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R V Lewis

Publications and source records attributed to R V Lewis.

At least 37 records · Page 2Linked to original sources
PubMed1992

Detection of proteins and phenol in DNA samples with second-derivative absorption spectroscopy.

We have employed near-uv second-derivative spectra of DNA, N-acetyl-L-tryptophanamide, N-acetyl-L-tyrosinamide, N-acetyl-L-phenylalanine ethyl ester, and phenol in a matrix least-squares multicomponent analysis algorithm to detect the presence of tryptophan, tyrosine, phenylalanine, and/or phenol in DNA preparations. With this method, each of these compounds can be detected in a DNA sample (absorbance, 0.1) at absorbance levels of less than 0.002. In practice, the presence of proteins can be detected at absorbance levels of less than 0.003. Using second-derivative spectra of proteins, contents of mixtures of proteins and DNA can be determined with less than 1% error. Mixtures of DNA and RNA can also be quantitatively analyzed with an error of approximately 2%. This technique can be easily implemented with computer-controlled spectrophotometers equipped with standard spectral analysis software. With prerecorded standard spectra, the time of analysis does not exceed a few seconds.

Animals↗
PubMed1992

Statistical determination of the average values of the extinction coefficients of tryptophan and tyrosine in native proteins.

Spectroscopic measurement of protein concentration requires knowledge of the value of the relevant extinction coefficient. If the amino acid composition of a protein is known, however, extinction coefficients can be calculated approximately, provided that the values of the molar absorptivities for tryptophan and tyrosine residues in the protein are known. We have applied a matrix linear regression procedure and a mapping of average absolute deviations between experimental and calculated values to find molar extinction coefficients (epsilon M, 1 cm, 280 nm) of 5540 M-1 cm-1 for tryptophan and 1480 M-1 cm-1 for tyrosine residues in an "average" protein, as defined by a set of experimentally determined extinction coefficients for more than 30 proteins. Use of these values provides a significant improvement in extinction coefficient estimation over that obtained with the commonly used values obtained from solutions of model compounds in guanidine-HCl. The consistency of these results when compared to the large deviations often observed between experimentally determined extinction coefficients suggest that this method may offer acceptable accuracy in the initial estimation of molar absorptivities of globular proteins.

Animals↗
PubMed1991

Examination of phenylalanine microenvironments in proteins by second-derivative absorption spectroscopy.

We have employed near ultraviolet derivative absorption spectroscopy to study the microenvironments of phenylalanine residues in proteins. The use of second-derivative uv spectra in the 250- to 270-nm range effectively suppresses spectral contributions from tryptophan and tyrosine residues. Fitting a polynomial to the numerically calculated second-derivative spectrum allows precise determination of the position of the negative derivative peak near 258 nm. This position is shown to be correlated with the polarity of the microenvironments of phenylalanine residues. This approach allows monitoring of changes in the state of phenylalanine side chains during folding/unfolding of the proteins. In addition, this method permits perturbation of protein samples with ethylene glycol to be used to establish the relative degree of solvent exposure of protein phenylalanine.

Animals↗
PubMed1991

Conformational stability of Cu,Zn-superoxide dismutase, the apoprotein, and its zinc-substituted derivatives: second-derivative spectroscopy of phenylalanine and tyrosine residues.

The relative stabilities of bovine copper-zinc superoxide dismutase (SOD), its apoprotein form, and zinc-substituted derivatives were investigated by denaturation in guanidine-HCl solutions. Analysis of the kinetics of changes in the second-derivative spectral bands of both phenylalanine and tyrosine residues was simultaneously performed. It was found that reduction of the cupric site increases the stability of the enzyme. The apoprotein appears to be the least stable form, while addition of zinc ions not only increases stability, but appears to induce a native-like conformation from a disordered form at pH 3.8. By perturbing the solvent with up to 20% ethylene glycol, at pH 6.8, it was determined that the only tyrosyl side chain appears to be about 50% solvent-exposed in the apoprotein, 65% exposed in the zinc derivative, and 75% exposed in the native copper-zinc form. In contrast, all four phenylalanine residues appear to be fully buried in all of these species in the mid-pH range. At pH 2.5, as the apoprotein unfolds, the apparent solvent-exposure of the tyrosyl side chain approaches 100%, while the phenylalanyl side chains become only 70% exposed. Substantial differences in the unfolding rate constants of tyrosine and phenylalanine residues of native and zinc-substituted SOD, but not the apoprotein, suggest the presence of metal-stabilized unfolding intermediates. Unfolding as monitored by the exposure of phenylalanine residues follows first-order kinetics, indicating that Phe 48 located at the interface between the two subunits is being exposed to the solvent simultaneously with the remaining three phenylalanine residues buried in the protein core.

Animals↗
PubMed1991

The interaction of gamma-crystallins with model surfaces.

Three biophysical techniques were employed to study the structure and thermal stability of a series of homologous bovine lens gamma-crystallins upon binding to three model surfaces. The surfaces in order of increasing hydrophobicity were silica, methyl silica, and diphenyl silica. Secondary structure was analyzed by deconvolution Fourier transform infrared spectroscopy, while tertiary structure alterations were probed by front surface fluorescence spectroscopy. The effect of surface binding on protein thermal stability was analyzed by fluorescence and differential scanning calorimetry. The comparison of free and surface-bound protein with variations in the electrostatic and hydrophobic character of both the protein and the adsorbent surface with these techniques demonstrated that: (i) destabilization on hydrophobic surfaces is greater than on a more hydrophilic interface, (ii) detectable conformational changes tend to increase as the hydrophobicity of the surface increases, and (iii) subtle structural differences among proteins can play an important role in determining differences in protein stability and structure upon surface adsorption.

Animals↗
PubMed1991

Molecular mechanism of spider silk elasticity.

Spider major ampullate (drag-line) silk is an extracellular fibrous protein which has impressive characteristics of strength and elasticity. This silk has been hypothesized to predominantly consist of a single protein, containing regions of antiparallel beta-sheets which are interspersed with amorphous segments responsible for its elastic properties. A rubber-like mechanism has been suggested to account for this elasticity, but the specific molecular mechanism is unknown. Using Fourier transform infrared spectroscopy (FTIR) we found evidence of either helix formation or reorientation of preexisting helices when axial tension is applied to the spider silk fiber. CD studies of a peptide derived from the silk gene repeat sequence show that it can form beta-sheets at high temperatures while alpha-helices are induced in 2,2,2-trifluoroethanol. These results suggest a possible molecular mechanism for the elasticity of spider silk fibers. It is proposed that the elastic process involves the formation and disruption of alpha-helical Ala-rich regions which are interspersed among stable beta-sheet domains.

Amino Acid Sequence↗
PubMed1991

Influence of debrisoquine oxidation phenotype on exercise tolerance and subjective fatigue after metoprolol and atenolol in healthy subjects.

1. The effects of single doses of metoprolol 50 mg, metoprolol 100 mg and atenolol 100 mg on exercise tolerance were compared with placebo in a double-blind random cross-over study in 12 healthy subjects. Nine subjects were extensive metabolisers of debrisoquine, and three were poor metabolisers. 2. Three hours after dosing beta-adrenoceptor blocker treatments significantly reduced exercise heart rate, prolonged time to complete exercise, and increased subjective fatigue measured by visual analogue scale. 3. Scores for subjective fatigue did not correlate with reduction in exercise heart rate or prolongation of exercise time. Exercise time prolongation was weakly but not significantly correlated with exercise heart rate reduction. 4. When compared with placebo, prolongation of exercise time and increased fatigue with metoprolol were not significantly related to debrisoquine oxidation phenotype or to the debrisoquine/4-hydroxydebrisoquine (D/4OH-D) ratio. 5. When metoprolol responses were compared with those for atenolol, changes in exercise time and fatigue scores were significantly related to oxidation phenotype. For metoprolol 100 mg, poor metabolisers required 20.8 s longer to complete exercise (P less than 0.05) and had higher fatigue scores by 78% (P less than 0.05) as compared with extensive metabolisers.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1990

Inhibition of alpha-crystallin aggregation by gamma-crystallin.

The transparency of the mammalian lens is primarily maintained by short range order among the major proteins of the lens fiber cells, the crystallins. Although these proteins are highly conserved at the amino acid sequence level, it has proven difficult to establish that they possess other than structural functions. We find that when non-lens proteins are added to concentrated solutions of alpha-crystallin, aggregation is induced, presumably through excluded volume effects. In contrast, the monomeric gamma-crystallins and the low molecular weight form of beta-crystallin (beta L) cause a decrease in the size of alpha-crystallin. When the naturally aggregated form of alpha-crystallin is examined, gamma- and beta L-crystallin, as well as a reducing agent, also cause partial dissociation as detected by dynamic light scattering and size exclusion chromatography, while no effect is seen with non-crystallin proteins. Furthermore, the chemical cross-linking of alpha-crystallin is inhibited by gamma- and beta L-crystallin but not by other proteins. The ability of gamma-crystallin to inhibit the association of alpha-crystallin is primarily localized to the gamma-II form which contains a high degree of exposed thiols. Only small amounts of gamma- and beta L-crystallin, however, can be cross-linked to alpha-crystallin in mixtures of the three proteins even at very high protein concentrations. These results suggest that one possible role for the lower molecular weight crystallins may be to minimize through a reductive effect the intrinsic tendency of alpha-crystallin to aggregate, an association reaction implicated in the loss of lens transparency.

Animals↗
PubMed1990

Presence of pancreatic trypsin inhibitor in adrenal medullary chromaffin cells.

A bovine pancreatic trypsin inhibitor was isolated from bovine adrenal medullary chromaffin granules. Its N-terminal sequence is: arg-pro-asp-phe-cys-leu-glu-pro-pro-tyr-thr-gly-pro-cys-lys-ala-arg-ile- arg-tyr- phe-tyr-asn-ala-lys-ala-gly-leu-cys-gln-thr-phe-val-tyr-gly-gly-cys-arg- ala-lys-arg-asn-asn-phe-lys- which corresponds precisely with the N-terminus of Bovine Pancreatic Trypsin inhibitor. The presence of this inhibitor in these granules suggests another method of regulating the prohormone proteases present there.

Adrenal Medulla↗
PubMed1990

Oral magnesium reduces ventricular ectopy in digitalised patients with chronic atrial fibrillation.

We have examined the effects of magnesium replacement therapy upon post-exercise heart rate and incidence of ventricular premature beats (VPB) in digitalised patients with AF. In 11 such patients, all of whom had serum magnesium concentrations of less than 0.85 mmol/l, treatment with magnesium glycerophosphate was associated with a significant reduction in number of VPBs (982 v. 416 VPB/24 h). Five patients had a high prevalence of ventricular ectopy (greater than 300 VPB/24 h) and these subjects showed particularly marked decreases in VPBs during magnesium treatment (1998 v. 690 VPB/24 h). Three patients had slightly increased QTc intervals but these did not change during magnesium replacement. No significant changes were seen in the mean post-exercise heart rate although 2 subjects did show falls of 25% or more during magnesium replacement. We conclude that treatment with magnesium glycerophosphate may be associated with a decreased prevalence of ventricular ectopy in some digitalised patients with chronic AF and mild-moderate hypomagnesaemia.

Aged↗
PubMed1990

Secondary structure characteristics of proenkephalin peptides E, B, and F.

The conformations of three adrenal medullary enkephalin containing polypeptides (ECPs) were investigated to gain an understanding of their potential structure-activity relationships. Secondary structure characteristics of peptides E, B, and F were examined by circular dichrosim (CD) under conditions designed to mimic both the soluble state and the anisotropic environment which exists at the biological effector site. Conformational differences between the three peptides were further examined by Fourier Transform Infrared Spectroscopy (FTIR) and by empirical predictions for conformation and hydrophobic periodicity. Although all three peptides have a similar structure, existing in random configurations in aqueous solutions, they do exhibit unique individual potentials to assume secondary structure in less polar environments. These conformational differences may be important factors in determining their unique individual biological activities.

Adrenal Medulla↗
PubMed1990

Structure of a protein superfiber: spider dragline silk.

Spider major ampullate (dragline) silk is an extracellular fibrous protein with unique characteristics of strength and elasticity. The silk fiber has been proposed to consist of pseudocrystalline regions of antiparallel beta-sheet interspersed with elastic amorphous segments. The repetitive sequence of a fibroin protein from major ampullate silk of the spider Nephila clavipes was determined from a partial cDNA clone. The repeating unit is a maximum of 34 amino acids long and is not rigidly conserved. The repeat unit is composed of three different segments: (i) a 6 amino acid segment that is conserved in sequence but has deletions of 3 or 6 amino acids in many of the repeats; (ii) a 13 amino acid segment dominated by a polyalanine sequence of 5-7 residues; (iii) a 15 amino acid, highly conserved segment. The latter is predominantly a Gly-Gly-Xaa repeat with Xaa being alanine, tyrosine, leucine, or glutamine. The codon usage for this DNA is highly selective, avoiding the use of cytosine or guanine in the third position. A model for the physical properties of fiber formation, strength, and elasticity, based on this repetitive protein sequence, is presented.

Amino Acid Sequence↗
PubMed1990

Putative N-terminal sequence of murine soluble immune response suppressor (SIRS): significant homology with short neurotoxin 1.

Soluble immune response suppressor (SIRS) is a low-molecular-weight protein (approximately 10,000 daltons) produced by mitogen- or interferon-activated T lymphocytes that can block development of humoral or cell-mediated immune responses in vivo or in vitro. As previously reported, murine SIRS is heterogeneous, eluting in two broad peaks on high performance reverse phase chromatography as well as displaying several broad isoelectric point forms. A putative N-terminal 21 amino acid sequence has been obtained for one of the less hydrophobic isoforms, SIRS-alpha 7. The sequence of SIRS-alpha 7 is unique in mammals but shows a remarkable homology to the family of short neurotoxins (short neurotoxin 1) found in sea snake, adder, and cobra species. A degenerate oligonucleotide probe based on the protein sequence was synthesized and was shown to hybridize to SIRS messenger RNA as measured by SIRS synthesis in a rabbit reticulocyte lysate system. A synthetic polypeptide based on the 21-residue sequence was also prepared and coupled to thyroglobulin or keyhole limpet hemocyanin. These were used to prepare rabbit antisera that neutralize SIRS bioactivity and precipitate authentic SIRS.

Amino Acid Sequence↗
PubMed1990

Central effects of the angiotensin-converting enzyme inhibitor, captopril. I. Performance and subjective assessments of mood.

1. Central effects of single doses of captopril (12.5, 25 and 50 mg) were studied in fourteen healthy male subjects. Two placebos and an active control drug, oxazepam (15 mg), were included, together with a single dose of atenolol (100 mg). The drugs were administered double-blind at 11.00 h, and performance and subjective feelings were assessed before and from 1.5-2.5 h and 3.5-4.5 h after ingestion. 2. Performance was assessed using digit symbol substitution, continuous attention, letter cancellation, choice reaction time, finger tapping, immediate and short-term memory, together with critical flicker fusion and two flash fusion. Subjects assessed their mood and well-being on a series of 12 visual analogue scales. 3. Captopril did not impair performance on any of the tests, but improved short-term memory (P less than 0.05) and increased the number of letters cancelled (P less than 0.05). Oxazepam reduced the number of substitutions completed in the digit symbol test (P less than 0.01), accuracy on continuous attention (P less than 0.05), number of letters cancelled (P less than 0.05), and rate of finger tapping (P less than 0.05), and increased choice reaction time (P less than 0.001). Atenolol reduced the rate of finger tapping (P less than 0.05), but increased the number of letters cancelled (P less than 0.05). 4. No effects on mood or on subjective feelings were evident with captopril. Oxazepam reduced subjective alertness (P less than 0.05), and atenolol increased feelings of sleepiness (P less than 0.05). 5. Although these observations suggest that central effects may exist with captopril, no adverse consequences have been established on performance or on subjective assessment of mood. Captopril may, therefore, be an appropriate drug for hypertensive patients engaged in skilled activity.

Adult↗
PubMed1990

Atrial fibrillation. The therapeutic options.

Atrial fibrillation (AF) is a common cardiac arrhythmia which is particularly prevalent among the elderly. In patients with AF of recent onset, restoration of sinus rhythm may be feasible and this can be achieved by DC cardioversion, or by the use of one of a number of drugs including amiodarone, flecainide or propafenone. Neither digoxin nor the calcium antagonists facilitate the restoration of sinus rhythm. Recurrence of AF is common after successful cardioversion and, although long term antiarrhythmic drug therapy may help to maintain sinus rhythm, all such drugs are potentially toxic and can have important proarrhythmic actions. In patients with chronic AF, restoration of sinus rhythm is rarely possible and treatment is directed towards control of the ventricular response rate, which may be achieved with digoxin and/or a rate-limiting calcium antagonist such as verapamil or diltiazem; beta-blockers may also be used although they appear to impair effort tolerance. In addition, long term anticoagulation may be indicated to reduce the risks of systemic embolisation, even in patients with 'nonrheumatic' AF; antiplatelet drugs are of no apparent value in this context. A minority of patients present with AF associated with ventricular pre-excitation; in these individuals both digoxin and the calcium antagonists are contraindicated and the ventricular response rate should be controlled with flecainide, amiodarone or propafenone.

Atrial Fibrillation↗
PubMed1989

Kinetics of chromium transformations in the environment.

Kinetics of chromium transformations under typical environmental conditions were systematically investigated using batch, microcosm and column experiments. Oxidation and reduction rates were evaluated in single phase water systems and in two phase water-solid systems. Natural and reference waters, sediments and soils representing a broad range of characteristics were used. Some of the investigated reactions, such as the reduction of Cr(VI) by S2- or Fe2+ ions under anaerobic conditions, were instantaneous and the experimental results exactly followed the stoichiometric and equilibrium calculations. Other reactions, such as the reduction of Cr(VI) by organics in sediments and soils, or the oxidation of Cr(III) by MnO2, were much slower and clearly exhibited kinetic controls. Reduction of Cr(VI) by organics was dependent on both the type and amount of organic material. Dissolved oxygen by itself did not induce measurable oxidation of Cr(III), spiked to the experimental waters, after 128 days. Slow Cr(III) oxidation to Cr(VI) was only noted in one of the natural waters and sediments with half-lives ranging from 2 to 9 years. In all cases the extent of Cr(III) oxidation did not exceed 15% of the initial Cr(III) present. The overall results indicate that the transport and transformation of chromium can be predicted using kinetics data derived from a simplified laboratory experiment, the Eh-pH data and the aquatic system characteristics. Results of the microcosm and laboratory experiments were in good agreement with the predicted concentrations.

Chemical Phenomena↗