Cloning of a Phenol Oxidase Gene from Acremonium murorum and Its Expression in Aspergillus awamori

Fungal multicopper oxidases have many potential industrial applications, since they perform reactions under mild conditions. We isolated a phenol oxidase from the fungus Acremonium murorum var. murorum that was capable of decolorizing plant chromophores (such as anthocyanins). This enzyme is of interest in laundry-cleaning products because of its broad specificity for chromophores. We expressed an A. murorum cDNA library in Saccharomyces cerevisiae and subsequently identified enzyme-producing yeast colonies based on their ability to decolor a plant chromophore. The cDNA sequence contained an open reading frame of 1,806 bp encoding an enzyme of 602 amino acids. The phenol oxidase was overproduced by Aspergillus awamori as a fusion protein with glucoamylase, cleaved in vivo, and purified from the culture broth by hydrophobic-interaction chromatography. The phenol oxidase is active at alkaline pH (the optimum for syringaldazine is pH 9) and high temperature (optimum, 60°C) and is fully stable for at least 1 h at 60°C under alkaline conditions. These characteristics and the high production level of 0.6 g of phenol oxidase per liter in shake flasks, which is equimolar with the glucoamylase protein levels, make this enzyme suitable for use in processes that occur under alkaline conditions, such as laundry cleaning.     

Heat inactivation of staphylococcus epidermidis at various water activities.

Members of the family Micrococcaceae play an important role in food spoilage and even in food poisoning. In contrast to members of the family Enterobacteriaceae, these bacteria can grow in media with low water activities. Therefore, the heat resistance of Staphylococcus epidermidis, a rather resistant member of the family Micrococcaceae, was studied at water activities between 0.87 and 1.00. The heat inactivation curves were clearly biphasic at all temperatures and water activities tested. Especially at low water activities, the D-values of the tail phase were extremely high (at 0.87 water activity, a D-value at 70 degrees C of 500 s was recorded).     

Identification and grouping of Clostridium botulinum strains by numerical analysis of their electrophoretic protein patterns

Strains of Clostridium botulinum type A, type E and both non-proteolytic and proteolytic types B and F were characterized by their electrophoretic protein patterns. As the protein pattern changes during sporulation, special attention was paid to the prevention of sporulation by selecting an appropriate medium (Strasdine's medium plus 1% w/v glucose) and a scheme of repeated subculturing. Ribosomal proteins, evolutionarily conservative and hence relatively similar in all types of bacteria, were removed to optimize the resolving power of the electrophoretic technique. Protein patterns were compared by computing correlation coefficients of normalized densitometric tracings. The method is highly reproducible and its resolving power is high: all protein patterns found were specific. The strains tested fall into two main groups: the proteolytic and the non-proteolytic cluster. Type A strains form a separate subgroup within the proteolytic cluster, the same applies to type E strains within the non-proteolytic group. Although time-consuming for spore-forming bacteria, this method is, to our knowledge, the only technique that recognizes individual strains of Cl. botulinum. For non-spore-forming micro-organisms the method is certainly much simpler and hence even more valuable.     

Rapid identification of target genes for 3-methyl-1-butanol production in Saccharomyces cerevisiae

Extracellular conditions determine the taste of fermented foods by affecting metabolite formation by the micro-organisms involved. To identify targets for improvement of metabolite formation in food fermentation processes, automated high-throughput screening and cDNA microarray approaches were applied. Saccharomyces cerevisiae was cultivated in 96-well microtiter plates, and the effects of salt concentration and pH on the growth and synthesis of the fusel alcohol-flavoured substance, 3-methyl-1-butanol, was evaluated. Optimal fermentation conditions for 3-methyl-1-butanol concentration were found at pH 3.0 and 0% NaCl. To identify genes encoding enzymes with major influence on product formation, a genome-wide gene expression analysis was carried out with S. cerevisiae cells grown at pH 3.0 (optimal for 3-methyl-1-butanol formation) and pH 5.0 (yeast cultivated under standard conditions). A subset of 747 genes was significantly induced or repressed when the pH was changed from pH 5.0 to 3.0. Expression of seven genes related to the 3-methyl-1-butanol pathway, i.e. LAT1, PDX1, THI3, ALD4, ILV3, ILV5 and LEU4, strongly changed in response to this switch in pH of the growth medium. In addition, genes involved in NAD metabolism, i.e. BNA2, BNA3, BNA4 and BNA6, or those involved in the TCA cycle and glutamate metabolism, i.e. MEU1, CIT1, CIT2, KDG1 and KDG2, displayed significant changes in expression. The results indicate that this is a rapid and valuable approach for identification of interesting target genes for improvement of yeast strains used in industrial processes.     

A review of recent non-target toxicity testing of vertebrate pesticides: establishing generic guidelines

Abstract

An analysis of the range, extent and importance of New Zealand-based ecotoxicology studies has been completed to better understand the hazards and non-target risks associated with new toxins and baits. This review focuses on compounds that have been recently developed for incorporation into bait for terrestrial vertebrate pest control, namely cholecalciferol, para-aminopropiophenone (PAPP) and zinc phosphide and the effects of these toxins on non-target species. Testing of these compounds has included cage and pen toxicity and bait acceptance studies. Locally conducted acute toxicity studies clarify overseas data and enhance risk assessments. Consistency in approach and selection of surrogate species, and careful selection of native non-target species for acute toxicity testing in cage and pen trials are recommended as important steps before field trials. We propose a systematic approach to cage and pen trials and offer guidelines on species selection.     

Remission in psoriatic arthritis: is it possible and how can it be predicted?

Introduction

Since remission is now possible in psoriatic arthritis (PsA) we wished to examine remission rates in PsA patients following anti tumour necrosis factor alpha (TNFα) therapy and to examine possible predictors of response.

Methods

Analysis of a prospective patient cohort attending a biologic clinic, between November 2004 and March 2008, was performed prior to commencing therapy and at regular intervals. Baseline clinical characteristics including demographics, previous disease-modifying antirheumatic drug (DMARD) response, tender and swollen joint counts, early morning stiffness, pain visual analogue score, patient global assessment, C reactive protein (CRP) and health assessment questionnaire (HAQ) were collected.

Results

A total of 473 patients (152 PsA; 321 rheumatoid arthritis (RA)) were analyzed. At 12 months remission, defined according to the disease activity score using 28 joint count and CRP (DAS28-CRP), was achieved in 58% of PsA patients compared to 44% of RA patients, significant improvement in outcome measures were noted in both groups (P < 0.05). Analysis of a subgroup of PsA and RA patients matched for DAS28-CRP at baseline also showed higher numbers of PsA patients achieving remission. Linear regression analysis identified the HAQ at baseline as the best predictor of remission in PsA patients (P < 0.001).

Conclusions

DAS28 remission is possible in PsA patients at one year following anti-TNF therapy, at higher rates than in RA patients and is predicted by baseline HAQ.     

Llama antibody fragments recognizing various epitopes of the CD4bs neutralize a broad range of HIV-1 subtypes A, B and C

Many of the neutralising antibodies, isolated to date, display limited activities against the globally most prevalent HIV-1 subtypes A and C. Therefore, those subtypes are considered to be an important target for antibody-based therapy. Variable domains of llama heavy chain antibodies (VHH) have some superior properties compared with classical antibodies. Therefore we describe the application of trimeric forms of envelope proteins (Env), derived from HIV-1 of subtype A and B/C, for a prolonged immunization of two llamas. A panel of VHH, which interfere with CD4 binding to HIV-1 Env were selected with use of panning. The results of binding and competition assays to various Env, including a variant with a stabilized CD4-binding state (gp120(Ds2)), cross-competition experiments, maturation analysis and neutralisation assays, enabled us to classify the selected VHH into three groups. The VHH of group I were efficient mainly against viruses of subtype A, C and B'/C. The VHH of group II resemble the broadly neutralising antibody (bnmAb) b12, neutralizing mainly subtype B and C viruses, however some had a broader neutralisation profile. A representative of the third group, 2E7, had an even higher neutralization breadth, neutralizing 21 out of the 26 tested strains belonging to the A, A/G, B, B/C and C subtypes. To evaluate the contribution of certain amino acids to the potency of the VHH a small set of the mutants were constructed. Surprisingly this yielded one mutant with slightly improved neutralisation potency against 92UG37.A9 (subtype A) and 96ZM651.02 (subtype C). These findings and the well-known stability of VHH indicate the potential application of these VHH as anti-HIV-1 microbicides.     

Llama-derived single domain antibodies to build multivalent, superpotent and broadened neutralizing anti-viral molecules

For efficient prevention of viral infections and cross protection, simultaneous targeting of multiple viral epitopes is a powerful strategy. Llama heavy chain antibody fragments (VHH) against the trimeric envelope proteins of Respiratory Syncytial Virus (Fusion protein), Rabies virus (Glycoprotein) and H5N1 Influenza (Hemagglutinin 5) were selected from llama derived immune libraries by phage display. Neutralizing VHH recognizing different epitopes in the receptor binding sites on the spikes with affinities in the low nanomolar range were identified for all the three viruses by viral neutralization assays. By fusion of VHH with variable linker lengths, multimeric constructs were made that improved neutralization potencies up to 4,000-fold for RSV, 1,500-fold for Rabies virus and 75-fold for Influenza H5N1. The potencies of the VHH constructs were similar or better than best performing monoclonal antibodies. The cross protection capacity against different viral strains was also improved for all three viruses, both by multivalent (two or three identical VHH) and biparatopic (two different VHH) constructs. By combining a VHH neutralizing RSV subtype A, but not subtype B with a poorly neutralizing VHH with high affinity for subtype B, a biparatopic construct was made with low nanomolar neutralizing potency against both subtypes. Trivalent anti-H5N1 VHH neutralized both Influenza H5N1 clade1 and 2 in a pseudotype assay and was very potent in neutralizing the NIBRG-14 Influenza H5N1 strain with IC(50) of 9 picomolar. Bivalent and biparatopic constructs against Rabies virus cross neutralized both 10 different Genotype 1 strains and Genotype 5.The results show that multimerization of VHH fragments targeting multiple epitopes on a viral trimeric spike protein is a powerful tool for anti-viral therapy to achieve "best-in-class" and broader neutralization capacity.     

The β-1,6-glucan containing side-chain of cell wall proteins of Saccharomyces cerevisiae is bound to the glycan core of the GPI moiety

Abstract Cell wall proteins of Saccharomyces cerevisiae are anchored by means of a β-1,6-glucan-containing side-chain. It is not known whether this chain is linked to the protein part (e.g. through carbohydrate side-chains) or to the glycosylphosphatidylinositol (GPI) moiety of cell wall proteins. An IgA protease recognition site was introduced in Cwp2p, a β-1,6-glucosylated cell wall protein, immediately N-terminal from the omega amino acid (the attachment site of the GPI moiety). Proteolytic cleavage of this site revealed that the β-1,6-glucan epitope was not linked to the protein part. We conclude that neither N - or O -glycosylation is involved in β-glucosylation of cell wall proteins. This confirms that the glycan core of the GPI moiety is the probable β-1,6-glucan attachment site.