Biomedical subjects

T L Beattie

Publications and source records attributed to T L Beattie.

14 recordsLinked to original sources

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↗

PubMed2001

Brain activity and cognitive status in pediatric patients: development of a clinical assessment protocol.

The purpose of this study was to test the validity of a new computerized task to assess children's cognitive problem-solving skills using the brain event-related potentials. This event-related potential-computerized cognitive problem-solving task does not require a child to give a verbal or motor (ie, pointing) response. The event-related potential waveforms were recorded from 20 typically developing children. Two nonverbal, problem-solving tasks (tasks 1 and 2) were developed for each of two age groups (5 and 6 years). For each task, single pictures, taken from an existing standardized test of nonverbal problem solving, were individually and sequentially presented on a computer screen. One of the seven pictures was classified as incongruent or outside category; it did not belong with the other pictures. As predicted, the event-related potential amplitudes were significantly larger to the outside- versus within-category pictures. This effect was found for tasks 1 and 2 for the 5- and 6-year-old children. Children as young as 5 years of age reliably exhibit brain activity, which can be used to infer cognitive problem-solving skill. This assessment paradigm may eventually serve as a clinically useful adjunct to a thorough neurologic and neurodevelopmental assessment of selected pediatric populations, such as those presenting with moderate-severe cerebral palsy whose expressive language and motor skills are notably impaired.

Age Factors↗

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

Preschoolers classified as having attention-deficit hyperactivity disorder (ADHD): DSM-IV symptom endorsement pattern.

In the present study, 50 preschoolers were formally and independently classified using both the Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV) and third edition-revised (DSM-III-R) criteria for attention-deficit hyperactivity disorder (ADHD). The sample consisted of 25 preschoolers classified as having ADHD and 25 typically developing preschoolers for comparison; the sample was matched on gender, age, and socioeconomic status. All 50 preschoolers were without neurologic or neurodevelopmental disorders, oppositional defiant disorder, or language delay. There were four key findings: first, of the 25 preschoolers with ADHD, DSM-IV classification was as follows: hyperactive-impulsivity type (68%), combined type (28%), and inattentive type (4%). Second, the DSM-IV profiles showed that several symptoms were either infrequently endorsed by parents, reflecting limited applicability to preschoolers with ADHD, or frequently endorsed by parents of typically developing preschoolers, thus reducing their diagnostic value. Third, of the 25 preschoolers classified as having ADHD using DSM-IV criteria, 16% would not have been classified as having ADHD using the DSM-III-R criteria. The DSM-IV criteria therefore appear to be more lenient than the DSM-III-R criteria for this age group. Fourth, two symptoms that were not included in the DSM-IV, but were part of the DSM-III-R, were found to have clinical value for differentiating preschoolers with ADHD from their typically developing peers.

Attention↗

PubMed1999

Brain activity and language assessment using event-related potentials: development of a clinical protocol.

To test the validity of a new computerized task to assess children's receptive vocabulary, event-related potentials (ERPs) were recorded from 56 typically developing children ranging in age from 5 to 12 years. This ERP-computerized vocabulary task does not require a child to give a verbal or motor (i.e. pointing) response. Single pictures, from an existing standardized test of receptive vocabulary, were presented on a computer screen and simultaneously named either correctly (congruent) or incorrectly (incongruent) via a computer. As predicted, the N400 amplitude was found to be significantly higher to the incongruent picture-word pair (i.e. the child knew it was an incorrect pairing) than to the congruent picture-word pair (i.e. the child knew it was a correct pairing). This effect was found for each of the four age groups (5 to 6 years, 7 to 8 years, 9 to 10 years, 11 to 12 years). This task accurately estimated current receptive vocabulary in typically developing children. Although still in the development stage, it may eventually serve as an adjunct to a thorough neurological and neurodevelopmental assessment of some children presenting with moderate to severe cerebral palsy.

Brain↗

PubMed1998

Identification of phosphate groups involved in metal binding and tertiary interactions in the core of the Neurospora VS ribozyme.

We have used ethylation protection experiments and modification interference using phosphorothioate nucleosides to identify phosphate groups involved in the magnesium-dependent tertiary structure and function of the VS ribozyme, a small, self-cleaving RNA. Phosphorothioate interference-rescue experiments in the presence of the thiophilic manganese ion implicate four phosphate groups in direct metal ion binding. Phosphorothioate substitution also creates a new manganese binding site that increases the cis cleavage rate of the ribozyme, possibly by disrupting an inhibitory structure. Interpreting these data in the context of a recently developed structural model shows that almost all of the important phosphate groups are located in the central core of the ribozyme. The model suggests roles for certain phosphate groups in particular steps of RNA folding and identifies a candidate region for the active site of the ribozyme.

Base Sequence↗

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

Clinical assessment of psychopharmacological treatment of preschoolers with ADHD.

Sixteen preschoolers, (8 with Attention-Deficit/Hyperactivity Disorder [ADHD], 8 matched controls) were assessed twice, 5 months apart. Preschoolers with ADHD were rated by their parents as significantly more inattentive, exhibited more behavior problems, fewer age-appropriate social skills, made more errors of omission on both the visual and auditory attention tests, and more errors of commission on both the visual attention and the visual-search cancellation tests. Preschoolers with ADHD were then treated with stimulant medication and exhibited improved behavior as well as significantly reduced errors of omission on visual and auditory preschool vigilance tests, and fewer errors of commission on the visual-search preschool cancellation test. Developmentally appropriate direct measures of attention, in conjunction with parental ratings of child behavior, can be used to assess the efficacy of pharmacological treatment of preschoolers with ADHD.

Attention↗

PubMed1997

Identification of functional domains in the self-cleaving Neurospora VS ribozyme using damage selection.

Varkud Satellite (VS) RNA contains a small self-cleaving RNA motif that is distinct in its sequence and secondary structure from the hammerhead, hairpin, and hepatitis delta virus ribozymes, which are found in other natural RNAs. We have used a base specific chemical damage selection (modification interference) assay to identify functionally important nucleotides and structural elements in VS RNA. Many modified bases interfered with self-cleavage and most of these clustered at helix junctions, certain internal loops, and in a long-range pseudoknot; these correspond to previously determined sites of magnesium-dependent protection from chemical modification. The clustering suggests that these bases are important not only for a large number of individual interactions, but because they form a smaller number of structural elements that are important for activity. Modification of bases in other single-stranded regions, which did not exhibit magnesium-dependent protection, generally did not interfere with activity, suggesting that some of these regions might be dispensable for function. Surprisingly, we found a separate cluster of bases whose modification significantly enhanced cleavage. These bases appear to form a structural element that naturally attenuates the self-cleavage reaction. In natural VS RNA this attenuator structure may affect the cleavage/ligation equilibrium by inhibiting circle re-opening, thereby helping to maintain the RNA in a circular form, which is the predominant form of VS RNA in vivo. Taken together, the results of the damage selection experiments localize the catalytic core of VS RNA to a small subset of the previously determined minimal contiguous sequence.

Base Sequence↗

PubMed1996

A long-range pseudoknot is required for activity of the Neurospora VS ribozyme.

Four small RNA self-cleaving domains, the hammerhead, hairpin, hepatitis delta virus and Neurospora VS ribozymes, have been identified previously in naturally occurring RNAs. The secondary structures of these ribozymes are reasonably well understood, but little is known about long-range interactions that form the catalytically active tertiary conformations. Our previous work, which identified several secondary structure elements of the VS ribozyme, also showed that many additional bases were protected by magnesium-dependent interactions, implying that several tertiary contacts remained to be identified. Here we have used site-directed mutagenesis and chemical modification to characterize the first long-range interaction identified in VS RNA. This interaction contains a 3 bp pseudoknot helix that is required for tertiary folding and self-cleavage activity of the VS ribozyme.

Diethyl Pyrocarbonate↗

PubMed1996

Model for the factor VIIIa-dependent decay of the intrinsic factor Xase. Role of subunit dissociation and factor IXa-catalyzed proteolysis.

The intrinsic factor Xase complex (FXase) is comprised of a serine protease, FIXa, and a protein cofactor, FVIIIa, assembled on a phospholipid surface. Activity of FXase decays with time and reflects the lability of FVIIIa. Two mechanisms potentially contribute to this decay: (i) a weak affinity interaction between the FVIIIa A2 subunit and Al/A3-Cl-C2 dimer and (ii) FVIIIa inactivation resulting from FIXa-catalyzed proteolysis of the Al subunit. At low reactant concentrations (0.5 nm FVIIIa; 5 nm FIXa), FXase decay is governed by the inter-FVIIIa subunit affinity and residual activity approaches a value consistent with this equilibrium, as judged by reactions containing exogenous A2 subunit. Analysis using a mutant form of FVIII (FVIIIR336I) possessing an altered FIXa cleavage site, showed similar rates of FXase decay (0.12 min(-1)) and confirmed the lack of contribution of proteolysis under these conditions. When the concentration of FIXa was increased 10-fold, the initial rate of decay of FXase containing native FVIIIa increased (0.82 min(-1)) and paralleled the rate of proteolysis of Al subunit. However, the rate of decay of FXase containing the FVIIIaR336I was reduced (0.048 min(-1)) consistent with the elevated concentration of FIXa stabilizing the labile subunit structure of the cofactor. Reconstitution of FVIII with FIXa-cleaved light chain showed that cleavage at the alternate FIXa site (A3 domain) was not inhibitory to FXase. The presence of substrate FX resulted in a 10-fold reduction in the rate of FIXa-catalyzed proteolysis of FVIIIa. These results suggest a model whereby decay of FXase results from both FVIIIa subunit dissociation and FIXa-catalyzed cleavage, dependent upon the relative concentration of reactants, with greater contribution of the former at low values and, in the absence of substrate, greater contribution of the latter at high values.

Amino Acid Sequence↗

PubMed1996

Activated protein C-catalyzed proteolysis of factor VIIIa alters its interactions within factor Xase.

Factor VIIIa, the cofactor for the factor IXa-dependent conversion of factor X to factor Xa, is proteolytically inactivated by activated protein C (APC). APC cleaves at two sites in factor VIIIa, Arg336, near the C terminus of the A1 subunit; and Arg562, bisecting the A2 subunit (Fay, P., Smudzin, T., and Walker, F. (1991) J. Biol. Chem. 266, 20139-20145). Factor VIIIa increased the fluorescence anisotropy of fluorescein-Phe-Phe-Arg factor IXa (Fl-FFR-FIXa; Kd = 42.4 nM), whereas cleavage of factor VIIIa by APC eliminated this property. Isolation of the APC-cleaved A1/A3-C1-C2 dimer (A1336/A3-C1-C2), and the fragments derived from cleaved A2 subunit (A2N/A2C), permitted dissection of the roles of individual cleavages in cofactor inactivation. Intact A1/A3-C1-C2 dimer increased Fl-FFR-FIXa anisotropy and bound factor X in a solid phase assay, while these activities were absent in the A1336/A3-C1-C2. However, the residues removed by this cleavage, Met337 Arg372, did not directly participate in these functions since neither a synthetic peptide to this sequence nor an anti-peptide polyclonal antibody blocked these activities using intact dimer. CD spectral analysis of the intact and truncated dimers indicated reduced alpha and/or beta content in the latter. The A1/A3-C1-C2 dimer plus A2 subunit reconstitutes cofactor activity and produced a factor VIIIa-like effect on the anisotropy of Fl-FFR-FIXa. However, when A2 was replaced by the A2N/A2C fragments, the resulting fluorescence signal was equivalent to that observed with the dimer alone. These results indicate that APC inactivates the cofactor at two levels within the intrinsic factor Xase complex. Cleavage of either subunit modulates the factor IXa active site, suggesting an essential synergy of interactive sites in factor VIIIa. Furthermore, cleavage of the A1 site alters the conformation of a factor X binding site within that subunit, thereby reducing the affinity of cofactor for substrate.

Amino Acid Sequence↗

PubMed1995

Enhancement of Neurospora VS ribozyme cleavage by tuberactinomycin antibiotics.

Several examples of inhibition of the function of a ribozyme or RNA-protein complex have shown that certain antibiotics can interact specifically with RNA. There are, however, few examples of antibiotics that have a positive, rather than a negative, effect on the function of an RNA. We have found that micromolar concentrations of viomycin, a basic, cyclic peptide antibiotic of the tuberactinomycin group, enhance the cleavage of a ribozyme derived from Neurospora VS RNA. Viomycin decreases by an order of magnitude the concentration of magnesium required for cleavage. It also stimulates an otherwise insignificant transcleavage reaction by enhancing interactions between RNA molecules. The ability of viomycin to enhance some RNA-mediated reactions but inhibit others, including translation and Group I intron splicing, demonstrates the potential for natural selection by small molecules during evolution in the 'RNA world' and may have broader implications with respect to ribozyme expression and activity in contemporary cells.

Enviomycin↗

PubMed1995

A secondary-structure model for the self-cleaving region of Neurospora VS RNA.

Neurospora VS RNA performs an RNA-mediated self-cleavage reaction whose products contain 2',3'-cyclic phosphate and 5'-hydroxyl termini. This reaction is similar to those of hammerhead, hairpin, and hepatitis delta virus ribozymes; however, VS RNA is not similar in sequence to these other self-cleaving motifs. Here we propose a model for the secondary structure of the self-cleaving region of VS RNA, supported by site-directed mutagenesis and chemical modification structure probing data. The secondary structure of VS RNA is distinct from those of the other naturally occurring RNA self-cleaving domains. In addition to a unique secondary structure, several Mg-dependent interactions occur during the folding of VS RNA into its active tertiary conformation.

Cloning, Molecular↗