Directed evolution of a fungal peroxidase.

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.

DNA shuffling of subgenomic sequences of subtilisin.

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.

Directed evolution of microbial oxidative enzymes.

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.

A comparative study of beclomethasone dipropionate aqueous nasal spray with terfenadine tablets in seasonal allergic rhinitis.

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.

Subjective tinnitus in children with otological disorders.

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.

Current conservative treatment of childhood chronic secretory otitis media (a survey and discussion).

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.

Relief of severe hyperacusis and diplacusis in a deafened ear by cochlear labyrinthectomy.

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.

Lingual abscess: the value of ultrasound in diagnosis.

A case of lingual abscess is presented. The sparsity of physical signs made ultrasound imaging an invaluable investigation in the diagnosis.

Primary cholesteatoma of the mastoid presenting as cervical abscess.

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.

Transient quadriplegia following re-puncture of tracheo-oesophageal fistula.

We present a case of a laryngectomized patient who underwent re-puncture of tracheo-oesophageal fistula for speaking valve voice restoration, resulting in a previously unreported inherent complication of this procedure: extradural abscess of the cervical spine with transient quadriplegia.

Overexpression of the yeast transcriptional activator ADR1 induces mutation of the mitochondrial genome.

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.

Double-blind cross-over trial comparing beclomethasone dipropionate and terfenadine in perennial rhinitis.

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.

Characterization of Tobacco Expressing Functional Oat Phytochrome : Domains Responsible for the Rapid Degradation of Pfr Are Conserved between Monocots and Dicots.

Constitutive expression of a chimeric oat phytochrome gene in tobacco (Nicotiana tabacum) results in the accumulation of a functional 124-kilodalton photoreceptor that markedly alters the phenotype of light-grown tobacco (Keller et al. [1989] EMBO J 8: 1005-1012). Here, we provide a detailed phenotypic and biochemical characterization of homozygous tobacco expressing high levels of oat phytochrome. Phenotypic changes include a substantial inhibition of stem elongation, decreased apical dominance, increased leaf chlorophyll content, and delayed leaf senescence. Oat phytochrome synthesized in tobacco is indistinguishable from that present in etiolated oats, having photoreversible difference spectrum maxima at 665 and 730 nanometers, exhibiting negligible dark reversion of phytochrome-far red-absorbing form (Pfr) to phytochrome-red-absorbing form (Pr), and existing as a dimer with an apparent size of approximately 300 kilodaltons. Heterodimers between the oat and tobacco chromoproteins were detected. Endogenous tobacco phytochrome and transgenically expressed oat phytochrome are rapidly degraded in vivo upon photoconversion of Pr to Pfr. Breakdown of both oat and tobacco Pfr is associated with the accumulation of ubiquitin-phytochrome conjugates, suggesting that degradation occurs via the ubiquitin-dependent proteolytic pathway. This result indicates that the factors responsible for selective recognition of Pfr by the ubiquitin pathway are conserved between monocot and dicot phytochromes. More broadly, it demonstrates that the domain(s) within a plant protein responsible for its selective breakdown can be recognized by the degradation machinery of heterologous species.

Cloning of the Aspergillus oryzae 5-aminolevulinate synthase gene and its use as a selectable marker.

The hemA gene encoding 5-aminolevulinate synthase, the first enzyme in heme biosynthesis, was cloned from Aspergillus oryzae and evaluated as a selectable marker for the transformation of filamentous fungi. Deletion of the hemA gene resulted in a lethal phenotype that could be rescued either by the supplementation of culture media with 5-aminolevulinic acid (ALA) or by transformation with the wild-type hemA gene, but not by growth on rich media, nor by the addition of exogenous heme. Transformation of a hemA deletion strain with the hemA gene linked to a lipase expression cassette yielded ALA prototrophs expressing lipase. The hemA gene can therefore be used as a selectable marker for the transformation of A. oryzae.

Phytochrome requires the 6-kDa N-terminal domain for full biological activity.

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.