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Biomedical subjects

R G Whittaker

Publications and source records attributed to R G Whittaker.

At least 19 recordsLinked to original sources
PubMed2006

Myotonic dystrophy: practical issues relating to assessment of strength.

BACKGROUND: Myotonic dystrophy type 1 is a slowly progressive multisystem disease in which skeletal muscle involvement is prominent. As novel physical and pharmacological treatments become available, it is crucial to be able to measure their efficacy accurately. METHODS: 158 consecutive patients with myotonic dystrophy were assessed annually in a specialist muscle clinic. Strength was measured using both the Medical Research Council (MRC) scale and a hand-held dynamometer. Dynamometer readings were obtained from 108 normal subjects (controls). RESULTS: The movements showing the greatest rate of change in strength were ankle dorsiflexion and pinch grip. Both of these showed a decline of only 0.06 points/year on the MRC scale. Using a hand-held dynamometer, a change in strength of 1.18 kgN/year for women and 1.61 kgN/year for men was detected. CONCLUSIONS: The MRC scale is unsuitable for detecting the small changes in strength seen in a slowly progressive disease such as myotonic dystrophy. Dynamometry provides a simple alternative that can give meaningful data over the duration of a typical clinical trial.

Adult↗
PubMed2000

Substrate preference profiles of proteases released by allergenic pollens.

BACKGROUND: Pollens are important triggers for allergic asthma and seasonal rhinitis. We have recently reported that proteases released by major allergenic pollens can injure airway epithelial cells in vitro. Disruption of epithelial integrity by proteases released following deposition of pollens on mucosal surfaces could promote sensitization and induce inflammation. OBJECTIVE: To compare protease activities released by allergenic pollens of various genera. METHODS: We used a rapid microassay which quantifies cleavage of dipeptide ester substrates to characterize the substrate preference profiles of serine proteases in diffusates of the pollens of perennial ryegrass (Lolium perenne), Kentucky blue grass (Poa pratensis), Bermuda grass (Cynodon dactylon), Western ragweed (Ambrosia spp.), white birch (Betula spp.) and Sydney golden wattle (Acacia longifolia). RESULTS: Comparison of the profiles revealed notable differences as well as similarities between serine protease activities released by these pollens. Diffusates of Kentucky blue grass pollen exhibited very high substrate preference for arginine and lysine. For other pollens, cleavage of the cysteine substrate was usually the most rapid and was associated with marked preference for leucine and methionine. There was considerable variation between these pollens in the rates of cleavage of the histidine substrate. In addition, we observed high rates of cleavage of arginine and lysine substrates by Acacia pollen diffusate. CONCLUSION: At least two dominant patterns of substrate preference are identifiable in the mixtures of proteases released by hydrated pollens. Purification of the proteases responsible for these patterns of activity will facilitate investigation of their role in airway epithelial injury and allergic disease.

Allergens↗
PubMed1999

A transfection compound series based on a versatile Tris linkage.

The family of cationic lipid transfection reagents described here demonstrates a modular design that offers potential for the ready synthesis of a wide variety of molecular variants. The key feature of these new molecules is the use of Tris as a linker for joining the hydrophobic domain to a cationic head group. The molecular design offers the opportunity to conveniently synthesise compounds differing in charge, the number and nature of hydrophobic groups in the hydrophobic domain and the characteristics of the spacer between the cationic and hydrophobic moieties. We show that prototype reagents of this design can deliver reporter genes into cultured cells with efficiencies rivaling those of established cationic lipid transfection reagents. A feature of these reagents is that they are not dependent on formulation with a neutral lipid for activity.

Animals↗
PubMed1998

Efficacy of fusion peptide homologs in blocking cell lysis and HIV-induced fusion.

Contrary to earlier reports, we have found that tri- and hexapeptides analogous or homologous with segments of the 23-residue N-terminal fusion sequence (FS) of the viral transmembrane glycoprotein gp41 (residues 517-539) did not significantly inhibit HIV-1-induced syncytium formation, using an uninfected cell-infected cell fusion assay. In contrast, we found that the high molecular weight apolipoprotein A-1 and a 23-residue analog of the FS, with the phenylalanine residues at positions 524 and 527 replaced with alanine residues, were effective inhibitors. Although the tripeptides were ineffective as inhibitors of syncytium formation, we found a number of them inhibited red cell lysis induced by the synthetic peptide AVGIGALFLGFLGAAGSTMGARS (based on the HIV-1 gp41 FS). This effect was also seen with apolipoprotein A-1. The Ala524,527 analog of the fusion sequence could not be tested in this system because it was hemolytic. We concluded that the smaller peptides were effective inhibitors of hemolysis because they interfered with pore formation by the fusion sequence peptide, either by disrupting the pores or by preventing the peptide from adopting the alpha-helical conformation found in the pores. On the other hand, membrane fusion, which is a prelude to syncytium formation, has been shown to require the fusion sequence in the beta-strand conformation. We argue that small peptides would be unable to block interaction between such strands, although larger molecules, such as apolipoprotein A-1 and the Ala524,527 analog, would be able to do so and thus inhibit fusion. It seems, therefore, that a successful drug directed against the FS-cell membrane interaction stage of syncytium formation would need to be of relatively high molecular weight and complexity.

Giant Cells↗
PubMed1994

A microtiter plate assay for the characterization of serine proteases by their esterase activity.

The action of serine (and cysteine) proteases on peptide esters proceeds, as a generalization, orders of magnitude faster than the corresponding enzymatic hydrolysis of peptide bonds or peptide amides. Esterolysis liberates an alcohol while generating a free carboxyl group on the peptide; the proton produced can be detected by the use of an appropriate indicator. The action of trypsin on benzyloxycarbonylalanylarginine methyl ester was used as a model for the development of a simple microtiter plate assay procedure that takes advantage of the speed of these reactions and the ease of detection afforded by the color change of the indicator. A family of ester substrates of the form benzyloxycarbonylalanyl-X-methyl ester, in which X is one of the 20 common amino acids, was synthesized to allow the determination of the primary specificity profiles of serine proteases. Using a 96-well microtiter plate the specificity profiles of four enzymes with all 20 substrates can be carried out in approximately 4 h per enzyme, including setting up and data processing. The primary substrate preferences of trypsin, chymotrypsin, thrombin, pancreatic elastase, alpha-lytic protease, subtilisin, and proteinase K were determined to demonstrate the method and were found to be in good general agreement with reported specificities established by more conventional means.

Chymotrypsin↗
PubMed1994

Purification and characterization of S1 mutants of alpha-lytic protease having altered catalytic properties.

A procedure is described for purifying alpha-lytic protease and its mutants from culture supernatants of recombinant Escherichia coli. The method affords substantial amounts (approx. 80 mg) of homogeneous enzyme. We compared the cleavage preferences of wild-type alpha-lytic protease and of mutants containing the substitutions Ala190 ("parent"), Ala190/Val192/His213/Met218 (mutant 1), Ala190/His213/Leu218 (mutant 9), and Ala190/Thr213/Leu218 (mutant 55), and for each enzyme we found broad agreement between the results obtained with synthetic ester and amide substrates. Kinetic constants were determined for the purified enzymes using selected tetrapeptide p-nitroanilide substrates. Mutant 55 had broad specificity and high activity. In terms of kcat/Km it cleaved at Met and Phe residues two to three times as effectively as the Ala190 enzyme and cleaved at Ala 7 times more effectively than the wild-type protease. The Ala190/His213 enzymes showed a preference for cleavage at His and Met residues. Not only were their kcat values for cleavage at His increased (in relation to the Ala190 enzyme) by an order of magnitude, but they also exhibited large decreases in kcat/Km for cleavage at other residues; for example, the value for cleavage at Phe was 400- to 600-fold lower. Mutant 9 cleaved a recombinant IGF-II fusion protein at a unique His residue and also at a nearby Asn residue.

Amides↗
PubMed1994

A new library of alpha-lytic protease S1 mutants generated by combinatorial random substitution.

Four positions in the S1 site of alpha-lytic protease (positions 190, 192, 213 and 218) were subjected to targeted random mutagenesis. In the resulting library we found active mutant proteases whose cleavage preferences could be grouped into three distinct families. Some potentially useful enzymes (such as one that prefers to cleave at Asn and Cys residues) were identified. In addition, we discuss instances where it was possible to relate changes in substrate specificity to alterations in the structure of the substrate binding site.

Amino Acid Sequence↗
PubMed1993

Random mutagenesis of the substrate-binding site of a serine protease can generate enzymes with increased activities and altered primary specificities.

In the past, several point mutations have been introduced individually into the substrate-binding site of alpha-lytic protease (EC 3.4.21.12) and shown to affect its specificity in a predictable manner [Bone, R., Silen, J. L., & Agard, D. A. (1989) Nature 339, 191-195]. One of the resulting mutant enzymes (Met190Ala in the numbering system of Fujinaga et al.) [Fujinaga, M., Delbaere, L. T. J., Brayer, G. D., & James, M. N. G. (1985) J. Mol. Biol. 183, 479-502] cleaves at large hydrophobic residues. We chose this enzyme as the parent for a library of mutant proteases. The library was constructed by effecting combinatorial random substitution of up to four other residues (Gly191, Arg192, Met213, and Val218) thought likely to influence the primary specificity of the protease. Active enzymes in the library were screened with a range of synthetic substrates (encompassing 19 different amino acids in the P1 position) in order to evaluate their primary cleavage preferences. The amino acid sequences of active mutants revealed a strong preference for the replacement of Met213 with a His residue. This substitution also had the greatest observed effect on specificity, conferring a greatly increased and, in some cases, dominant ability to cleave at His residues in synthetic amide substrates. Mutant enzymes with greatly increased proteolytic activity were also found in the library.

Amino Acid Sequence↗
PubMed1991

Health screening in elderly Oklahomans.

A total of 550 males and 457 females in their 60s and 70s were screened for height and weight, blood pressure, glucose, cholesterol, and hemoglobin. Statistical analysis was performed using SAS software. Male values were abnormal for all screening parameters except for cholesterol. Statistically significant lower hemoglobin in males suggests that blood loss may be a problem, and in males increases in body weight and glucose may herald a higher frequency of cardiovascular disease. Control of blood pressure, weight reduction, decreased consumption of fat and salt, and regular exercise may be the health imperatives in this group of elderly Oklahomans.

Aged↗
PubMed1987

Effect of Eperythrozoon ovis on the lysis of sheep erythrocytes in the complement fixation test.

When erythrocytes from sheep experimentally infected with Eperythrozoon ovis were used in the titration of reagents for a standardised complement fixation test, increased amounts of both haemolysin and complement were required for erythrocyte lysis compared with preinfection titrations. The haemolysin requirement increased by up to 125% at 55 days post-infection and complement requirement increased by up to 40% at 40 days post-infection. These changes appeared to correlate with the development of a macrocytic anaemia in affected sheep rather than E. ovis parasitaemia. The results emphasise the need to carefully monitor the haematological parameters of sheep used as sources of erythrocytes for the complement fixation test.

Anaplasmataceae Infections↗
PubMed1985

The acquired immune deficiency syndrome and related complex. A report of 2 confirmed cases in Cape Town with comments on human T-cell lymphotropic virus type III infections.

We report a case of acquired immune deficiency syndrome (AIDS) and one of AIDS-related complex presenting in Cape Town. The first patient was probably infected in the USA. In turn he infected the second patient by regular homosexual contact. Human T-cell lymphotropic virus type III (HTLV-III) was cultured, we believe for the first time in Africa, from the peripheral blood lymphocytes and a lymph node of our patient with AIDS. HTLV-III infection and high-risk groups in South Africa are discussed in comparison with those in the USA. It is suggested that HTLV-III infection and AIDS will increasingly affect women. Prevention of the spread of HTLV-III infection and AIDS is discussed in relation to close medical surveillance and the protection of blood and blood products from contamination. Counselling of patients with AIDS and persons infected with HTLV-III, general health education, and the protection of health care staff are important in preventing spread but beyond the scope of this article.

Acquired Immunodeficiency Syndrome↗
PubMed1984

Malaria 1984. Part II. Drug-resistant malaria.

Despite the one-time hope that malaria would be eradicated, this disease is today still the world's most common severe parasitic disease involving man. The emergence of drug-resistant malaria, especially that due to Plasmodium falciparum, in 1961 therefore caused great concern, particularly among those seeking to control this scourge. Initially P. falciparum species were resistant to a varying degree to chloroquine. Subsequently chloroquine resistance has been followed by resistance to almost all antimalarial agents to which the organism has been exposed by man. Guidance in the diagnosis and treatment of chloroquine-resistant malaria is outlined, as well as the difficulties involved. Further problems which accompany pregnancy complicated by drug-resistant malaria are briefly mentioned. Ways of approaching and possibly retarding the development of drug-resistant malaria are discussed.

Animals↗
PubMed1980

A mechanism for influenza subtype disappearance.

We propose that changes in the antigenic region/s of influenza haemagglutinin can also cause changes in the binding site for host cells. If these changes have an overall deleterious effect on virus binding and hence on viral infectivity and reproduction one has a simple mechanism to explain the limited time span of influenza subtypes. Evidence is presented from the published results of other workers to support this hypothesis.

Epitopes↗