Biomedical subjects

L Harrington

Publications and source records attributed to L Harrington.

At least 19 recordsLinked to original sources

PubMed2006

Pharmacology of airways and vessels in lung slices in situ: role of endogenous dilator hormones.

Small airway and vessels play a critical role in chronic airway and pulmonary vascular diseases, but their pharmacology has not been well characterised. We have studied airway and vascular responses in rat lung slices and separately in vitro using myography. In lung slices, under basal conditions, acetylcholine contracted airways, but had no vascular effect. The thromboxane mimetic, U46619 contracted both vessels and airways. In the presence of U46619, acetylcholine dilated vessels, but further contracted airways, an effect that was blocked by the nitric oxide synthase inhibitor L-NG-nitro-L-arginine or apamin plus charybdotoxin, which inhibit endothelial-derived hyperpolarising factor. Airway responses in lung slices were unaffected by L-NGnitro-L-arginine methyl ester, indomethacin or apamin plus charybdotoxin. By contrast, apamin plus charybdotoxin contracted bronchi studied in isolation. Our observations are the first to identify mechanisms of endothelium dependent dilations in precision cut lung slices and the potential for transverse hormonal communication between airways and vessels.

Acetylcholine↗

PubMed2005

Dissociation of telomerase activity and telomere length maintenance in primitive human hematopoietic cells.

Primitive human hematopoietic cells have low endogenous telomerase activity, yet telomeres are not maintained. In contrast, ectopic telomerase expression in fibroblasts and other cells leads to telomere length maintenance or elongation. It is unclear whether this disparity can be attributed to telomerase level or stems from fundamentally different telomere biology. Here, we show that telomerase overexpression does not prevent proliferation-associated telomere shortening in human hematopoietic cells, pointing to the existence of cell type-specific differences in telomere dynamics. Furthermore, we observed eventual stabilization of telomere length without detectable changes in telomerase activity during establishment of two leukemic cell lines from normal cord blood cells, indicating that additional cooperating events are required for telomere maintenance in immortalized human hematopoietic cells.

Cell Proliferation↗

PubMed2005

Direct evidence for cooperating genetic events in the leukemic transformation of normal human hematopoietic cells.

Although genetic abnormalities associated with hematological malignancies are readily identified, the natural history of human leukemia cannot be observed because initiating and subsequent transforming events occur before clinical presentation. Furthermore, it has not been possible to study leukemogenesis in vitro as normal human cells do not spontaneously transform. Thus, the nature and sequence of genetic changes required to convert human hematopoietic cells into leukemia cells have never been directly examined. We have developed a system where the first step in the leukemogenic process is an engineered disruption of differentiation and self-renewal due to expression of the TLS-ERG oncogene, followed in some cases by overexpression of hTERT. In two of 13 experiments, transduced cells underwent step-wise transformation and immortalization through spontaneous acquisition of additional changes. The acquired karyotypic abnormalities and alterations including upregulation of Bmi-1 and telomerase all occur in acute myeloid leukemia (AML), establishing the relevance of this system. One resultant cell line studied in depth exhibits cellular properties characteristic of AML, notably a hierarchical organization initiated by leukemic stem cells that differentiate abnormally. These findings provide direct evidence for multiple cooperating events in human leukemogenesis, and provide a foundation for studying the genetic changes that occur during leukemic initiation and progression.

Blotting, Western↗

PubMed2001

The Telomerase/vault-associated protein TEP1 is required for vault RNA stability and its association with the vault particle.

Vaults and telomerase are ribonucleoprotein (RNP) particles that share a common protein subunit, TEP1. Although its role in either complex has not yet been defined, TEP1 has been shown to interact with the mouse telomerase RNA and with several of the human vault RNAs in a yeast three-hybrid assay. An mTep1(-/-) mouse was previously generated which resulted in no apparent change in telomere length or telomerase activity in six generations of mTep1-deficient mice. Here we show that the levels of the telomerase RNA and its association with the telomerase RNP are also unaffected in mTep1(-/-) mice. Although vaults purified from the livers of mTep1(-/-) mice appear structurally intact by both negative stain and cryoelectron microscopy, three-dimensional reconstruction of the mTep1(-/-) vault revealed less density in the cap than previously observed for the intact rat vault. Furthermore, the absence of TEP1 completely disrupted the stable association of the vault RNA with the purified vault particle and also resulted in a decrease in the levels and stability of the vault RNA. Therefore, we have uncovered a novel role for TEP1 in vivo as an integral vault protein important for the stabilization and recruitment of the vault RNA to the vault particle.

Animals↗

PubMed2001

Functional multimerization of the human telomerase reverse transcriptase.

The telomerase enzyme exists as a large complex (approximately 1,000 kDa) in mammals and at minimum is composed of the telomerase RNA and the catalytic subunit telomerase reverse transcriptase (TERT). In Saccharomyces cerevisiae, telomerase appears to function as an interdependent dimer or multimer in vivo (J. Prescott and E. H. Blackburn, Genes Dev. 11:2790-2800, 1997). However, the requirements for multimerization are not known, and it remained unclear whether telomerase exists as a multimer in other organisms. We show here that human TERT (hTERT) forms a functional multimer in a rabbit reticulocyte lysate reconstitution assay and in human cell extracts. Two separate, catalytically inactive TERT proteins can complement each other in trans to reconstitute catalytic activity. This complementation requires the amino terminus of one hTERT and the reverse transcriptase and C-terminal domains of the second hTERT. The telomerase RNA must associate with only the latter hTERT for reconstitution of telomerase activity to occur. Multimerization of telomerase also facilitates the recognition and elongation of substrates in vitro and in vivo. These data suggest that the catalytic core of human telomerase may exist as a functionally cooperative dimer or multimer in vivo.

Catalysis↗

PubMed2000

The telomerase reverse transcriptase is limiting and necessary for telomerase function in vivo.

Mammalian telomerase is essential for the maintenance of telomere length [1-5]. Its catalytic core comprises a reverse transcriptase component (TERT) and an RNA component. While the biochemical role of mammalian TERT is well established [6-11], it is unknown whether it is sufficient for telomere-length maintenance, chromosome stability or other cellular processes. Cells from mice in which the mTert gene had been disrupted showed progressive loss of telomere DNA, a phenotype similar to cells in which the gene encoding the telomerase RNA component (mTR) has been disrupted [1,12]. On prolonged growth, mTert-deficient embryonic stem (ES) cells exhibited genomic instability, aneuploidy and telomeric fusions. ES cells heterozygous for the mTert disruption also showed telomere attrition, a phenotype that differs from heterozygous mTR cells [12]. Thus, telomere maintenance in mammals is carried out by a single, limiting TERT.

Animals↗

PubMed2000

Antibody responses to Yersinia pestis F1-antigen expressed in Salmonella typhimurium aroA from in vivo-inducible promoters.

Attenuated mutants of Salmonella typhimurium are being evaluated as delivery systems for a variety of heterologous vaccine antigens. Gene promoters which are induced in vivo can direct the stable expression of genes encoding these antigens. We have investigated the utility of the phoP, ompC, pagC and lacZ gene promoters for expression of the Y. pestis F1-antigen in S. typhimurium SL3261 (aroA). After i.g. (intragastric) dosing the highest level of spleen colonisation was found with recombinant Salmonella expressing F1-antigen from the phoP gene promoter, and this recombinant was most effective in inducing serum and mucosal antibody responses. The use of the pagC gene promoter to direct expression of F1-antigen resulted in the induction of serum and mucosal antibody responses even though the recombinant Salmonella were unable to colonise spleen tissues suggesting that colonisation of these tissues is not essential for the induction of antibody responses.

Animals↗

PubMed2000

Polymerization defects within human telomerase are distinct from telomerase RNA and TEP1 binding.

The minimal, active core of human telomerase is postulated to contain two components, the telomerase RNA hTER and the telomerase reverse transcriptase hTERT. The reconstitution of human telomerase activity in vitro has facilitated the identification of sequences within the telomerase RNA and the RT motifs of hTERT that are essential for telomerase activity. However, the precise role of residues outside the RT domain of hTERT is unknown. Here we have delineated several regions within hTERT that are important for telomerase catalysis, primer use, and interaction with the telomerase RNA and the telomerase-associated protein TEP1. In particular, certain deletions of the amino and carboxy terminus of hTERT that retained an interaction with telomerase RNA and TEP1 were nonetheless completely inactive in vitro and in vivo. Furthermore, hTERT truncations lacking the amino terminus that were competent to bind the telomerase RNA were severely compromised for the ability to elongate telomeric and nontelomeric primers. These results suggest that the interaction of telomerase RNA with hTERT can be functionally uncoupled from polymerization, and that there are regions outside the RT domain of hTERT that are critical for telomerase activity and primer use. These results establish that the human telomerase RT possesses unique polymerization determinants that distinguish it from other RTs.

Animals↗

PubMed2000

Telomeres, telomerase, and cancer: life on the edge of genomic stability.

The presence of telomerase activity in most human tumors, but not in many normal somatic tissues, has raised considerable interest in telomerase as a possible anticancer therapy. Recent advances in the cloning and characterization of mammalian telomerase components have paved the way for a more detailed understanding of the role of telomerase and telomere length maintenance in cell proliferation. Here, we summarize the most recent biochemical and genetic evidence suggesting that telomere length maintenance by telomerase is critical to the proliferative ability of some immortalized mammalian cells in culture and in vivo.

Animals↗

PubMed2000

Telomerase-associated protein TEP1 is not essential for telomerase activity or telomere length maintenance in vivo.

TEP1 is a mammalian telomerase-associated protein with similarity to the Tetrahymena telomerase protein p80. Like p80, TEP1 is associated with telomerase activity and the telomerase reverse transcriptase, and it specifically interacts with the telomerase RNA. To determine the role of mTep1 in telomerase function in vivo, we generated mouse embryonic stem (ES) cells and mice lacking mTep1. The mTep1-deficient (mTep1(-/-)) mice were viable and were bred for seven successive generations with no obvious phenotypic abnormalities. All murine tissues from mTep1(-/-) mice possessed a level of telomerase activity comparable to that in wild-type mice. In addition, analysis of several tissues that normally lack telomerase activity revealed no reactivation of telomerase activity in mTep1(-/-) mice. Telomere length, even in later generations of mTep1(-/-) mice, was equivalent to that in wild-type animals. ES cells deficient in mTep1 also showed no detectable alteration in telomerase activity or telomere length with increased passage in culture. Thus, mTep1 appears to be completely dispensable for telomerase function in vivo. Recently, TEP1 has been identified within a second ribonucleoprotein (RNP) complex, the vault particle. TEP1 can also specifically bind to a small RNA, vRNA, which is associated with the vault particle and is unrelated in sequence to mammalian telomerase RNA. These results reveal that TEP1 is an RNA binding protein that is not restricted to the telomerase complex and that TEP1 plays a redundant role in the assembly or localization of the telomerase RNP in vivo.

Animals↗

PubMed1999

Vaults and telomerase share a common subunit, TEP1.

Vaults are large cytoplasmic ribonucleoprotein complexes of undetermined function. Mammalian vaults have two high molecular mass proteins of 193 and 240 kDa. We have identified a partial cDNA encoding the 240-kDa vault protein and determined it is identical to the mammalian telomerase-associated component, TEP1. TEP1 is the mammalian homolog of the Tetrahymena p80 telomerase protein and has been shown to interact specifically with mammalian telomerase RNA and the catalytic protein subunit hTERT. We show that while TEP1 is a component of the vault particle, vaults have no detectable telomerase activity. Using a yeast three-hybrid assay we demonstrate that several of the human vRNAs interact in a sequence-specific manner with TEP1. The presence of 16 WD40 repeats in the carboxyl terminus of the TEP1 protein is a convenient number for this protein to serve a structural or organizing role in the vault, a particle with eight-fold symmetry. The sharing of the TEP1 protein between vaults and telomerase suggests that TEP1 may play a common role in some aspect of ribonucleoprotein structure, function, or assembly.

Animals↗

PubMed1999

Telomere shortening and apoptosis in telomerase-inhibited human tumor cells.

Despite a strong correlation between telomerase activity and malignancy, the outcome of telomerase inhibition in human tumor cells has not been examined. Here, we have addressed the role of telomerase activity in the proliferation of human tumor and immortal cells by inhibiting TERT function. Inducible dominant-negative mutants of hTERT dramatically reduced the level of endogenous telomerase activity in tumor cell lines. Clones with short telomeres continued to divide, then exhibited an increase in abnormal mitoses followed by massive apoptosis leading to the loss of the entire population. This cell death was telomere-length dependent, as cells with long telomeres were viable but exhibited telomere shortening at a rate similar to that of mortal cells. It appears that telomerase inhibition in cells with short telomeres lead to chromosomal damage, which in turn trigger apoptotic cell death. These results provide the first direct evidence that telomerase is required for the maintenance of human tumor and immortal cell viability, and suggest that tumors with short telomeres may be effectively and rapidly killed following telomerase inhibition.

Apoptosis↗

PubMed1998

Reconstitution of human telomerase activity in vitro.

Telomerase is a ribonucleoprotein enzyme complex that adds single-stranded telomere DNA to chromosome ends [1]. The RNA component of telomerase contains the template for telomeric DNA addition and is essential for activity [1,2]. Telomerase proteins have been identified in ciliates, yeast and mammals [3-12]. In Saccharomyces cerevisiae, the Est2 protein is homologous to the 123 kDa reverse transcriptase subunit of Euplotes telomerase, and is essential for telomerase activity [8]. In humans, telomerase activity is associated with the telomerase RNA hTR [13], the telomerase RNA-binding protein TP1/TLP1 [5,12] and the TP2 protein encoded by the human EST2 homolog [12] (also known as TRT1, hEST2 or TCS1 [9-11]). The minimal complex sufficient for activity is, however, unknown. We have reconstituted human telomerase activity in reticulocyte lysates and find that only exogenous hTR and TP2 are required for telomerase activity in vitro. Recognition of telomerase RNA by TP2 was species specific, and nucleotides 10-159 of hTR were sufficient for telomerase activity. Telomerase activity immunoprecipitated from the reticulocyte lysate contained hTR and recombinant TP2. Substitution of conserved amino acid residues in the reverse transcriptase domain of TP2 completely abolished telomerase activity. We suggest that TP2 and hTR might represent the minimal catalytic core of human telomerase.

Carrier Proteins↗

PubMed1998

Ultrasonographic and clinical predictors of intussusception.

OBJECTIVE: The objective of this study was to determine the positive and negative clinical predictors of intussusception and the correlation of ultrasonography and air enema in establishing this diagnosis. STUDY DESIGN: This was a prospective descriptive cohort study. SETTING: This study was performed in a tertiary care pediatric emergency department. PARTICIPANTS: Eighty-eight of 245 candidates were assessed for clinical predictors of intussusception. All 245 cases were examined for correlation between ultrasonography and air enema. INTERVENTIONS: A questionnaire, ultrasonography, and air enema were used. RESULTS: Thirty-five of the 88 patients assessed for clinical predictors were positive for intussusception. Significant positive predictors were right upper quadrant abdominal mass (positive predictive value [PPV] 94%), gross blood in stool (PPV 80%), blood on rectal examination (PPV 78%), the triad of intermittent abdominal pain, vomiting, and right upper quadrant abdominal mass (PPV 93%, p = 0.0001), and the triad with occult or gross blood per rectum (PPV 100%, p = not significant). Significant negative predictors were a combination of > or = 3 of 10 clinically significant negative features (negative predictive value 77%, p = 0.035). Of the total 245 cases, intussusception (as confirmed by doughnut, target, or pseudokidney sign) was ruled out by ultrasonography in 97.4%. Alternate ultrasound findings comprised 27% of negative cases. CONCLUSIONS: Excellent positive predictors of intussusception were identified prospectively. Although no reliable negative predictors were found, patients at low risk may be screened by ultrasonography.

Air↗

PubMed1998

Refined genetic mapping of the darier locus to a <1-cM region of chromosome 12q24.1, and construction of a complete, high-resolution P1 artificial chromosome/bacterial artificial chromosome contig of the critical region.

Darier disease (DD) (MIM 124200) is an autosomal dominant skin disorder characterized by loss of adhesion between epidermal cells and by abnormal keratinization. We present linkage analysis showing, in four families, key recombination events that refine the location of the DD locus on chromosome 12q23-24.1 to a region of <1 cM. We have constructed a YAC/P1 artificial chromosome (PAC)/bacterial artificial chromosome (BAC)-based physical map that encompasses this refined DD region. The map consists of 35 YAC, 69 PAC, 16 BAC, and 2 cosmid clones that were ordered by mapping 54 anonymous sequence-tagged sites. The critical region is estimated to be 2.4 Mb in size, with an average marker resolution of 37.5 kb. The refinement of the critical interval excludes the ALDH2, RPL6, PTPN11, and OAS genes, as well as seven expressed sequence tags (ESTs) previously mapped in the DD region. The three known genes (ATP2A2, PPP1CC, and SCA2) and the 10 ESTs mapped within the critical region are not obvious candidates for the DD gene. Therefore, this detailed integrated physical, genetic, and partial transcript map provides an important resource for the isolation of the DD gene and, possibly, other disease genes.

Chromosome Mapping↗

PubMed1997

Human telomerase contains evolutionarily conserved catalytic and structural subunits.

We have cloned and characterized a human gene encoding TP2 (telomerase-associated protein 2), a protein with similarity to reverse transcriptases and the catalytic telomerase subunits from Saccharomyces cerevisiae and Euplotes aediculatus. Indirect immunofluorescence revealed that TP2 was localized to the nucleus. Using antibodies to endogenous and epitope-tagged TP2, we found that TP2 was associated specifically with human telomerase activity and the recently identified telomerase-associated protein TP1. Mutation of conserved residues within the reverse transcriptase domain of TP2 severely reduced associated telomerase activity. These results suggest that telomerase is an evolutionarily conserved multisubunit complex composed of both structural and catalytic subunits.

Amino Acid Sequence↗

PubMed1997

A mammalian telomerase-associated protein.

The telomerase ribonucleoprotein catalyzes the addition of new telomeres onto chromosome ends. A gene encoding a mammalian telomerase homolog called TP1 (telomerase-associated protein 1) was identified and cloned. TP1 exhibited extensive amino acid similarity to the Tetrahymena telomerase protein p80 and was shown to interact specifically with mammalian telomerase RNA. Antiserum to TP1 immunoprecipitated telomerase activity from cell extracts, suggesting that TP1 is associated with telomerase in vivo. The identification of TP1 suggests that telomerase-associated proteins are conserved from ciliates to humans.

Amino Acid Sequence↗

PubMed1997

Complications of Fleet enema administration and suggested guidelines for use in the pediatric emergency department.

INTRODUCTION: Hypertonic sodium phosphate enema solutions are commonly used for the treatment of acute constipation in the pediatric emergency department. The potential for severe metabolic derangement and death in children with gastrointestinal and/or renal abnormalities and these reported as normal has been documented in the literature. OBJECTIVE: To develop guidelines for the safe and effective use of hypertonic sodium phosphate enema solutions such as Fleet enema, in the pediatric emergency department setting. RESOURCES: A comprehensive review of the literature was conducted using the MEDLINE database for the time period 1966 to September 1995. Resources used within our institution, the Hospital for Sick Children, Toronto, Ontario, included Staff of the Emergency and General Surgery Departments. SUMMARY: The guidelines provided will promote safe use of hypertonic sodium phosphate enema solutions for the treatment of acute constipation in children presenting to the emergency department.

Acute Disease↗