The role of CRE1 in nucleosome positioning within the cbh1 promoter and coding regions of Trichoderma reesei.

Nucleosome positioning within the promoter and coding regions of the cellobiohydrolase-encoding cbh1 gene of Trichoderma reesei was investigated. T. reesei is a filamentous fungus that is able to degrade dead plant biomass by secreting enzymes such as cellulases, a feature which is exploited in industrial applications. In the presence of different carbon sources, regulation of one of these cellulase-encoding genes, cbh1, is mediated by various transcription factors including CRE1. Deletion or mutation of cre1 caused an increase in cbh1 transcript levels under repressing conditions. CRE1 was shown to bind to several consensus recognition sequences in the cbh1 promoter region in vitro. Under repressing conditions (glucose), the cbh1 promoter and coding regions are occupied by several positioned nucleosomes. Transcription of cbh1 in the presence of the inducer sophorose resulted in a loss of nucleosomes from the coding region and in the re-positioning of the promoter nucleosomes which prevents CRE1 from binding to its recognition sites within the promoter region. Strains expressing a non-functional CRE1 (in strains with mutated CRE1 or cre1-deletion) exhibited a loss of positioned nucleosomes within the cbh1 coding region under repressing conditions only. This indicates that CRE1 is important for correct nucleosome positioning within the cbh1 coding region under repressing conditions.

Yeast identification: reassessment of assimilation tests as sole universal identifiers.

AIMS: To assess whether assimilation tests in isolation remain a valid method of identification of yeasts, when applied to a wide range of environmental and spoilage isolates. METHODS AND RESULTS: Seventy-one yeast strains were isolated from a soft drinks factory. These were identified using assimilation tests and by D1/D2 rDNA sequencing. When compared to sequencing, assimilation test identifications (MicroLog™) were 18·3% correct, a further 14·1% correct within the genus and 67·6% were incorrectly identified. The majority of the latter could be attributed to the rise in newly reported yeast species. CONCLUSIONS: Assimilation tests alone are unreliable as a universal means of yeast identification, because of numerous new species, variability of strains and increasing coincidence of assimilation profiles. Assimilation tests still have a useful role in the identification of common species, such as the majority of clinical isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: It is probable, based on these results, that many yeast identifications reported in older literature are incorrect. This emphasizes the crucial need for accurate identification in present and future publications.

SOD1-targeted gene disruption in the ericoid mycorrhizal fungus Oidiodendron maius reduces conidiation and the capacity for mycorrhization.

The genome sequences of mycorrhizal fungi will provide new opportunities for studying the biology and the evolution underlying this symbiotic lifestyle. The generation of null mutants at the wild-type loci is one of the best methods for gene-function assignment in the post-genomic era. To our knowledge, the generation of superoxide dismutase 1 (SOD1)-null mutants in the ericoid mycorrhizal fungus Oidiodendron maius is the first example of a gene-targeted disruption via homologous recombination in a mycorrhizal fungus. The disruption of OmSOD1 by Agrobacterium-mediated transformation resulted in the presence of oxidative stress markers, even in the absence of external superimposed stresses, and an increased sensitivity to reactive oxygen species (ROS)-generating substances, especially to menadione. A reduction in conidiation and in the percentage of mycorrhization of Vaccinium myrtillus roots was also observed. The latter findings establish the pivotal role of SOD1 as an important factor in the relationship between O. maius and its symbiotic partner. The lack of this ROS-scavenger may cause an imbalance in the redox homeostasis during host colonization and an alteration in the delicate dialogue between the fungus and its host plant.

Modulation of chaperone gene expression in mutagenized Saccharomyces cerevisiae strains developed for recombinant human albumin production results in increased production of multiple heterologous proteins.

The yeast Saccharomyces cerevisiae has been successfully established as a commercially viable system for the production of recombinant proteins. Manipulation of chaperone gene expression has been utilized extensively to increase recombinant protein production from S. cerevisiae, focusing predominantly on the products of the protein disulfide isomerase gene PDI1 and the hsp70 gene KAR2. Here we show that the expression of the genes SIL1, LHS1, JEM1, and SCJ1, all of which are involved in regulating the ATPase cycle of Kar2p, is increased in a proprietary yeast strain, developed by several rounds of random mutagenesis and screening for increased production of recombinant human albumin (rHA). To establish whether this expression contributes to the enhanced-production phenotype, these genes were overexpressed both individually and in combination. The resultant strains showed significantly increased shake-flask production levels of rHA, granulocyte-macrophage colony-stimulating factor, and recombinant human transferrin.

Spore germination in Mortierella alpina is associated with a transient depletion of arachidonic acid and induction of fatty acid desaturase gene expression.

Mortierella alpina is an oleaginous filamentous fungus whose vegetative mycelium is known to accumulate triglyceride oil containing large amounts of arachidonic acid (ARA 20:4, n - 6). We report that the spores of Mortierella alpina also contain a large proportion of ARA, comprising 50% of total fatty acid. Fatty acid desaturase genes were not expressed in dormant spores but were induced during germination, following a significant drop in the level of ARA (down from 50% of total fatty acid to 12%) prior to germ-tube emergence. We propose that ARA serves as a reserve supply of carbon and energy that is utilised during the early stages of spore germination in Mortierella alpina.

UPR-independent dithiothreitol stress-induced genes in Aspergillus niger.

A subtraction library was prepared from cultures of Aspergillus niger that had or had not been exposed to dithiothreitol (DTT), in order to identify genes involved in the unfolded protein response (UPR) or in the response to reductive stress. A large fraction of the clones in the library (40%) encoded two putative methyltransferases (MTs) whose function has yet to be determined. Other stress-responsive genes included a homologue of the Mn2+-containing superoxide dismutase gene (sodB) and a number of genes predicted to code for products that function in protein turnover and in intra- and extracellular transport of molecules. Transcriptional microarray analysis was carried out with a group of 15 genes, comprising 11 from the cDNA library, two genes linked to the putative MT genes but not represented in the library, and two UPR control genes (bipA and pdiA). Eleven of the 15 genes were inducible with DTT. This was either reflected by the presence of transcripts in cells subjected to DTT stress compared to absence under control conditions, or by an induction ratio of between 1.4 and 8.0 in cases where transcripts were already detectable under control conditions. The MT genes were among the four most highly induced. None of the genes, apart from bipA and pdiA, showed significant induction in response to other stresses that are known to induce the UPR in fungi. We conclude that DTT alone does not provide for specific induction of UPR genes and that other stress conditions must also be examined.

A preliminary analysis of the process of protein secretion and the diversity of putative secreted hydrolases encoded in Aspergillus fumigatus: insights from the genome.

The genome sequence of Aspergillus fumigatus has enabled the annotation of genes likely to encode secreted enzymes that may be important in underpinning the natural lifestyle of the fungus and its pathogenicity. We summarize the data from the genome sequence relevant to both the process of protein secretion and the predicted hydrolase enzymes secreted by A. fumigatus.

Chloroquine causes lysosomal dysfunction in neural retina and RPE: implications for retinopathy.

Chronic use of chloroquine has been shown to induce numerous pathophysiological defects in the retina. This drug has the ability to alter pH of intracellular compartments and lysosomal function of the retinal pigment epithelium (RPE) and retinal neurons may constitute the basis of chloroquine retinopathy. The aim of the current study was to investigate pathogenic alterations in retinal cells continuously exposed to chloroquine using appropriate in vivo and in vitro models. Male hooded Lister rats were implanted with osmotic mini pumps which released chloroquine continuously over a period of seven days. The eyes were processed for electron microscopy and ultrastructural abnormalities determined in the neural retina and quantified using stereology in the retinal pigment epithelium (RPE). RPE were also exposed to chloroquine in vitro and lysosomal pH changes were investigated using a pH sensitive probe. Degradative capacity was also analysed using FITC labeled rod outer segments (ROS). Chloroquine-treated animals displayed several ultrastructural abnormalities including numerous membranous cytoplasmic bodies (MCBs) in retinal neurons. Cone photoreceptors displayed numerous MCBs although rods did not. The RPE of the treated groups all showed significantly higher numbers of lysosomal associated organelles (LAO) than the control group (p < 0.001). The in vitro experiments demonstrated chloroquine-mediated rises in lysosomal pH and an increase in lysosome/phagosome accumulation of ROS in the chloroquine treated group (p < 0.01). The current study demonstrates that chloroquine disrupts lysosomal function in retinal neurons and RPE. The evidence presented provides a clear pathogenic basis for the functional defects experienced by patients with chloroquine retinopathy.

In vitro stability and immunoreactivity of the native and recombinant plant food 2S albumins Ber e 1 and SFA-8.

BACKGROUND: The ability of an intact protein to reach the circulatory system may be a prerequisite to allergenicity and many allergens, particularly those from plant foods, have been found to be consistently more resistant to digestion by pepsin than other proteins. OBJECTIVE: This study assessed the pepsinolytic stability of native 2S albumins from Brazil nut and sunflower seed and their recombinant versions produced in Pichia pastoris. The physicochemical stability of native and recombinant Brazil nut 2S albumins and recombinant sunflower seed 2S albumin was also assessed. The immunoreactivity of native Brazil nut 2S albumin and recombinant 2S albumins was compared using serum from patients allergic to Brazil nuts and animals immunized with native 2S albumins. METHODS: Digestibility was measured in simulated gastric fluid followed by SDS-PAGE. Circular dichroism spectra were used to analyse unfolding, as proteins were denatured by temperature, pH and guanidinium chloride. Immunoreactivity was assessed by immunoblot, RAST and ELISA. RESULTS: Brazil nut 2S albumin was significantly more resistant to proteolytic digestion than other Brazil nut proteins. It was also resistant to thermally and chemically induced denaturation. Equally high resistance to proteolytic digestion was observed with sunflower seed 2S albumin. The recombinant albumins mirrored their native counterparts in stability and immunoreactivity. CONCLUSION: The important food allergen Brazil nut 2S albumin is as stable to digestion as is sunflower seed 2S albumin, whose allergenicity has yet to be determined. The 2S albumins and their recombinant counterparts could not be easily denatured by physicochemical treatments. The results suggest that 2S albumin is the only Brazil nut protein to reach the gut immune system intact. The production of properly folded recombinant proteins will facilitate mechanistic studies as well as diagnostic testing and antigen-based therapies.

Prevention of retinal capillary basement membrane thickening in diabetic dogs by a non-steroidal anti-inflammatory drug.

AIMS/HYPOTHESIS: To investigate the effect of treatment with the non-steroidal anti-inflammatory drug Sulindac on the early vascular pathology of diabetic retinopathy in the dog, and it's effect on recognised biochemical indices of hyperglycaemia-related pathophysiology. METHODS: Experimental diabetes (streptozotocin/alloxan) was induced in 22 male beagle dogs and 12 of the animals were assigned at random to receive oral Sulindac (10 mg/kg daily). Age- and sex-matched control animals were maintained as non-diabetic controls. After 4 years, several morphological parameters were quantified in the retinal microvasculature of each animal group using an established stereological method. Also, the following diabetes-associated biochemical parameters were analysed: accumulation of advanced glycation end products (AGEs), red blood cell polyol levels and antioxidant status. RESULTS: Diabetes increased red blood cell sorbitol levels when compared to non-diabetic controls (p< or =0.05), however, there was no difference in sorbitol levels between the untreated and the treated diabetic animals. No significant differences were found in red blood cell myoinositol levels between the three groups of animals. Pentosidine and other AGEs were increased two- to three-fold in the diabetic animals (p< or =0.001) although treatment with Sulindac did not affect their accumulation in diabetic skin collagen or alter diabetes-induced rises in plasma malondialdehyde. Retinal capillary basement membrane volume was significantly increased in the untreated diabetic dogs compared to non-diabetic controls or Sulindac-treated diabetic animals (p< or =0.0001). CONCLUSION/INTERPRETATION: This study has confirmed the beneficial effect of a non-steroidal anti-inflammatory drug on the early vascular pathology of diabetic retinopathy. However the treatment benefit was not dependent on inhibition of polyol pathway activity, advanced glycation, or oxidative stress.

Comparison of modular and non-modular xylanases as carrier proteins for the efficient secretion of heterologous proteins from Penicillium funiculosum.

Genes encoding three enzymes with xylanase activity from the filamentous fungus Penicillium funiculosum are described. Two of the encoded xylanases are predicted to be modular in structure with catalytic and substrate-binding domains separated by a serine and threonine-rich linker region; the other had none of these properties and was non-modular. In order to develop P. funiculosum as a host for the secreted production of heterologous proteins, each of the xylanases was assessed for use as a carrier protein in a fusion strategy. We show that one of the modular xylanases (encoded by xynA) was an effective carrier protein but the other (encoded by xynB) and the non-modular xylanase (encoded by xynC) were not effective as secretion carriers. We show that the beta-glucuronidase (GUS) protein from Escherichia coli is secreted by P. funiculosum when expressed as an XYNA fusion but that the secreted GUS protein, cleaved in vivo from XYNA, is glycosylated and enzymatically inactive.

Isolation and characterisation of a calnexin homologue, clxA, from Aspergillus niger.

We describe the isolation of a gene (clxA) encoding calnexin from laboratory and industrial strains of Aspergillus niger. Calnexin is a chaperone, which specifically recognises monoglucosylated glycoproteins in the endoplasmic reticulum, and is thus an essential component of the process that assesses the folded state of nascent secreted glycoproteins. Manipulation of chaperones has previously been adopted in attempts to overcome some of the problems associated with the secretion of heterologous proteins from filamentous fungi. The A. niger clxA gene encodes a 562-residue protein with strong homology to the calnexin of Schizosaccharomyces pombe. The clxAgene product complements a S. pombe cnx1 mutant. Motifs associated with genes controlled via the Unfolded Protein Response (UPR) were identified by sequence homology in the promoter of clxA. Steady-state levels of clxA mRNA were elevated in a strain expressing bovine prochymosin fused to the catalytic domain of glucoamylase. The ORF is punctuated by four introns, and contains two sets of four repeated peptide motifs that are characteristic of the calnexin family, together with a putative membrane-spanning domain. Deletion studies indicate that clxA is not an essential gene in A. niger.

Use of a histone H4 promoter to drive the expression of homologous and heterologous proteins by Penicillium funiculosum.

Two genes encoding histone H4 (H4.1 and H4.2) from Penicillium funiculosum have been cloned and characterised. Structurally, the histone H4.1 gene is divergently linked to the histone H3 gene and the two genes are separated by approximately 800 bp. The transcription of the histone H4.1 and H4.2 genes in P. funiculosum appears to be distinctively regulated. Histone H4.1 mRNA showed a high steady-state level during the early stages of batch culture that decreased as growth reached the stationary phase. In contrast, the expression of the histone H4.2 gene was lower than that of H4.1 throughout batch growth and increased gradually with time. In order to expand the industrial application of P. funiculosum as a host for the production of heterologous proteins, the promoter of the histone H4.1 gene was successfully used to drive the expression of an intracellular bacterial enzyme, beta-glucuronidase, and a secreted homologous enzyme, xylanase C. The constitutive secretion of xylanase C was achieved in the absence of other xylanases by batch fermentation in the presence of glucose.

Stability of recombinant 2 S albumin allergens in vitro.

Two well known 2 S albumins, Ber e 1 from brazil nut and sunflower 2 S albumin 8 (SFA-8), have been expressed in a eukaryotic system and purified. Analysis of recombinant versions of Ber e 1 and SFA-8 revealed them to be significantly more resistant to digestion by pepsin than BSA, and to be stable for up to 30 min in simulated gastric fluid. Unfolding monitored by CD indicated that both proteins were also very resistant to denaturation induced by heat and low pH. These results suggest that, although the ability of 2 S albumins to reach the circulatory system may be a prerequisite for the allergenicity of this group of proteins, stability is just one of a number of characteristics that provoke a selective immune response.

A defined level of protein disulfide isomerase expression is required for optimal secretion of thaumatin by Aspegillus awamori.

Thaumatin, a 22-kDa protein containing eight disulfide bonds, is secreted by the filamentous fungus Aspergillus awamori at levels which are dependent upon the extent of overexpression of protein disulfide isomerase (PDIA). Additional copies of the PDIA-encoding gene pdiA were introduced into a strain of A. awamori that expresses a cassette encoding thaumatin. Transformants with different levels of pdiA mRNA and measured PDIA levels were chosen for examination of the impact that PDIA levels had on thaumatin secretion. The secretion of two native proteins, alpha-amylase and acid phosphatase, was also examined in relation to varying levels of PDIA. Over a range of PDIA levels of 1-8, relative to the native level in strains with just one copy of the pdiA gene, the fraction of alpha-amylase and acid phosphatase in the total secreted protein was unaffected. In contrast, a peak level of thaumatin, about 5-fold higher than in the strain with one copy of pdiA, was found in strains with a relative PDIA level of between two and four. Improved thaumatin production was confirmed in 5-1 fermenters using a strain of A. awamori with six pdiA gene copies, containing 3.2-fold higher levels of PDIA than wild-type strains.

Ocular surface reconstruction in LOGIC syndrome by amniotic membrane transplantation.

PURPOSE: To determine whether preserved amniotic membrane can be used to reconstruct the ocular surface after excision of the invading granulation material typical of LOGIC syndrome (laryngeal and ocular granulation tissue in children from the Indian subcontinent). METHODS: Granulation tissue was dissected and excised from both eyes of a 10-year-old boy with LOGIC syndrome. This procedure was followed by coverage of the corneal, scleral, and subtarsal regions of each eye with amniotic membrane, which had been stored for 6 months at -70 degrees C. RESULTS: Initial 2.5-month follow up demonstrated complete disappearance of granulation tissue. The fornices were patent, there was no recurrence of symblepharon, ocular inflammation was suppressed, and the patient reported markedly increased comfort. Both eyelids remained ptotic because of levator muscle atrophy secondary to many years of inability to open either eye. No residual scarring or evidence of granulation tissue was observed in that period. The 10-month follow up demonstrated limited recurrence, particularly where there was an intraoperative break in the amniotic membrane. CONCLUSIONS: After 24 operations to treat the ocular complications induced by LOGIC syndrome, amniotic membrane transplantation was the first effective treatment. In the early follow up period (2-3 months), there was complete cessation of the proliferation of granulation tissue and reepithelialization of the corneal surface. Longer follow up (10 months) demonstrated limited recurrence, which will require retreatment.

Residual visual functions of guide dog owners in the UK.

Numbers of guide dog owners (GDOs) in the United Kingdom reached 4700 by the end of 1998. Despite this growing trend, little is known about the nature of their visual loss. This paper reports the results of a national three-centre investigation into the residual visual functions and ophthalmic conditions of guide dog owners. Random samples of GDOs (Scotland n = 82, England n = 77, and Northern Ireland n = 87) underwent a detailed visual analysis and interview. GDOs had an overall median age of 53 years. Nationally, they make up just 2.4% of the registered blind population. All GDOs were found to have profound loss of visual acuity, contrast sensitivity or visual fields, but only 43% were totally blind. GDOs in Scotland retained higher levels of residual visual function than those in the other two regions. The main causes of visual loss were congenital and early onset degenerative eye disease (retinitis pigmentosa 18%, optic atrophy 10%). Results taken in conjunction with epidemiological registration trends suggest that the past growth in numbers of GDOs is unlikely to be sustainable. Implications for mobility service providers are discussed. It is suggested that increased optometric input and a multidisciplinary approach could assist present and potential guide dog owners.

Protein-glutaminase from Chryseobacterium proteolyticum, an enzyme that deamidates glutaminyl residues in proteins. Purification, characterization and gene cloning.

A novel protein-deamidating enzyme was purified to homogeneity from Chryseobacterium proteolyticum and the gene encoding it was cloned. The enzyme is a monomer with a pI of 10.0, a measured M(r) of approximately 20,000 and a calculated M(r) of 19,860. Extensive comparison with Streptoverticillium transglutaminase showed that the protein-deamidating enzyme lacked transglutaminase activity in terms of hydroxamate-formation between benzyloxycarbonyl-Gln-Gly and hydroxylamine, or monodansylcadaverine incorporation into casein. The enzyme deamidated the two glutaminyl residues in the oxidized insulin A chain and deamidated both casein and the oxidized insulin B chain with higher catalytic efficiencies (k(cat)/K(m)) than with short peptides. The enzyme was active against several proteins, including insoluble wheat gluten, but did not deamidate asparaginyl residues in peptides, free glutamine or other amides. The enzyme was therefore named protein-glutaminase (EC 3.5.1). The gene encoding the protein was cloned and, when expressed in Escherichia coli, the protein product had protein-glutaminase activity and cross-reacted with antiserum raised against the purified enzyme. The protein-glutaminase was shown to be expressed as a prepro-protein with a putative signal peptide of 21 amino acids and a pro-sequence of 114 amino acids. The amino-acid sequence had no obvious homology to any published sequence and is therefore a novel protein-glutaminase.

Nerve pathology in the type 1 diabetic dog: effects of treatment with sulindac.

The purpose of this study was to define pathological abnormalities in the peripheral nerve of a large animal model of long-duration type 1 diabetes and also to determine the effects of treatment with sulindac. Detailed morphometric studies were performed to define nerve fiber and endoneurial capillary pathology in 6 control dogs, 6 type 1 diabetic dogs treated with insulin, and 6 type 1 diabetic dogs treated with insulin and sulindac for 4 years. Myelinated fiber and regenerative cluster density showed a non-significant trend toward a reduction in diabetic compared to control animals, which was prevented by treatment with sulindac. Unmyelinated fiber density did not differ among groups. However, diabetic animals showed a non-significant trend toward an increase in axon diameter (p < 0.07), with a shift of the size frequency distribution towards larger axons, which was not prevented by treatment with sulindac. Endoneurial capillary density and luminal area showed a non-significant trend toward an increase in diabetic animals, which was prevented with sulindac treatment. Endoneurial capillary basement membrane area was significantly increased (p < 0.05) in diabetic animals, but was not prevented with sulindac treatment. We conclude that the type 1 diabetic dog demonstrates minor structural abnormalities in the nerve fibers and endoneurial capillaries of the sciatic nerve, and treatment with sulindac ameliorates some but not all of these abnormalities.

A modular esterase from Penicillium funiculosum which releases ferulic acid from plant cell walls and binds crystalline cellulose contains a carbohydrate binding module.

An esterase was isolated from cultures of the filamentous fungus Penicillium funiculosum grown on sugar beet pulp as the sole carbon source. The enzyme (ferulic acid esterase B, FAEB) was shown to be a cinnamoyl esterase (CE), efficiently releasing hydroxycinnamic acids from synthetic ester substrates and plant cell walls, and bound strongly to microcrystalline cellulose. A gene fragment was obtained by PCR using partial amino-acid sequences obtained from the pure enzyme and used to a probe a P. funiculosum genomic DNA library. A clone containing a 1120-bp ORF, faeB, was obtained which encoded a putative 353-residue preprotein including an 18-residue signal peptide, which when expressed in Eschericia coli produced CE activity. Northern analysis showed that transcription of faeB was tightly regulated, being stimulated by growth of the fungus on sugar beet pulp but inhibited by free glucose. The faeB promoter sequence contains putative motifs for binding an activator protein, XLNR, and a carbon catabolite repressor protein, CREA. FAEB was comprised of two distinct domains separated by a 20 residue Thr/Ser/Pro linker region. The N-terminal domain comprised 276 amino acids, contained a G-X-S-X-G motif typical of serine esterases, and was shown to be a member of a family comprising serine esterases, including microbial acetyl xylan esterases, poly (3-hydroxyalkanoate) depolymerases and CEs, and proteins of unknown function from Mycobacterium spp. and plants. The C-terminal domain comprised 39 amino acids and closely resembled the family 1 cellulose binding carbohydrate-binding modules (CBM) of fungal glycosyl hydrolases. This is the first report of a fungal CE with a CBM.