Biomedical subjects
Publications and source records attributed to J R Cherry.
In the past year, a number of oxidative enzymes have been the target of directed evolution. Catalase reaction specificity has been shifted to peroxidase, the high pH, thermal and oxidative stability of a fungal peroxidase has been dramatically improved, and the substrate specificity of cytochrome P450 has been altered to include substrates that the wild-type enzymes are incapable of oxidizing.
Novamyl is a thermostable five-domain maltogenic alpha-amylase that shows sequence and structural homology with the cyclodextrin glycosyltransferases (CGTases). Comparing X-ray crystal structures of Novamyl and CGTases, two major differences in the active site cleft were observed: Novamyl contains a loop insertion consisting of five residues (residues 191-195) and the location of an aromatic residue known to be essential to obtain an efficient cyclization reaction. To convert Novamyl into a cyclodextrin (CD)-producing enzyme, the loop was deleted and two substitutions, F188L and T189Y, were introduced. Unlike the parent Novamyl, the obtained variant is able to produce beta-CD and showed an overall conversion of starch to CD of 9%, compared with CGTases which are able to convert up to 40%. The lower conversion compared with the CGTase is probably due to additional differences in the active site cleft and in the starch-binding E domain. A variant with only the five-residue loop deleted was not able to form beta-CD.
DNA family shuffling of 26 protease genes was used to create a library of chimeric proteases that was screened for four distinct enzymatic properties. Multiple clones were identified that were significantly improved over any of the parental enzymes for each individual property. Family shuffling, also known as molecular breeding, efficiently created all of the combinations of parental properties, producing a great diversity of property combinations in the progeny enzymes. Thus, molecular breeding, like classical breeding, is a powerful tool for recombining existing diversity to tailor biological systems for multiple functional parameters.
The Coprinus cinereus (CiP) heme peroxidase was subjected to multiple rounds of directed evolution in an effort to produce a mutant suitable for use as a dye-transfer inhibitor in laundry detergent. The wild-type peroxidase is rapidly inactivated under laundry conditions due to the high pH (10.5), high temperature (50 degrees C), and high peroxide concentration (5-10 mM). Peroxidase mutants were initially generated using two parallel approaches: site-directed mutagenesis based on structure-function considerations, and error-prone PCR to create random mutations. Mutants were expressed in Saccharomyces cerevisiae and screened for improved stability by measuring residual activity after incubation under conditions mimicking those in a washing machine. Manually combining mutations from the site-directed and random approaches led to a mutant with 110 times the thermal stability and 2.8 times the oxidative stability of wild-type CiP. In the final two rounds, mutants were randomly recombined by using the efficient yeast homologous recombination system to shuffle point mutations among a large number of parents. This in vivo shuffling led to the most dramatic improvements in oxidative stability, yielding a mutant with 174 times the thermal stability and 100 times the oxidative stability of wild-type CiP.
A professional musician with intolerable hyperacusis and dysharmonic diplacusis in a severely deafened ear was successfully relieved of his symptoms by deliberate destruction of the cochlea.
The N-terminus of phytochrome A is important for the structural integrity and biological activity of the photoreceptor. Mutational analysis of the N-terminus by two different strategies created two distinct photoreceptors, one inactive and the other hyperactive when expressed in transgenic tobacco, suggesting that this region has multiple functional domains. To identify critical residues within this N-terminal region, a series of smaller deletions of oat phytochrome A were created, designated NB (delta49-62), NC (delta6-47), ND (delta7-21), NE (delta2-5), and NF (delta6-12), and compared with a previously characterized deletion mutant NA (delta7-69) and full-length oat phytochrome A. Using photochemical properties as a measure of chromoprotein structure, it was found that the region between residues 13 and 62 was important for the spectral integrity of the photoreceptor. These deletion mutants were also biologically inactive when expressed in both mature tobacco plants and seedlings grown under continuous far-red or red light. In contrast, deletion of the serine-rich region between residues 6 and 12 did not alter the photochemical properties but did produce a hyperactive photoreceptor, indicating this region may be involved in down-regulating phytochrome A activity. The data show that the N-terminus of phytochrome A contains two functional domains, one necessary for conformational stability and biological activity (residues 13-62), and the other involved in attenuating phytochrome responses (residues 6-12).
A series of seven carboxy-terminal deletion mutants of oat phytochrome A were stably expressed in transgenic tobacco to localize phytochrome domains involved in chromophore attachment, spectral integrity, photoreversibility between the red light (Pr)- and far-red light (Pfr)-absorbing forms, dimerization, and biological activity. Amino acids necessary for chromophore attachment in vivo were localized to the amino-terminal 398 residues because mutant proteins this small had covalently bound chromophore. Deletion mutants from the carboxy terminus to residue 653 were spectrally indistinguishable from the full-length chromoprotein. In contrast, further truncation to residue 399 resulted in a chromoprotein with a bleached Pfr absorbance spectrum, Pr and Pfr absorbance maxima shifted toward shorter wavelengths, and reduced Pfr to Pr phototransformation efficiency. Thus, residues between 399 ad 652 are required for spectral integrity but are not essential for chromophore attachment. The sequence(s) between residues 919 and 1093 appears to be necessary for dimerization. Carboxy-terminal mutants containing this region behaved as dimers under nondenaturing conditions in vitro, whereas truncations without this region behaved as monomers. None of the plants expressing high levels of deletion mutants lacking the 35 carboxy-terminal amino acids displayed the light-exaggerated phenotype characteristic of plants expressing biologically active phytochrome A, even when the truncated phytochromes were expressed at levels 6- to 15-fold greater than that effective for the full-length chromoprotein. Collectively, these data show that the phytochrome protein contains several separable carboxy-terminal domains required for structure/function and identify a domain within 35 residues of the carboxy terminus that is critical for the biological activity of the photoreceptor in vivo.
Phytochrome is a red/far-red-absorbing photoreceptor that controls many aspects of plant photomorphogenesis. Because proteolytic removal of approximately 6 kDa from the N terminus of 124-kDa oat phytochrome substantially alters many physicochemical properties of the chromoprotein, it has been proposed that the N terminus is required for biological activity. Here we test this hypothesis by comparing tobacco plants expressing full-length oat phytochrome (FL) with plants expressing a 118-kDa oat phytochrome lacking amino acids 7-69 (NA phytochrome). NA phytochrome, like its FL counterpart, exists as a homodimer in solution, is capable of covalently binding chromophore to form a red/far-red-photoreversible product, and is rapidly degraded in vivo after photoconversion to the far-red-absorbing form. However, like proteolytically degraded phytochrome missing the N terminus, the absorption maxima of the red- and far-red-light-absorbing forms of NA phytochrome are blue shifted relative to the maxima of the FL chromoprotein, and the rate of dark reversion of the far-red- to red-light-absorbing form is substantially increased. Tobacco plants producing high levels of NA phytochrome do not exhibit the light-exaggerated phenotype characteristic of FL phytochrome overexpression. By comparison of phytochrome-dose-phenotype-response curves generated by using a series of transgenic lines expressing various levels of FL or NA phytochrome, we demonstrate that NA phytochrome has less than 1/5th the biological activity of FL phytochrome expressed in tobacco. Furthermore, the shape of the dose-response curve for plants expressing FL phytochrome indicates that there is a sharp transition between phenotypically normal and abnormal plants over a relatively narrow range of phytochrome content, demonstrating that precise control of phytochrome levels is critical to photomorphogenesis.
It has been proposed in several eukaryotic systems that the regulation of gene transcription involves phosphorylation of specific transcription factors. We report here that the yeast transcriptional activator ADR1 is phosphorylated in vitro by cyclic AMP-dependent protein kinase and that mutations which enhance the ability of ADR1 to activate ADH2 expression decrease ADR1 phosphorylation. We also show that increased kinase activity in vivo inhibits ADH2 expression in an ADR1 allele-specific manner. Our data suggest that glucose repression of ADH2 is in part mediated through a cAMP-dependent phosphorylation-inactivation of the ADR1 regulatory protein.
It was previously observed that increased dosages of the ADR1 gene, which encodes a yeast transcriptional activator required for alcohol dehydrogenase II (ADH II) expression, cause a decreased rate of growth in medium containing ethanol as the carbon source. Here we show that observed reduction in growth rate is mediated by the ADR1 protein which, when overexpressed, increases the frequency of cytoplasmic petites. Unlike previously characterized mutations known to potentiate petite formation, the ADR1 effect is dominant, with the petite frequency rising concomitantly with increasing ADR1 dosage. The ability of ADR1 to increase the frequency of mitochondrial mutation is correlated with its ability to activate ADH II transcription but is independent of the level of ADH II being expressed. Based on restoration tests using characterized mit- strains, ADR1 appears to cause non-specific deletions within the mitochondrial genome to produce rho- petites. Pedigree analysis of ADR1-overproducing strains indicates that only daughter cells become petite. This pattern is analogous to that observed for petite induction by growth at elevated temperature and by treatment with the acridine dye euflavine. One strain resistant to ADR1-induced petite formation displayed cross-resistance to petite mutation by growth at elevated temperature and euflavine treatment, yet was susceptible to petite induction by ethidium bromide. These results suggest that ADR1 overexpression disrupts the fidelity of mitochondrial DNA replication or repair.
Primary cholesteatoma has been described in a number of sites within the temporal bone. We report an unusual case of primary cholesteatoma, confined to the mastoid, presenting with Bezold's abscess of the anterior cervical triangle, in an otherwise asymptomatic elderly man with normal hearing.
A case of lingual abscess is presented. The sparsity of physical signs made ultrasound imaging an invaluable investigation in the diagnosis.
Eighteen patients with perennial rhinitis were evaluated in this double-blind cross-over trial comparing beclomethasone dipropionate (BDP) aqueous nasal spray with terfenadine tablets. Both treatments were effective in reducing symptom scores but BDP was significantly better than terfenadine in relieving running nose and sneezing (P less than 0.05). BDP also had a greater effect on reducing nasal inflammation than terfenadine. Although the clinicians and patients assessed both therapies to be equi-effective, significantly more patients preferred the BDP treatment (P less than 0.003). Overall, BDP therapy proved more beneficial than terfenadine therapy in this small group of perennial rhinitis sufferers.
A questionnaire on medical treatment of childhood secretory otitis was posted to all Consultant Otologists in the Thames Regions of the National Health Service. Questions dealt with indications for medical management, choice of treatment, and indications for omitting medical therapy in favour of immediate surgery, or giving no treatment at all. 86 questionnaires were completed. 81.4 per cent of respondents used medical treatment, but there was no consensus as to the drug or combination preferred. Oral decongestant preparations were the most popular. 87.1 per cent of users of medical therapy sometimes omitted it in favour of immediate surgery, most commonly because previous treatment by another practitioner appeared a failure. 63.1 per cent of all respondents left some cases completely untreated. The views of experienced otologists on the treatment of 'glue ear' vary widely, and there is no acceptable evidence to support any method of medical therapy. Large-scale trials are needed to clarify the position.
Forty-nine patients participated in a randomized double-blind, parallel group comparison of beclomethasone dipropionate aqueous nasal spray with terfenadine tablets in the treatment of hay fever. Symptom scores for nasal and ocular symptoms as well as grass pollen counts were registered daily for at least 1 month. Evaluation of daily symptom scores and the physicians' and patients' assessments of treatment demonstrated that both treatments were effective in controlling the symptoms of hay fever, with a similar incidence of side-effects. The beclomethasone dipropionate group, in general, had lower nasal symptom scores than the terfenadine group and this reached statistical significance on high pollen count days. In contrast, the terfenadine group had lower eye symptom scores than the other group and these were statistically significant during the first half of the study period. However, the use of additional medication for control of eye symptoms was similar in both groups. It is concluded that treatment with both beclomethasone dipropionate and terfenadine throughout the season was effective in controlling hay fever symptoms, but beclomethasone dipropionate is likely to provide better overall control since it prevented breakthrough of troublesome nasal symptoms during high pollen count days.
Children attending ENT and audiology clinics and a further group interviewed in the partially hearing units of their schools have been questioned about tinnitus. A total of 79 children reported tinnitus during the course of the study. A wide variety of descriptions were given. Only 10 of the subjects found the symptom annoying. Only 17 of them could say when their tinnitus began. Of a group of 66 children with secretory otitis media, 43.9% reported tinnitus, while amongst 44 children with sensorineural hearing loss the incidence was 29.5%. Tinnitus is a common phenomenon in children with ear disease, but it is seldom reported spontaneously.
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