Showing papers by "Kettering University published in 1994"

Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease.

Abstract: To maintain genomic integrity, double-strand breaks (DSBs) in chromosomal DNA must be repaired. In mammalian systems, the analysis of the repair of chromosomal DSBs has been limited by the inability to introduce well-defined DSBs in genomic DNA. In this study, we created specific DSBs in mouse chromosomes for the first time, using an expression system for a rare-cutting endonuclease, I-SceI. A genetic assay has been devised to monitor the repair of DSBs, whereby cleavage sites for I-SceI have been integrated into the mouse genome in two tandem neomycin phosphotransferase genes. We find that cleavage of the I-SceI sites is very efficient, with at least 12% of stably transfected cells having at least one cleavage event and, of these, more than 70% have undergone cleavage at both I-SceI sites. Cleavage of both sites in a fraction of clones deletes 3.8 kb of intervening chromosomal sequences. We find that the DSBs are repaired by both homologous and nonhomologous mechanisms. Nonhomologous repair events frequently result in small deletions after rejoining of the two DNA ends. Some of these appear to occur by simple blunt-ended ligation, whereas several others may occur through annealing of short regions of terminal homology. The DSBs are apparently recombinogenic, stimulating gene targeting of a homologous fragment by more than 2 orders of magnitude. Whereas gene-targeted clones are nearly undetectable without endonuclease expression, they represent approximately 10% of cells transfected with the I-SceI expression vector. Gene targeted clones are of two major types, those that occur by two-sided homologous recombination with the homologous fragment and those that occur by one-sided homologous recombination. Our results are expected to impact a number of areas in the study of mammalian genome dynamics, including the analysis of the repair of DSBs and homologous recombination and, potentially, molecular genetic analyses of mammalian genomes.

Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells

Abstract: Double-strand breaks introduced into DNA in vivo have been shown to enhance homologous recombination in a variety of chromosomal and extrachromosomal loci in Saccharomyces cerevisiae. To introduce double-strand breaks in DNA at defined locations in mammalian cells, we have constructed a mammalian expression vector for a modified form of I-Sce I, a yeast mitochondrial intron-encoded endonuclease with an 18-bp recognition sequence. Expression of the modified I-Sce I endonuclease in COS1 cells results in cleavage of model recombination substrates and enhanced extrachromosomal recombination, as assayed by chloramphenicol acetyltransferase activity and Southern blot analysis. Constitutive expression of the endonuclease in mouse 3T3 cells is not lethal, possibly due to either the lack of I-Sce I sites in the genome or sufficient repair of them. Expression of an endonuclease with such a long recognition sequence will provide a powerful approach to studying a number of molecular processes in mammalian cells, including homologous recombination.

A 13-amino-acid motif in the cytoplasmic domain of FcγRIIB modulates B-cell receptor signalling

Abstract: The Fc receptor on B lymphocytes, Fc gamma RIIB (beta 1 isoform), helps to modulate B-cell activation triggered by the surface immunoglobulin complex. Crosslinking of membrane immunoglobulin by antigen or anti-Ig F(ab')2 antibody induces a transient increase in cytosolic free Ca2+, a rise in inositol-3-phosphate, activation of protein kinase C, and enhanced protein tyrosine phosphorylation. Crosslinking Fc gamma RIIB with the surface immunoglobulin complex confers a dominant signal that prevents or aborts lymphocyte activation triggered through the ARH-1 motifs of the signal transduction subunits Ig-alpha and Ig-beta. Here we show that Fc gamma RIIB modulates membrane immunoglobulin-induced Ca2+ mobilization by inhibiting Ca2+ influx, without changing the pattern of tyrosine phosphorylation. A 13-amino-acid motif in the cytoplasmic domain of Fc gamma RIIB is both necessary and sufficient for this effect. Tyrosine at residue 309 in this motif is phosphorylated upon co-crosslinking with surface immunoglobulin; mutation of this residue aborts the inhibitory effect of Fc gamma RIIB. This inhibition is directly coupled to signalling mediated through Ig-alpha and Ig-beta as evidenced by chimaeric IgM/alpha and IgM/beta molecules. The 13-residue motif in Fc gamma RIIB controls lymphocyte activation by inhibiting a Ca2+ signalling pathway triggered through ARH-1 motifs as a result of recruitment of novel SH2-containing proteins that interact with this Fc gamma RIIB cytoplasmic motif.

Fc receptors initiate the Arthus reaction: Redefining the inflammatory cascade

Abstract: Antibody-antigen complexes initiate the inflammatory response and are central to the pathogenesis of tissue injury. The classical model for this immunopathological cascade, the Arthus reaction, was reinvestigated with a murine strain deficient in Fc receptor expression. Despite normal inflammatory responses to other stimuli, the inflammatory response to immune complexes was markedly attenuated. These results suggest that immune complex-triggered inflammation is initiated by cell bound Fc receptors and is then amplified by cellular mediators and activated complement. These results redefine the inflammatory cascade and may offer other approaches for the study and treatment of immunological injury.

CD28 is associated with and induces the immediate tyrosine phosphorylation and activation of the Tec family kinase ITK/EMT in the human Jurkat leukemic T-cell line

Abstract: T lymphocytes require two signals to be activated. The antigen-specific T-cell receptor can deliver the first signal, while ligation of the T-cell surface molecule CD28 by antibodies or its cognate ligands B7-1 (CD80) or B7-2 has been demonstrated to be sufficient for the delivery of the second signal. Signaling via CD28 and the T-cell receptor results (i) in their costimulation of T cells to produce numerous lymphokines including interleukin 2 and (ii) in the prevention of anergy induction. Little is known about the pathway by which CD28 mediates its signals except that protein-tyrosine phosphorylation is involved. We show here in human Jurkat cells that the Tec-family protein-tyrosine kinase ITK/EMT (p72ITK/EMT) is associated with CD28 and becomes tyrosine-phosphorylated and activated within seconds of CD28 ligation. This tyrosine phosphorylation of p72ITK/EMT is rapid (within 30 sec), occurs in the absence of LCK activation, and precedes tyrosine phosphorylation of the guanine nucleotide exchange factor VAV. Secondary crosslinking of CD28 is unnecessary for the induced tyrosine phosphorylation of p72ITK/EMT. Thus, tyrosine phosphorylation of p72ITK/EMT may represent one of the earliest events in CD28 signaling. This demonstrates that a member of the Tec family of protein tyrosine kinases, similar to members of the Src and Syk families, plays a role in the activation of T cells. Furthermore, the data demonstrate that p72ITK/EMT, and by analogy other members of the Tec family, responds to extracellularly generated signals.

Total synthesis of taxol

Abstract: The present invention provides two basic routes for the total synthesis of taxol having the structure: ##STR1## The present invention also provides the intermediates produced in the above processes, processes for synthesizing these intermediates as well as analogs to taxol. Both the intermediates and analogs to taxol may prove to be valuable anticancer agents.

Breast cancer during pregnancy

Abstract: Ten to 20% of the 178,700 new cases of breast cancer occurring yearly (1998 estimates) are found in women of childbearing age (1). The issue of pregnancy-associated breast cancer is very important, particularly as more women delay childbearing for personal or professional reasons. The delay in childbearing to the 30s or 40s occurs concordantly with an increasing incidence of breast cancer in those ages. Pregnancy-associated breast cancer has been traditionally defined as the diagnosis of breast cancer made during pregnancy or within 1 year afterward. It is estimated to have an incidence of 0.2–3.8% (2) and is reported to occur in 1/10,000–1/3000 pregnancies (3,4).

Role of kit-ligand in proliferation and suppression of apoptosis in mast cells: basis for radiosensitivity of white spotting and steel mutant mice.

Abstract: The receptor tyrosine kinase Kit and its cognate ligand KL/steel factor are encoded at the white spotting (W) and Steel (Sl) loci of the mouse, respectively. Mutations at both the W and the Sl loci affect hematopoiesis including the stem cell hierarchy, erythropoiesis, and mast cells, as well as gametogenesis and melanogenesis. In addition, mutant mice display an increased sensitivity to lethal doses of irradiation. The role of KL/c-kit in cell proliferation and survival under conditions of growth factor-deprivation and gamma-irradiation was studied by using bone marrow-derived mast cells (BMMC) as a model. Whereas apoptosis induced by growth factor deprivation in BMMC is a stochastic process and follows zero order kinetics, gamma-irradiation-induced apoptosis is an inductive process and follows higher order kinetics. In agreement with these results, gamma-irradiation-induced apoptosis in BMMC was shown to be dependent on p53 whereas apoptosis induced by deprivation is partly dependent on p53, implying that there are other mechanisms mediating apoptosis in KL-deprived BMMC. In the presence and in the absence of serum, KL stimulated proliferation by promoting cell cycle progression. The presence of KL was required only during the early part of the G1 phase for entry into the S phase. At concentrations lower than those required for proliferation, KL suppressed apoptosis induced by both growth factor-deprivation and gamma-irradiation, and internucleosomal DNA fragmentation characteristic of apoptosis. The ability of KL to suppress apoptosis was independent of the phase of the cell cycle in which the cells were irradiated and suppression of apoptosis was a prerequisite for subsequent cell cycle progression. Moreover, addition of KL to gamma-irradiated and growth factor-deprived cells could be delayed for up to 1 h after irradiation or removal of growth factors when cells became irreversibly committed to apoptosis. KL and IL-3 induce suppression of apoptosis in mast cells by different mechanisms based on the observations of induction of bcl-2 gene expression by IL-3 but not by KL. It is proposed that the increased sensitivity of W and Sl mutant mice to lethal irradiation results from paucity of the apoptosis suppressing and proliferative effects of KL.

CD28 of T lymphocytes associates with phosphatidylinositol 3-kinase.

Abstract: T lymphocyte activation requires recognition of antigen by the antigen specific TCR as well as second co-stimulatory signals. This recognition event results in the activation of non-TCR linked protein tyrosine kinases (PTKs). The mechanism of co-stimulation of T cells is unknown except for the involvement of PTKs. The T cell surface molecule CD28 is effective in delivering co-stimulatory signals and prevents T cell anergy by inducing T cell proliferation in TCR stimulated T cells, primarily due to an increase in IL-2 production. The mechanism by which CD28 mediates this effect is currently unknown. Some conventional receptor molecules possess intrinsic tyrosine kinase and as a consequence of cross-linking or ligand binding, phosphorylate numerous tyrosines within their cytoplasmic tail, leading these tyrosines to become 'activated' and bind cytoplasmic effector molecules possessing Src homology 2 domains which specifically recognize phosphorylated tyrosines. One such cytoplasmic effector molecule is the phosphatidylinositol-3-phosphate kinase (PI3 kinase) which recognizes the motif phosphotyrosine-methione/valine-X-methionine (X being any amino acid) within the cytoplasmic tails of numerous receptor tyrosine kinases. As CD28 contains a copy of the PI3 kinase binding motif within its cytoplasmic tail, we investigated CD28 signaling and PI3 kinase activation. Here we demonstrate using the Jurkat cell line that CD28 becomes tyrosine phosphorylated following CD28 cross-linking and associates with PI3 kinase. Furthermore, a synthetic peptide representing the YM/VXM motif within the cytoplasmic tail of CD28 also interacts with PI3 kinase only when the tyrosine is phosphorylated.(ABSTRACT TRUNCATED AT 250 WORDS)

Covalent catalysis in nucleotidyl transfer reactions: essential motifs in Saccharomyces cerevisiae RNA capping enzyme are conserved in Schizosaccharomyces pombe and viral capping enzymes and among polynucleotide ligases

Abstract: Formation of the 5' cap structure of eukaryotic mRNAs occurs via transfer of GMP from GTP to the 5' terminus of the primary transcript. RNA guanylyltransferase, the enzyme that catalyzes this reaction, has been isolated from many viral and cellular sources. Though differing in molecular weight and subunit structure, the various guanylyltransferases employ a common catalytic mechanism involving a covalent enzyme-(Lys-GMP) intermediate. Saccharomyces cerevisiae CEG1 is the sole example of a cellular capping enzyme gene. In this report, we describe the identification and characterization of the PCE1 gene encoding the capping enzyme from Schizosaccharomyces pombe. PCE1 was isolated from a cDNA library by functional complementation in Sa. cerevisiae. Induced expression of PCE1 in bacteria and in yeast confirmed that the 47-kDa Sc. pombe protein was enzymatically active. The amino acid sequence of PCE1 is 38% identical (152 of 402 residues) to the 52-kDa capping enzyme from Sa. cerevisiae. Comparison of the two cellular capping enzymes with guanylyltransferases encoded by DNA viruses revealed local sequence similarity at the enzyme's active site and at four additional collinear motifs. Mutational analysis of yeast CEG1 demonstrated that four of the five conserved motifs are essential for capping enzyme function in vivo. Remarkably, the same motifs are conserved in the polynucleotide ligase family of enzymes that employ an enzyme-(Lys-AMP) intermediate. These findings illuminate a shared structural basis for covalent catalysis in nucleotidyl transfer and suggest a common evolutionary origin for capping enzymes and ligases.

PU.1 and an HLH family member contribute to the myeloid-specific transcription of the Fc gamma RIIIA promoter.

Abstract: Expression of the low-affinity Fc receptor for IgG (murine Fc gamma RIIIA) is restricted to cells of myelomonocytic origin. We report here the promoter structure, the proximal DNA sequences responsible for transcription of Fc gamma RIIIA in macrophages and the protein factors which interact with these sequences. A 51 bp sequence, termed the myeloid restricted region (MRR), was both necessary and sufficient for conferring cell type-specific expression in macrophages. Reporter constructs containing mutations in this sequence result in the loss of MRR activity upon transfection into the macrophage cell line, RAW264.7. Two cis-acting elements have been identified and are required for full promoter function. These same elements analyzed by EMSA define two binding sites recognized by nuclear factors derived from macrophages. A 3' purine tract (-50 to -39) within the MRR binds the macrophage and B cell-specific factor, PU.1, and a second E box-like element, termed MyE, upstream of the PU.1 box (-88 to -78) binds the HLH factors TFE3 and USF. EMSA studies using RAW cell extracts suggest that both PU.1 and MyE factors may bind simultaneously to the MRR resulting in a ternary complex that is responsible, in part, for the myeloid-specific activity of the Fc gamma RIIIA promoter.

Ex vivo expansion of cord blood-derived stem cells and progenitors.

Abstract: Cord blood-derived CD34+ cells have proved superior to adult marrow or elicited peripheral blood in ex vivo cell and progenitor expansion. In addition, cord blood-derived cells with a phenotype and function of early stem cells (e.g., long-term culture-initiating cells) can be expanded in vitro in the presence of a combination of cytokines by 15- to 20-fold over 7-14 days. These observations suggest that ex vivo expansion of cord blood CD34+ cells will permit improved engraftment of adults and will prove effective in retroviral-mediated gene transfection of early stem cell populations.

The polymorphic subtelomeric regions of Plasmodium falciparum chromosomes contain arrays of repetitive sequence elements.

Abstract: The human malaria parasite Plasmodium falciparum exhibits a high degree of chromosomal polymorphism, which may contribute to its ability to evade host defenses The analysis of parasite chromosomes has revealed that these polymorphisms are confined to the subtelomeric regions, which are transcriptionally silent and contain repetitive sequence elements Several subtelomeric repetitive elements have been isolated and mapped by using P falciparum yeast artificial chromosome (YAC) clones Structural analysis of parasite telomeric and subtelomeric YAC clones demonstrated that these repetitive elements are conserved between P falciparum chromosome ends We suggest that these subtelomeric elements promote chromosome pairing in P falciparum and facilitate meiotic recombination and gene conversion between telomere-proximal genes

Detection of psychiatric disorders in elderly medical inpatients.

Abstract: In a psychiatric census of a 196-bed acute inpatient Medicine for the Elderly unit, 76.1% of patients resident during 1 week were screened and interviewed in a two-stage diagnostic procedure. Of 153 patients studied, 11.1% were delirious, 26.8% were demented, and 9.2% were depressed. Overall, 56.9% of the cases were identified by ward nurses, and 55.5% by the ward doctors; taken together, ward staff identified 75.0% of the cases (kappa = 0.46), indicating that detection of psychiatric disorder in this population might be improved if doctors and nurses pooled their observations on this aspect of patient assessment.

Process and device to reduce interstitial fluid pressure in tissue

Abstract: A method and apparatus are provided for reducing interstitial fluid pressure in tissues, particularly in tumors, by applying suction to the interior of the tissue. The method comprises inserting into the tissue one or more needle-like, elongated tubes, each having at least one hole at or near the end that is inserted into the tissue and each having means to apply suction to the protruding end. The reduced pressure produced inside the tissue by this method is useful to: facilitate penetration by drugs and therapeutic macromolecules into the tissue; enhance radiation treatment of tumors by increasing the oxygen supply in the tumor; and remove fluid to reduce edema in tissues, e.g. brain edema. Means may be provided to measure the pressure within the tissue and to use this measurement to control the suction applied to the tissue through the tubes.

High level of Nm23-H1 gene expression is associated with local colorectal cancer progression not with metastases.

Abstract: This study aimed to determine the expression of Nm23-H1 in colorectal cancer and liver metastases and to correlate Nm23-H1 expression with clinicopathological variables. Specimens from 59 primary colorectal cancers and five liver metastases were studied using Northern blot hybridisation. The mean +/- s.e. of tumour/normal (T/N) ratio of Nm23-H1 RNA expression was 4.3 +/- 0.4 (P or = 3.0 cm (P = 0.05). There appeared to be a trend between increasing relative Nm23-H1 RNA and bowel wall invasion, irrespective of metastatic status (T1 = 1.9 +/- 0.3, T2 = 4.1 +/- 0.6, T3 = 4.1 +/- 0.5 and T4 = 6.4 +/- 1.6). This difference was statistically significant when T1 was compared against > or = T2 lesions (P = 0.01). Western blot analysis reveals two Nm23H-1 bands (17.0 kDa and 18.5 kDa). In 16 colorectal patients, the T/N fold-increase in protein expression was 2.66 +/- 0.46 (P < 0.001) and 2.40 +/- 0.32 (P < 0.001) for the 17.0 and 18.5 kDa band respectively. Both Nm23-H1 RNA and protein levels in primary colorectal cancers do not appear to correlate with synchronous regional or distant metastases. Since Nm23-H1 RNA expression is associated with increasing tumour size and tumour local invasion, Nm23-H1 RNA expression may be associated with local disease progression.

Mutational analysis of the Drosophila snake protease: an essential role for domains within the proenzyme polypeptide chain.

Abstract: Two genes involved in the generation of dorsoventral asymmetry in the developing Drosophila melanogaster embryo, snake and easter, encode the zymogen form of serine proteases Mutant alleles of snake were cloned and sequenced revealing two types of lesions: point mutations which alter the amino acid sequence (snk073 and snkrm4) and point mutations which alter the splicing (snk229 or snk233) of intron 1 of the mRNA from the normal 3' end of the intron to a cryptic site snake mutant embryos derived from homozygous mothers can be fully rescued by injection of RNA transcripts of the wild-type snake cDNA RNA phenotypic rescue and site-directed mutagenesis experiments indicate that snake requires the serine, histidine and aspartic acid of the catalytic triad for normal activity Deletion experiments show that an acidic proenzyme domain is required for snake rescue activity to be uniformly distributed throughout the embryo A second proenzyme domain, called the disulfide knot, appears to be essential for normal regulation of activity of the snake catalytic chain Transcripts encoding only the proenzyme polypeptides of either snake or easter can dorsalize wild type embryos We propose a model in which the proenzyme determinants of both the snake and easter enzymes mediate interaction between the serine proteases and other components of the dorsal-ventral patterning system