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Biomedical subjects

L O'Connor

Publications and source records attributed to L O'Connor.

At least 19 recordsLinked to original sources
PubMed2000

CD95 (Fas/APO-1) and p53 signal apoptosis independently in diverse cell types.

The tumor suppressor p53 exerts its antioncogenic effects in cells chiefly by regulating their progression through the cell cycle and by inducing cell death. It has been claimed that p53-transduced apoptosis involves the death receptor CD95 (Fas/APO-1). We report that thymocytes from mice lacking functional Fas ligand (gld) show normal sensitivity to apoptosis transduced by p53, and that hepatocytes fromp53-/- mice have normal sensitivity to apoptosis triggered through ligation of CD95. p53 and CD95, therefore, function in independent pathways to cell death in these diverse cell types.

Animals↗
PubMed2000

Normal detrusor is more sensitive than hypertrophied detrusor to in vitro ischemia followed by re-oxygenation.

Partial outlet obstruction results in marked metabolic as well as contractile alterations. Specifically, the ratio of anaerobic to oxidative metabolism is significantly greater in hypertrophied than normal bladder smooth muscle, lactate dehydrogenase (LDH) and lactic acid production are increased, and the contractile apparatus is altered to allow for metabolically more efficient tension generation. In addition, contractile responses of hypertrophied bladder are apparently more resistant than those of normal bladder to hypoxia. In the current experiment, we studied the effects of in vitro ischemia (hypoxia + substrate deprivation) followed by an in vitro model of reperfusion (re-oxygenation + substrate replacement) on contractile responses of normal and hypertrophied urinary bladder strips. We used repetitive field stimulation (FS) during the hypoxic period as a model for hyperreflexia. The purpose of the current study was to compare the responses of normal and hypertrophied bladder smooth muscle to repetitive stimulation in the presence of in vitro ischemia followed by re-oxygenation and substrate replacement. Thirty-two rats were separated into four groups of eight each. The rats in groups 1 and 3 were subjected to partial outlet obstruction. Two weeks later, all rats were anesthetized; their bladders were isolated and cut into four strips. Each strip was mounted in an isolated bath, and after 1-hour incubation in Tyrode's solution containing glucose (in the presence of O(2)), contractile responses to FS, carbachol, and KCl were determined. After this first set of stimulations, the strips were incubated without glucose and in the presence of N(2) for 30 minutes and 1 hour (groups 1 and 2); and for 2 and 4 hours (groups 3 and 4). For groups 1 and 2, the tissues were stimulated at 5-minute intervals with FS at 32 Hz, 1-millisecond duration, 3-second trains (in vitro model of hyperreflexia). For groups 3 and 4, no stimulations were performed during the ischemic period. At the end of the ischemic period, all strips were washed and incubated for 1 hour in the presence of O(2) and with glucose. At the end of this incubation, all strips received a second set of stimulations. a) Partial outlet obstruction resulted in a significant increase in bladder weight. b) Responses to in vitro ischemia: After in vitro ischemia, contractile responses of both normal and hypertrophied tissues to FS were reduced to a significantly greater degree than were responses to carbachol and KCl. The rate of development of contractile dysfunction was significantly greater in normal bladder tissue strips than in hypertrophied bladder strips. c) Responses to repetitive stimulation: The rate of development of contractile dysfunction was significantly greater in all strips subjected to repetitive stimulation than in those not repetitively stimulated; in addition, normal bladder strips were more sensitive than hypertrophied strips to hypoxia and substrate deprivation-induced contractile dysfunction. The rate of contractile failure induced by in vitro ischemia followed by re-oxygenation and substrate replacement was significantly greater for normal bladder strips than for hypertrophied bladder strips. These results indicate that, after partial outlet obstruction, the hypertrophied tissue is more resistant than normal tissue to hypoxia and substrate deprivation.

Animals↗
PubMed2000

IGF-II and IL-2 act synergistically to alter HDAC1 expression following treatments with trichostatin a.

Histone deacetylases play key roles in the regulation of gene transcription. Studies have shown that expression of interleukins IL-2 and IL-8, and insulin-like growth factor 2 (IGF2) are affected by treatment with histone deacetylase inhibitors. We have previously shown that the gene for histone deacetylase 1 (HDAC1) is upregulated following treatment with TSA. The murine homologue of this gene has been reported to be inducible by IL-2. In this study, we have examined the effects IL-2, IGF-II and TSA have on HDAC1 expression in the human hepatocellular carcinoma derived cell line Hep3B. Our results indicate that in contrast to the mouse, HDAC1 is not inducible by IL-2. However, in TSA treated cells, IL-2 and IGF-II were found to act synergistically to reduce TSA induced HDAC1 mRNA levels almost to normal.

Acetylation↗
PubMed2000

Gamma-radiation-induced growth arrest and apoptosis in p53-null lymphoma cells is accompanied by modest transcriptional changes in many genes.

Damage to DNA produces cell cycle arrest, apoptosis, or both. The response in cells with p53 tumor suppressor function involves transcriptional changes, but whether that holds for cells lacking active p53, as in most tumors, is not known. Better characterization of the DNA damage response in tumors lacking p53 function is relevant to cytotoxic therapy. We have explored whether gamma-irradiated p53-null mouse T lymphoma cells undergo marked changes in transcription. Their arrest in G2/M prior to apoptosis required transcription. Transcripts whose abundance altered on irradiation were sought by subtractive hybridization, and 1010 candidate clones from two oppositely enriched cDNA populations were sequenced. Hybridization revealed small (<3-fold) increases or decreases in the transcripts of more than 15 genes, including some implicated in cell cycle control (e.g., BTG, Bap1) or apoptosis (e.g., STAT1, calpain), but no marked changes like those associated with other forms of T-cell death. Moreover, the expression of some critical apoptosis regulators, such as Bcl-2 family members, did not change. Hence, the G2/M arrest and apoptosis in the irradiated p53-null lymphoma appears to involve modest expression changes for many genes, but post-transcriptional alterations may be more critical.

Animals↗
PubMed2000

The role of bim, a proapoptotic BH3-only member of the Bcl-2 family in cell-death control.

Apoptosis is an evolutionarily conserved process for killing unwanted cells. Genetic and biochemical experiments have indicated that three groups of proteins are necessary for activation of the cell-death effector machinery: cysteine proteases, their adaptors, and proapoptotic Bcl-2 family members. Antiapoptotic Bcl-2 family members are needed for cell survival. We have cloned Bim, a proapoptotic Bcl-2 family member that shares with the family only a 9-16 aa region of homology [Bcl-3 homology region(BH3)], but is otherwise unique. Bim requires its BH3 region for binding to Bcl-2 and activation of apoptosis. Analysis of Bim-deficient mice has shown that Bim is essential for the execution of some but not all apoptotic stimuli that can be antagonized by Bcl-2. Bim-deficient mice have increased numbers of lymphocytes, plasma cells, and myeloid cells, and most develop fatal autoimmune glomerulonephritis. In healthy cells, Bim is bound to the microtubule-associated dynein motor complex, and is thereby sequestered from Bcl-2. Certain apoptotic signals unleash Bim and allow it to translocate to intracellular membranes, where it interacts with Bcl-2 or its homologues. These results indicate that BH3-only proteins are essential inducers of apoptosis that can be unleashed by certain death signals. Unleashed BH3-only proteins neutralize the prosurvival function of Bcl-2-like molecules, and this is thought to liberate Apaf-l-like adapters to activate caspase zymogens, which then initiate cell degradation.

Animals↗
PubMed2000

The role of the pro-apoptotic Bcl-2 family member bim in physiological cell death.

Apoptosis, an evolutionarily conserved process for killing unwanted cells in multicellular organisms, is essential for normal development, tissue homeostasis and as a defense against pathogens. The control of apoptosis is of considerable importance for clinical medicine, as its deregulation can lead to cancer, autoimmunity or degenerative diseases. We have disrupted the Bim gene in the mouse and demonstrated that it plays a major and non-redundant role in embryogenesis, in the control of hematopoietic cell death, and as a barrier against autoimmunity.

Animals↗
PubMed2000

Apoptosis signaling.

Apoptosis, a physiological process for killing cells, is critical for the normal development and function of multicellular organisms. Abnormalities in cell death control can contribute to a variety of diseases, including cancer, autoimmunity, and degenerative disorders. Signaling for apoptosis occurs through multiple independent pathways that are initiated either from triggering events within the cell or from outside the cell, for instance, by ligation of death receptors. All apoptosis signaling pathways converge on a common machinery of cell destruction that is activated by a family of cysteine proteases (caspases) that cleave proteins at aspartate residues. Dismantling and removal of doomed cells is accomplished by proteolysis of vital cellular constituents, DNA degradation, and phagocytosis by neighboring cells. This article reviews current knowledge of apoptosis signaling, lists several pressing questions, and presents a novel model to explain the biochemical and functional interactions between components of the cell death regulatory machinery.

Animals↗
PubMed2000

Rapid polymerase chain reaction/DNA probe membrane-based assay for the detection of Listeria and Listeria monocytogenes in food.

We describe the development of polymerase chain reaction (PCR)/DNA probe membrane-based colorimetric assays for the detection and identification of Listeria and L. monocytogenes. PCR primers designed from the 16S to 23S rRNA intergenic spacer region amplified products that were reverse hybridized to membrane-bound oligonucleotide probes specific for Listeria and L. monocytogenes with a detection limit of 1 to 10 CFU/25 ml in inoculated raw and pasteurized milk samples. These qualitative assays have the potential to be integrated into testing laboratories for monitoring the microbiological quality of foods.

Animals↗
PubMed1999

Expression, regulation, and mode of action of the AbiG abortive infection system of lactococcus lactis subsp. cremoris UC653

The abortive infection system AbiG is encoded by the lactococcal plasmid pCI750. The abiG locus (consisting of two genes, abiGi and abiGii) was examined by Northern blot analysis, revealing two transcripts of approximately 2.8 and 1.5 kb which were homologous to the two gene-specific probes. A transcriptional start site was mapped upstream of abiGi, and it appeared that the two genes were cotranscribed, resulting in the 2.8-kb transcript. The smaller transcript may be the result of independent transcription of abiGii within abiGi or of the presence of a weak terminator within abiGii. The locus was shown to be constitutively expressed. Evidence is presented for the possible existence of a second Abi mechanism on pCI750. Examination of phage sk1 RNA synthesis demonstrated that both the subcloned AbiG and, to a greater extent, pCI750 inhibited this process. pCI750 also severely inhibited synthesis of both early and late phage c2 transcripts, while the presence of the subclone resulted in a reduction in late transcript synthesis only.

Journal Article↗
PubMed1998

Bim: a novel member of the Bcl-2 family that promotes apoptosis.

Certain members of the Bcl-2 family inhibit apoptosis while others facilitate this physiological process of cell death. An expression screen for proteins that bind to Bcl-2 yielded a small novel protein, denoted Bim, whose only similarity to any known protein is the short (nine amino acid) BH3 motif shared by most Bcl-2 homologues. Bim provokes apoptosis, and the BH3 region is required for Bcl-2 binding and for most of its cytotoxicity. Like Bcl-2, Bim possesses a hydrophobic C-terminus and localizes to intracytoplasmic membranes. Three Bim isoforms, probably generated by alternative splicing, all induce apoptosis, the shortest being the most potent. Wild-type Bcl-2 associates with Bim in vivo and modulates its death function, whereas Bcl-2 mutants that lack survival function do neither. Significantly, Bcl-xL and Bcl-w, the two closest homologues of Bcl-2, also bind to Bim and inhibit its activity, but more distant viral homologues, adenovirus E1B19K and Epstein-Barr virus BHRF-1, can do neither. Hence, Bim appears to act as a 'death ligand' which can only neutralize certain members of the pro-survival Bcl-2 sub-family.

Amino Acid Sequence↗
PubMed1998

Emigration of mature T cells from the thymus is inhibited by the imidazole-based compound 2-acetyl-4-tetrahydroxybutylimidazole.

The primary role of the thymus is to provide mature T cells for the peripheral immune system. The mechanisms involved in the cellular export processes are as yet unknown. In this study, we examined the ability of 2-acetyl-4-tetrahydroxybutylimidazole (THI), an agent widely used as a component of ammonia caramel food colouring, to inhibit T-cell export from the thymus. BALB/c mice were maintained on drinking water containing THI for 5 days. The mice showed a twofold increase in the total number of mature medullary thymocytes (CD4+CD8- and CD4-CD8+) as well as a slight decrease in the total number of immature double-positive cells (CD4+CD8+). The mature single-positive thymocytes were found to express high levels of the homing molecule L-selectin, suggesting that these potential emigrants were prevented from leaving the thymus. To confirm this, THI-treated mice were injected intrathymically with fluorescein isothiocyanate and the number of labelled T cells appearing in the lymph nodes and spleen was determined 16 hr later. A 10-fold decrease in the number of CD4+ and CD8+ recent thymic emigrants in the lymph nodes and spleen of THI-treated mice was observed. Previous studies have shown that THI does not affect other aspects of thymocyte development, such as proliferation and differentiation. Taken together, these results suggest that the immunosuppressive effects of THI may be due, in part, to preventing of the final step of T-cell export out of the thymus.

Animals↗
PubMed1998

Rapid hybridoma screening method for the identification of monoclonal antibodies to low-abundance cytoplasmic proteins.

Screening assays are the most time-consuming and labor-intensive part of generating monoclonal antibodies (MAbs). Antibodies identified by enzyme-linked immunosorbent assay (ELISA) screening often are not suitable for their intended application such as immunofluorescence staining. We describe here a rapid and efficient flow cytometric screening procedure for the identification of MAbs directed against low-abundance cytoplasmic proteins, in our case, the pro-apoptotic molecule Bim. Cells from an equal mixture of a parental cell line and a subline expressing Bim were fixed, permeabilized and incubated with hybridoma supernatants. The supernatants were derived from a fusion of Sp2/0 plasmacytoma cells and spleen cells from a rat immunized with recombinant glutathione-S-transferase (GST)-BimL fusion protein. Secondary staining with fluorochrome-labeled anti-rat Ig antibodies allowed detection of clones expressing Bim-specific antibodies. The screening procedure was rapid and efficient, and most monoclonal antibodies identified were proven to be useful for immunofluorescence staining and other applications.

Animals↗