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Biomedical subjects

M P Robertson

Publications and source records attributed to M P Robertson.

At least 19 recordsLinked to original sources
PubMed2001

Optimization and optimality of a short ribozyme ligase that joins non-Watson-Crick base pairings.

A small ribozyme ligase (L1) selected from a random sequence population appears to utilize non-Watson-Crick base pairs at its ligation junction. Mutational and selection analyses confirmed the presence of these base pairings. Randomization of the L1 core and selection of active ligases yielded highly active variants whose rates were on the order of 1 min(-1). Base-pairing covariations confirmed the general secondary structure of the ligase, and the most active ligases contained a novel pentuple sequence covariation. The optimized L1 ligases may be optimal within their sequence spaces, and minimal ligases that span less than 60 nt in length have been engineered based on these results.

Base Pairing↗
PubMed2001

Concentration by evaporation and the prebiotic synthesis of cytosine.

The efficient prebiotic synthesis of cytosine from urea and cyanoacetaldehyde (CA) has recently been claimed to be invalid on the basis of possible side reactions of the starting materials and the inapplicability of prebiotic syntheses using drying beach conditions. We therefore have investigated the synthesis of cytosine and uracil from urea and cyanoacetaldehyde at 100 degrees C under dry-down conditions, and in solution at 4 degrees C and -20 degrees C. We find that cytosine is produced from the low temperature experiments more efficiently than calculated from the Arrhenius extrapolation from higher temperatures, i.e., 60-120 degrees C. In addition, we find that CA dimer is as efficient as the monomer in cytosine synthesis. We also studied whether evaporating very dilute solutions of nonvolatile organic compounds will concentrate according to theory. Solutions as dilute as 10(-4) M concentrate from pure water approximately according to theory. Similar solutions in 0.5 M NaCl have less than theoretical concentrations due to absorption, but concentrations near dryness were very high.

Acetaldehyde↗
PubMed2001

In vitro selection of nucleoprotein enzymes.

Natural nucleic acids frequently rely on proteins for stabilization or catalytic activity. In contrast, nucleic acids selected in vitro can catalyze a wide range of reactions even in the absence of proteins. To augment selected nucleic acids with protein functionalities, we have developed a technique for the selection of protein-dependent ribozyme ligases. After randomizing a previously selected ribozyme ligase, L1, we selected variants that required one of two protein cofactors, a tyrosyl transfer RNA (tRNA) synthetase (Cyt18) or hen egg white lysozyme. The resulting nucleoprotein enzymes were activated several thousand fold by their cognate protein effectors, and could specifically recognize the structures of the native proteins. Protein-dependent ribozymes can potentially be adapted to novel assays for detecting target proteins, and the selection method's generality may allow the high-throughput identification of ribozymes capable of recognizing a sizable fraction of a proteome.

Base Sequence↗
PubMed2000

Design and optimization of effector-activated ribozyme ligases.

A selected ribozyme ligase, L1, has been engineered to respond to small organic effectors. Residues important for ribozyme catalysis were mapped to a compact core structure. Aptamers that bound adenosine and theophylline were appended to the core structure, and the resultant aptazymes proved to be responsive to their cognate effectors. Rational sequence substitutions in the joining region between the aptamer and the ribozyme yielded aptazymes whose activities were enhanced from 800-1600-fold in the presence of 1 mM ATP or theophylline, respectively. However, when an anti-flavin aptamer was appended to the core ribozyme structure flavin-responsivity was minimal. The joining region between the aptamer and the ribozyme core was randomized and a series of negative and positive selection steps yielded aptazymes that were activated by up to 260-fold in the presence of 100 microM FMN. The selected joining regions proved to be 'communication modules' that could be used to join other aptamers to the ribozyme core to form aptazymes. These results show that ribozyme ligases can be readily engineered to function as allosteric enzymes, and reveal that many of the techniques and principles previously demonstrated during the development of hammerhead aptazymes may be generalizable.

Base Sequence↗
PubMed2000

In vitro selection of nucleic acids for diagnostic applications.

In vitro selection methods have proven to be extraordinarily adept at generating a wide variety of nucleic acid-binding species (aptamers) and catalysts (ribozymes). To date, selected nucleic acids have primarily been of academic interest. However, just as antibodies have proven utility as 'universal receptors' that can be crafted against a huge variety of ligands and can be readily adapted to diagnostic assays, aptamers may yet find application in assays. A new class of research reagents, aptazymes, are not mere mimics of antibodies but in fact allow the direct transduction of molecular recognition to catalysis. Aptamers and aptazymes may prove to be uniquely useful for the development of chip arrays for the detection and quantitation of a wide range of molecules in organismal proteomes and metabolomes.

Animals↗
PubMed1999

In vitro selection of an allosteric ribozyme that transduces analytes to amplicons.

We have selected an allosteric ribozyme ligase from a random sequence population that is activated up to 10,000-fold by oligonucleotide effectors. The ribozyme conforms to a classic two-state model for allostery in which the equilibrium between inactive and active conformers is dramatically altered by the presence of effector ligands. In the presence of the effector the allosteric ribozyme ligase generates templates that can subsequently be amplified using conventional amplification technologies, such as RT-PCR. Thus, the allosteric ribozyme can transduce (or convert) analytes into amplicons. We demonstrate two potential diagnostic applications of the selected allosteric ribozyme ligase: 'counting' short oligonucleotide effectors by RT-PCR, and counting a non-nucleic acid effector, ATP, by ligation.

Allosteric Site↗
PubMed1997

Ribozymes: red in tooth and claw.

A population of catalytic RNA molecules has been engineered to operate and evolve in vitro in a continuous manner. The novel continuity of the process allows the propagation of many generations in a very short time and without the manual manipulation necessary with traditional in vitro selection techniques.

Catalysis↗
PubMed1996

Prebiotic synthesis of diaminopyrimidine and thiocytosine.

The reaction of guanidine hydrochloride with cyanoacetaldehyde gives high yields (40-85%) of 2,4-diaminopyrimidine under the concentrated conditions of a drying lagoon model of prebiotic synthesis, in contrast to the low yields previously obtained under more dilute conditions. The prebiotic source of cyanoacetaldehyde, cyanoacetylene, is produced from electric discharges under reducing conditions. The effect of pH and concentration of guanidine hydrochloride on the rate of synthesis and yield of diaminopyrimidine were investigated, as well as the hydrolysis of diaminopyrimidine to cytosine, isocytosine, and uracil. Thiourea also reacts with cyanoacetaldehyde to give 2-thiocytosine, but the pyrimidine yields are much lower than with guanidine hydrochloride or urea. Thiocytosine hydrolyzes to thiouracil and cytosine and then to uracil. This synthesis would have been a significant prebiotic source of 2-thiopyrimidines and 5-substituted derivatives of thiouracil, many of which occur in tRNA. The applicability of these results to the drying lagoon model of prebiotic synthesis was tested by dry-down experiments where dilute solutions of cyanoacetaldehyde, guanidine hydrochloride, and 0.5 M NaCl were evaporated over varying periods of time. The yields of diaminopyrimidine varied from 1 to 7%. These results show that drying lagoons and beaches may have been major sites of prebiotic syntheses.

Acetaldehyde↗
PubMed1995

Rates of decomposition of ribose and other sugars: implications for chemical evolution.

The existence of the RNA world, in which RNA acted as a catalyst as well as an informational macromolecule, assumes a large prebiotic source of ribose or the existence of pre-RNA molecules with backbones different from ribose-phosphate. The generally accepted prebiotic synthesis of ribose, the formose reaction, yields numerous sugars without any selectivity. Even if there were a selective synthesis of ribose, there is still the problem of stability. Sugars are known to be unstable in strong acid or base, but there are few data for neutral solutions. Therefore, we have measured the rate of decomposition of ribose between pH 4 and pH 8 from 40 degrees C to 120 degrees C. The ribose half-lives are very short (73 min at pH 7.0 and 100 degrees C and 44 years at pH 7.0 and 0 degrees C). The other aldopentoses and aldohexoses have half-lives within an order of magnitude of these values, as do 2-deoxyribose, ribose 5-phosphate, and ribose 2,4-bisphosphate. These results suggest that the backbone of the first genetic material could not have contained ribose or other sugars because of their instability.

Biological Evolution↗
PubMed1995

An efficient prebiotic synthesis of cytosine and uracil.

In contrast to the purines, the routes that have been proposed for the prebiotic synthesis of pyrimidines from simple precursors give only low yields. Cytosine can be synthesized from cyanoacetylene and cyanate; the former precursor is produced from a spark discharge in a CH4/N2 mixture and is an abundant interstellar molecule. But this reaction requires relatively high concentrations of cyanate (> 0.1 M), which are unlikely to occur in aqueous media as cyanate is hydrolysed rapidly to CO2 and NH3. An alternative route that has been explored is the reaction of cyanoacetaldehyde (formed by hydrolysis of cyanoacetylene) with urea. But at low concentrations of urea, this reaction produces no detectable quantities of cytosine. Here we show that in concentrated urea solution--such as might have been found in an evaporating lagoon or in pools on drying beaches on the early Earth--cyanoacetaldehyde reacts to form cytosine in yields of 30-50%, from which uracil can be formed by hydrolysis. These reactions provide a plausible route to the pyrimidine bases required in the RNA world.

Acetaldehyde↗
PubMed1995

Prebiotic synthesis of 5-substituted uracils: a bridge between the RNA world and the DNA-protein world.

Under prebiotic conditions, formaldehyde adds to uracil at the C-5 position to produce 5-hydroxymethyluracil with favorable rates and equilibria. Hydroxymethyluracil adds a variety of nucleophiles, such as ammonia, glycine, guanidine, hydrogen sulfide, hydrogen cyanide, imidazole, indole, and phenol, to give 5-substituted uracils with the side chains of most of the 20 amino acids in proteins. These reactions are sufficiently robust that, if uracil had been present on the primitive Earth, then these substituted uracils would also have been present. The ribozymes of the RNA world would have included many of the functional groups found in proteins today, and their catalytic activities may have been considerably greater than presently assumed.

Amino Acids↗
PubMed1991

Ketanserin in the treatment of peripheral vascular disease or hypertension in patients with diabetes mellitus: a review.

The available data on the use of ketanserin in diabetes mellitus have been reviewed. Ketanserin does not worsen glucose tolerance or destabilize diabetic control. Ketanserin can improve impairment of peripheral blood flow, and especially skin blood flow; the healing of cutaneous ulcers is improved; trophic skin changes are fewer; and there is a trend towards less limb amputations. Walking distance in diabetic claudicants is unaltered by ketanserin. Controlled trials in hypertensive diabetics have provided variable results, with a significant fall in arterial pressure in only one of five studies. As in nondiabetics, ketanserin generally lowers serum cholesterol. The electrocardiographic QTc interval is prolonged. Symptomatic side effects in diabetics are as those in nondiabetic subjects.

Adult↗
PubMed1989

Comparison of enalapril and atenolol in mild to moderate hypertension.

PURPOSE: Short-term therapy with angiotensin converting enzyme (ACE) inhibitors for hypertension is effective and well tolerated, and compared with beta blockers, may cause fewer adverse reactions. Furthermore, enalapril has been observed to have a greater effect on systolic blood pressure than beta blockers. We therefore decided to compare the ACE inhibitor enalapril and the beta blocker atenolol as monotherapy in a double-blind study of patients with mild to moderate hypertension. PATIENTS AND METHODS: After a four-week placebo run-in period, 162 patients were allocated randomly to receive atenolol (50 to 100 mg daily) or enalapril (20 to 40 mg daily) for 12 weeks. To assess the influence of these drugs on quality of life, a series of psychologic tests was performed, and a subset of patients also underwent treadmill exercise and pulmonary function tests. RESULTS: In 147 patients who completed the study, enalapril reduced supine blood pressure by 19/12 mm Hg, compared with 9/7 mm Hg for atenolol (p less than 0.001/p less than 0.005). The modest blood pressure response to atenolol was not due to poor compliance. A target blood pressure of 140/90 mm Hg or less was achieved by 35 percent of enalapril-treated atenolol (p less than 0.01). The frequency and severity of adverse effects with the two drugs were similar, and few important differences emerged from the quality-of-life assessments. CONCLUSION: At the doses used, enalapril induced a greater short-term blood pressure response than atenolol; long-term studies of its safety and efficacy are required.

Adolescent↗
PubMed1985

A comparison of atenolol and long-acting trimazosin in mild to moderate essential hypertension.

In a 12-week double-blind randomised study the efficacy of atenolol and a new longer-acting formulation of trimazosin were compared when given once daily in patients with mild to moderate hypertension. Two parallel groups, each consisting of 18 patients, were studied. At randomisation the two groups were well matched for age and sex distribution. They were also well matched for blood pressure, pulse rate and body weight; these latter measurements were recorded at regular intervals during the 12 weeks of study. Atenolol produced substantial reduction in both systolic and diastolic blood pressure, and in heart rate, during 12 weeks of treatment. This therapeutic effect was maintained until the next dose after 24 hours. Trimazosin, by comparison, failed to reduce either systolic or diastolic pressure, or to alter heart rate. Side effects were minor with both agents and compliance with treatment was good. Atenolol caused significant elevation of plasma concentration of triglyceride, with reduction in high density lipoprotein concentration when compared with trimazosin. In conclusion, atenolol was confirmed as an effective agent for the treatment of mild to moderate hypertension. By comparison trimazosin in the longer-acting formulation was ineffective in this study. However, trimazosin may still find a place in treatment if used at higher dose.

Adolescent↗
PubMed1984

"Third drug" trial: comparative study of antihypertensive agents added to treatment when blood pressure remains uncontrolled by a beta blocker plus thiazide diuretic.

Hydralazine, labetalol, methyldopa, minoxidil, prazosin, and placebo were compared when added by random allocation to atenolol 100 mg and bendrofluazide 5 mg daily in a series of 238 hypertensive patients inadequately controlled by the beta blocker-diuretic combination. Atenolol was withdrawn in those allocated to labetalol, and minoxidil was given only to men. The order of acceptability was: placebo, hydralazine, prazosin, methyldopa, minoxidil, labetalol. Minoxidil was more effective than the other active drugs, which had similar potency to one another. All the active agents were more effective than placebo. Hydralazine was the most generally suitable third drug, with prazosin a close second. Minoxidil was especially effective in patients with less severe hypertension but the same regimen caused fluid retention in those with more severe disease. Labetalol should probably be introduced at a low dose (150 mg daily) even when replacing full doses of a previously administered beta blocker.

Adolescent↗