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Biomedical subjects

S E Holt

Publications and source records attributed to S E Holt.

30 records · Page 2Linked to original sources
PubMed1996

Refining the telomere-telomerase hypothesis of aging and cancer.

While there has been substantial experimental evidence in support of the telomere-telomerase hypothesis of aging and cancer, it has been suggested that the theory has been oversimplified to the exclusion of alternative mechanisms in the progression of cancer. This review strives to present an overview of some of the areas that have not been well explained and to indicate where multiple interpretations of the data are possible.

Aging↗
PubMed1996

Regulation of telomerase activity in immortal cell lines.

Telomerase is a ribonucleoprotein whose activity has been detected in germ line cells, immortal cells, and most cancer cells. Except in stem cells, which have a low level of telomerase activity, its activity is absent from normal somatic tissues. Understanding the regulation of telomerase activity is critical for the development of potential tools for the diagnosis and treatment of cancer. Using the telomeric repeat amplification protocol, we found that immortal, telomerase-positive, pseudodiploid human cells (HT1080 and HL60 cells) sorted by flow repressed in quiescent cells. This was true whether quiescence was induced by contact inhibition (NIH 3T3 mouse cells), growth factor removal (bromodeoxyuridine-blocked mouse myoblasts), reexpression of cellular senescence (the reversibly immortalized IDH4 cells), or irreversible cell differentiation (HL60 promyelocytic leukemia cells and C2C12 mouse myoblasts). Taken together, these results indicate that telomerase is active throughout the cell in dividing, immortal cells but that its activity is repressed in cells that exit the cell cycle. This suggests that quiescent stem cells that have the potential to express telomerase may remain unaffected by potential antitelomerase cancer therapies.

3T3 Cells↗
PubMed1995

Mutational analysis of the 18-base-pair inverted repeat element at the bovine papillomavirus origin of replication: identification of critical sequences for E1 binding and in vivo replication.

Replication of bovine papillomavirus requires two viral proteins, E1 and E2-TA. Previously we demonstrated that sequences within an imperfect 18-bp inverted repeat (IR) element were sufficient to confer specific binding of the E1 protein to the origin region (S. E. Holt, G. Schuller, and V. G. Wilson, J. Virol. 68:1094-1102, 1994). To identify critical nucleotides for E1 binding and origin function, a series of individual point mutations was constructed at each nucleotide position in the 18-bp IR. Binding of E1 to these point mutations established that both the position of the mutation and the specific nucleotide change were important for the E1-DNA interaction. Equivalent mutations from each half of the IR exhibited similar binding, suggesting that the halves were functionally symmetric for E1 interactions. Each of these mutations was evaluated also for origin function in vivo by a transient-replication assay. No single point mutation eliminated replication capacity completely, though many mutants were severely impaired, demonstrating an important functional contribution for the E1 binding site. Furthermore, E1 binding was not sufficient for replication, as several origin mutants bound E1 well in vitro but replicated poorly in vivo. This suggests that certain nucleotides within the 18-bp IR may be involved in postbinding events necessary for replication initiation. The results with the point mutations suggest that E1-E1 interactions are important for stable complex formation and also indicate that there is some flexibility with regard to formation of a functional E1 replication complex at the origin.

Adenovirus E1A Proteins↗
PubMed1995

Narrow complex tachycardias.

Narrow complex tachycardias are those cardiac rhythms with a ventricular rate of more than 100 beats per minute and a QRS complex width of less than 0.12 seconds. They originate either from the SA node, from atrial tissue itself, or from in or around the AV node. The term SVT is generally accurate for such tachycardias. By diagnosing the source of an SVT and appreciating its likely cause, therapy can be more precisely, safely, and effectively guided to treat these patients. Atrial and junctional rhythms can be treated with vagal maneuvers, drugs from classes I to IV and other antiarrythmic agents, magnesium, and cardioversion. Some patients may be candidates for surgical or catheter ablation.

Anti-Arrhythmia Agents↗
PubMed1994

The bovine papillomavirus type 1 genome contains multiple loci of static DNA bending, but bends are absent from the functional origin of replication.

Twenty-four overlapping restriction fragments spanning the entire bovine papillomavirus type 1 (BPV-1) genome were analyzed by electrophoresis to determine the extent of static DNA bending in the BPV-1 genome. Thirteen of 24 fragments contained static bends. Based on known locations of previously mapped bend loci and the overlapping pattern of these 13 fragments, we estimate that there are 8-11 distinct static bend loci in the BPV-1 genome. The bend loci were not uniformly distributed on the genome and with one exception, were clustered from nucleotides 5816 to 2621 on the BPV-1 map. This portion of the BPV-1 genome contains most of the transcriptional regulatory sequences as well as the origin of replication. The concordance between the genomic distribution of DNA bends and cis-active elements is consistent with the possibility that bent sequences may contribute to the function of at least some of these elements. However, unlike SV40, there was no static bend at that functional origin of replication for BPV-1. The nearest bends to the origin were approximately 120 bp to the 5' side and 300 bp to the 3' side. As both of these bends were outside of the sequences required for origin function, it is unlikely that static bending plays a critical role in BPV-1 replication.

Base Sequence↗
PubMed1994

DNA binding specificity of the bovine papillomavirus E1 protein is determined by sequences contained within an 18-base-pair inverted repeat element at the origin of replication.

Bovine papillomavirus type 1 (BPV-1) DNA replicates episomally and requires two virally expressed proteins, E1 and E2, for this process. Both proteins bind to the BPV-1 genome in the region that functions as the origin of replication. The binding sequences for the E2 protein have been characterized previously, but little is known about critical sequence requirements for E1 binding. Using a bacterially expressed E1 fusion protein, we examined binding of the BPV-1 E1 protein to the origin region. E1 strongly protected a 28-bp segment of the origin (nucleotides 7932 to 15) from both DNase I and exonuclease III digestion. Additional exonuclease III protection was observed beyond the core region on both the 5' and 3' sides, suggesting that E1 interacted with more distal sequences as well. Within the 28-bp protected core, there were two overlapping imperfect inverted repeats (IR), one of 27 bp and one of 18 bp. We show that sequences within the smaller, 18-bp IR element were sufficient for specific recognition of DNA by E1 and that additional BPV-1 sequences beyond the 18-bp IR element did not significantly increase origin binding by E1 protein. While the 18-bp IR element contained sequences sufficient for specific binding by E1, E1 did not form a stable complex with just the isolated 18-bp element. Formation of a detectable E1-DNA complex required that the 18-bp IR be flanked by additional DNA sequences. Furthermore, binding of E1 to DNA containing the 18-bp IR increased as a function of overall increasing fragment length. We conclude that E1-DNA interactions outside the boundaries of the 18-bp IR are important for thermodynamic stabilization of the E1-DNA complex. However, since the flanking sequences need not be derived from BPV-1, these distal E1-DNA interactions are not sequence specific. Comparison of the 18-bp IR from BPV-1 with the corresponding region from other papillomaviruses revealed a symmetric conserved consensus sequence, T-RY--TTAA--RY-A, that may reflect the specific nucleotides critical for E1-DNA recognition.

Base Sequence↗
PubMed1992

A second phenazine methosulphate-linked formate dehydrogenase isoenzyme in Escherichia coli.

A biochemical and immunological study has revealed a new formate dehydrogenase isoenzyme in Escherichia coli. The enzyme is an isoenzyme of the respiratory formate dehydrogenase (FDH-N) which forms part of the formate to nitrate respiratory pathway found in the organisms when it is grown anaerobically in the presence of nitrate. The new enzyme, termed FDH-Z, cross reacts with antibodies raised to FDH-N and possesses a similar polypeptide composition to FDH-N. FDH-Z catalyses the phenazine methosulphate-linked formate dehydrogenase activity present in the aerobically-grown bacterium. FDH-Z and FDH-N exhibit distinct regulation. Like formate dehydrogenase N, formate dehydrogenase Z is a membrane-bound molybdoenzyme. With nitrate reductase it can catalyse electron transfer between formate and nitrate. Quinones are required for the physiological electron transfer to nitrate. It seems likely that like FDH-N, FDH-Z functions physiologically as a formate: quinone oxidoreductase.

Electron Transport↗
PubMed1989

Nickel deficiency gives rise to the defective hydrogenase phenotype of hydC and fnr mutants in Escherichia coli.

Hydrogenase activity and other hydrogenase-related functions can be restored to hydC mutants by the specific addition of nickel salts to the growth medium. These mutants are defective in all three hydrogenase isoenzymes and the restoration is dependent upon protein synthesis. The cellular nickel content of the mutant when grown in LB medium is less than 1% of that of the parental strain. Partial suppression of the hydrogenase phenotype of hydC mutants occurs when growth takes place in a different medium. This correlates with an increased cellular nickel content. The phenotype of the mutant is also fully suppressed by growth in media of very low magnesium content. Such media facilitate nickel uptake via the magnesium transport system, which leads to the acquisition of a normal cellular nickel content. Mutations in the fnr gene, which encodes a transcriptional regulator for several anaerobically expressed enzymes, abolishes hydC expression and gives rise to a defective hydrogenase phenotype. The hydrogenase phenotype of fnr is closely similar to that of hydC in all respects examined. The hydrogenase activity of fnr strains can be restored by the presence of a functional hydC gene on a multicopy plasmid. The hydrogenase phenotype of fnr strains therefore arises indirectly via suppression of hydC, which leads to a low cellular nickel content. Nickel has no influence on fumarate reductase or nitrate reductase activities in fnr strains. The hydrogen-metabolism phenotype of fnr strains is, therefore, dependent upon their ability to acquire nickel from growth media. It is likely that hydC encodes a specific transport system for nickel.

Escherichia coli↗
PubMed1987

Cadmium resistance in Drosophila: a small cadmium binding substance.

A small cadmium-binding substance (CdBS) has been observed in adult Drosophila melanogaster that were raised for their entire growth cycle on a diet that contained 0.15 mM CdCl2. Induction of CdBS was observed in strains that differed widely in their sensitivity to CdCl2. This report describes the induction of CdBS and some of its characteristics.

Animals↗
PubMed1986

On the nature of the receptor mediating the action of 5-hydroxytryptamine in potentiating responses of the mouse urinary bladder strip to electrical stimulation.

Superfused mouse bladder strip responded to electrical stimulation (ES) by twitch contractions. These contractions were potentiated by 5-hydroxytryptamine (5-HT; 0.03-3.0 microM). Three tryptamine analogues were tested for their ability to potentiate the responses to ES and their relative activities were compared with values in the literature. 5-Carboxamidotryptamine was more potent than 5-HT, N-omega-methyl 5-HT was about equipotent, and alpha-methyl 5-HT was much less potent. Exogenous noradrenaline did not reduce the effect of 5-HT, although it reduced the response of the tissue to ES. The maximum increase in tissue tension evoked by 5-HT was unaltered by phentolamine (1 microM) or bretylium (25 microM). These results show that 5-HT did not act on the bladder by inhibiting the release of catecholamines from intrinsic nerves. The effect of 5-HT proved very resistant to attempts to block it with conventional 5-HT antagonists. Several antagonists were studied including ketanserin, methysergide, methiothepin, and MDL 72222 but even at concentrations as high as 1 microM none of them exhibited any marked inhibitory effect against the action of 5-HT. At 10 microM some compounds showed activity, but none abolished the effect except methiothepin - and its action was not surmountable by increasing the concentration of 5-HT. Thus the effect of 5-HT on the bladder was not mediated by 5-HT receptors of types 'M', 'D' or 5-HT2.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Evidence for purinergic transmission in mouse bladder and for modulation of responses to electrical stimulation by 5-hydroxytryptamine.

Electrical stimulation (ES) contracted superfused mouse bladder, and 10(-7) M tetrodotoxin (TTX) abolished the twitches without impairing responses to acetylcholine (ACh) or beta,gamma-methylene ATP. ES acted largely through nerves which were not cholinergic, adrenergic or histaminergic. They may be purinergic because the bladder was contracted by stable analogues of ATP, and after desensitisation by a high concentration of alpha,beta-methylene ATP the response to ES was selectively reduced. 5-Hydroxytryptamine (5-HT) at 0.03-3 X 10(-6) M and tetraethylammonium (TEA) at 0.1-10 X 10(-3) M potentiated responses to ES, on average by 64% and 182%. Pempidine had no effect on responses to ES. The action of TEA was different from that of 5-HT; potentiation of responses was greater than could be produced by 5-HT, and whereas 5-HT did not increase responses to ACh, TEA markedly increased twitch tensions. The mode of action of 5-HT is not clear.

Adenosine Triphosphate↗