stat5 in the IL-2-Jak-STAT signaling pathway was suppressed, and this correlated with inhibition of IL-2Rbeta subunit expression. In contrast to suppression of Stat5, proliferative responses to IL-2 were augmented in IL-4-cultured cells, and activation of proliferative pathways leading to activation of mitogen activated protein kinases, induction of expression of Myc, Fos, Pim-1, and cyclin D3, and decreased levels of the cyclin-dependent kinase inhibitor p27 were intact. These results identify molecular mechanisms underlying interactions between IL-4 and IL-2 in T cells and demonstrate that one mechanism of regulation of IL-2 activity is selective and differential modulation of signaling pathways.

A double blind, placebo controlled study of intracavernosal vasoactive intestinal polypeptide and phenotolamine mesylate in a novel auto-injector for the treatment of non-psychogenic erectile dysfunction

Abstract: Three hundred and four patients with non-psychogenic erectile dysfunction (ED) completed a dose assessment phase with intracavernosal injection utilizing 25 micrograms vasoactive intestinal polypeptide (VIP) combined with phentolamine mesylate 1.0 mg (VIP/P-1) or 2.0 mg (VIP/P-2) in an auto-injector for a response rate of 83.9%. In a sub-group of 183 patients who withdrew from one or more previous ED therapies, 82% responded with an erection suitable for intercourse. One hundred and ninety-five patients were subsequently treated in a placebo controlled phase. 75.1% responded to VIP/P-1, 12% to placebo (P < 0.001); 66.5% responded to VIP/P-2, 10.3% to placebo (P < 0.001), with the median duration of erection of 54 min. The principal adverse event was transient facial flushing in 2770 injections (33.9%). There was no pain post injection and two episodes of priapism (0.05%). Only nine patients withdrew because of adverse events. Over 85% and 95% of patients were satisfied with the drug and auto-injector, respectively. Over 81% of patients and 76% of partners reported an improved quality of life.

Activation conditions determine susceptibility of murine primary T-lymphocytes to retroviral infection.

Abstract: Background Genetically modified T-lymphocytes are potential therapeutic agents for the treatment of various disorders. Successful retroviral infection of primary murine T-lymphocytes is a prerequisite to the study of adoptive cell therapies in a small animal model. The definition of factors controlling retroviral infection of T-lymphocytes would also be useful to better understand retroviral diseases. However, retroviral-mediated gene transfer into murine primary T-cells has remained elusive. Methods In order to define the requirements for stable and efficient gene transfer in primary murine T-lymphocytes, we investigated factors capable of affecting retroviral infection. These include activation conditions (using mitogens or monoclonal antibodies), culture conditions (including media composition and cytokine addition), timing of viral exposure, retroviral receptor selection (ecotropic, amphotropic, or vesicular stomatitis virus G (VSV-G) glycoprotein receptor), and viral titer. Results We show that efficient gene transfer can be achieved in murine T-lymphocytes, provided that a number of favorable conditions are met, in particular optimized T-cell activation conditions, optimal timing of infection, adequate interleukin-2 concentration and T-cell density, and a high viral titer. On a particulate basis, we find that ecotropic particles are more effective than amphotropic or VSV-G-pseudotyped particles, and recommend the use of a specifically selected retroviral packaging cell line. CD4+ T-cells are equally or more infectible than CD8+ lymphocytes, depending on the activation conditions. The Th1 and Th2 subsets are comparably susceptible to retroviral infection, in contrast to what has been reported in some instances of human T-cell infection by human immunodeficiency virus (HIV). Conclusions We establish conditions that enable efficient retroviral-mediated gene transfer in murine primary T-lymphocytes. T-lymphocytes are not uniformly susceptible to retroviral infection depending on the mode of T-cell activation. These findings have implications for devising approaches to the study of T-cell biology, adoptive cell therapies, and the pathophysiology of retroviral diseases in mouse models. Copyright © 1999 John Wiley & Sons, Ltd.