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D B Archer

Publications and source records attributed to D B Archer.

At least 37 records · Page 2Linked to original sources
PubMed2000

A glucoamylase::GFP gene fusion to study protein secretion by individual hyphae of Aspergillus niger.

Although Aspergillus niger is used as a host for heterologous protein production, yields are generally lower than those obtained for homologous proteins. Mechanisms of protein secretion and the secretory pathway in filamentous fungi are poorly characterised, although there is evidence to suggest that secretion occurs by a mechanism similar to that in other eukaryotes, but with proteins destined for secretion being directed to the hyphal tip. We report on a method using a glucoamylase: GFP gene fusion which allows us for the first time to monitor, in vivo, protein secretion in A. niger at the single hyphal level. A synthetic green fluorescent protein (sGFP(S65T)) was fused to truncated A. niger glucoamylase (GLA:499). Southern blot analysis of transformants confirmed that the gene fusion had successfully integrated into the A. niger genome. Confocal and fluorescence microscopy revealed that the GLA::GFP fusion protein is fluorescent in A. niger and appears to be directed to the hyphal tip. In young mycelia, hyphal cell wall fluorescence is apparent and immunogold labelling of GFP confirmed that GFP was partially localised within the hyphal cell wall. Using Western blotting, extracellular GLA::GFP was detected only in culture filtrates of young mycelia grown in a soya milk medium. The actin inhibitor latrunculin B was used to disrupt the secretion process, and its effects on the distribution of GLA::GFP were monitored.

Actins↗
PubMed2000

Filamentous fungi as microbial cell factories for food use.

The knowledge base that will underpin the more efficient use of filamentous fungi as cell factories in food has increased during the past year in the areas of gene regulation, protein secretion, safety and synthesis of ingredients such as long-chain polyunsaturated fatty acids.

Biotechnology↗
PubMed2000

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.

Amino Acid Sequence↗
PubMed2000

Isolation and use of a homologous histone H4 promoter and a ribosomal DNA region in a transformation vector for the oil-producing fungus Mortierella alpina.

Mortierella alpina was transformed successfully to hygromycin B resistance by using a homologous histone H4 promoter to drive gene expression and a homologous ribosomal DNA region to promote chromosomal integration. This is the first description of transformation in this commercially important oleaginous organism. Two pairs of histone H3 and H4 genes were isolated from this fungus. Each pair consisted of one histone H3 gene and one histone H4 gene, transcribed divergently from an intergenic promoter region. The pairs of encoded histone H3 or H4 proteins were identical in amino acid sequence. At the DNA level, each histone H3 or H4 open reading frame showed 97 to 99% identity to its counterpart but the noncoding regions had little sequence identity. Unlike the histone genes from other filamentous fungi, all four M. alpina genes lacked introns. During normal vegetative growth, transcripts from the two histone H4 genes were produced at approximately the same level, indicating that either histone H4 promoter could be used in transformation vectors. The generation of stable, hygromycin B-resistant transformants required the incorporation of a homologous ribosomal DNA region into the transformation vector to promote chromosomal integration.

DNA, Fungal↗
PubMed2000

Characterization of a foldase, protein disulfide isomerase A, in the protein secretory pathway of Aspergillus niger.

Protein disulfide isomerase (PDI) is important in assisting the folding and maturation of secretory proteins in eukaryotes. A gene, pdiA, encoding PDIA was previously isolated from Aspergillus niger, and we report its functional characterization here. Functional analysis of PDIA showed that it catalyzes the refolding of denatured and reduced RNase A. pdiA also complemented PDI function in a Saccharomyces cerevisiae Deltapdi1 mutant in a yeast-based killer toxin assay. Levels of pdiA mRNA and PDIA protein were raised by the accumulation of unfolded proteins in the endoplasmic reticulum. This response of pdiA mRNA levels was slower and lower in magnitude than that of A. niger bipA, suggesting that the induction of pdiA is not part of the primary stress response. An increased level of pdiA transcripts was also observed in two A. niger strains overproducing a heterologous protein, hen egg white lysozyme (HEWL). Although overexpression of PDI has been successful in increasing yields of some heterologous proteins in S. cerevisiae, overexpression of PDIA did not increase secreted yields of HEWL in A. niger, suggesting that PDIA itself is not limiting for secretion of this protein. Downregulation of pdiA by antisense mRNA reduced the levels of microsomal PDIA activity by up to 50%, lowered the level of PDIA as judged by Western blots, and lowered the secreted levels of glucoamylase by 60 to 70%.

Actins↗
PubMed2000

Prevention of yeast spoilage in feed and food by the yeast mycocin HMK.

The yeast Williopsis mrakii produces a mycocin or yeast killer toxin designated HMK; this toxin exhibits high thermal stability, high pH stability, and a broad spectrum of activity against other yeasts. We describe construction of a synthetic gene for mycocin HMK and heterologous expression of this toxin in Aspergillus niger. Mycocin HMK was fused to a glucoamylase protein carrier, which resulted in secretion of biologically active mycocin into the culture media. A partial purification protocol was developed, and a comparison with native W. mrakii mycocin showed that the heterologously expressed mycocin had similar physiological properties and an almost identical spectrum of biological activity against a number of yeasts isolated from silage and yoghurt. Two food and feed production systems prone to yeast spoilage were used as models to assess the ability of mycocin HMK to act as a biocontrol agent. The onset of aerobic spoilage in mature maize silage was delayed by application of A. niger mycocin HMK on opening because the toxin inhibited growth of the indigenous spoilage yeasts. This helped maintain both higher lactic acid levels and a lower pH. In yoghurt spiked with dairy spoilage yeasts, A. niger mycocin HMK was active at all of the storage temperatures tested at which yeast growth occurred, and there was no resurgence of resistant yeasts. The higher the yeast growth rate, the more effective the killing action of the mycocin. Thus, mycocin HMK has potential applications in controlling both silage spoilage and yoghurt spoilage caused by yeasts.

Aerobiosis↗
PubMed1999

Effect of pH on hen egg white lysozyme production and evolution of a recombinant strain of Aspergillus niger.

An Aspergillus niger strain (B1) transformed to produce mature hen egg white lysozyme (HEWL) from a glucoamylase fusion protein under control of the A. niger glucoamylase promoter was grown in glucose-limited chemostat culture at a dilution rate of 0.07 h-1 at various pH values. Maximum HEWL production (9.3 mg g-1; specific production rate = 0.65 mg g-1 per h) was obtained at pH 4.5. However, in chemostat culture, HEWL production was not stable at any pH tested. After 240 h in steady state, specific production decreased to only 0.03 +/- 0.01 and 0.24 +/- 0.02 mg g-1 per h at pH 6.5 and 4.5, respectively. Some isolates removed from the chemostat cultures had lost copies of the HEWL gene and when grown in shake flask cultures all of the isolates produced less HEWL than the parental strain. Morphological mutants with similar phenotypes were isolated at all pHs, but their rate of increase in the population was pH dependent, with cultures at low pH (< 4.5) being more morphologically stable than cultures at high (> 4.5) pH. The selective advantage of these mutants was also generally dependent on pH. Both yellow pigment producing mutants and brown sporulation mutants had higher selective advantages over the parental strain at high than at low pH, regardless of the pH at which they were isolated. However, the selective advantage of densely sporulating mutants was independent of pH.

Animals↗
PubMed1999

Expression of the VEGF gene family during retinal vaso-obliteration and hypoxia.

Vascular insufficiency and retinal ischaemia precede many proliferative retinopathies and stimulate secretion of vasoactive growth factors. Vascular endothelial growth factor (VEGF) plays a major role and we therefore investigated the other members of the VEGF family: Placental growth factor (PlGF), VEGF-B, -C, and -D, and platelet derived growth factors (PDGF) A and B. Neonatal mice were exposed to hyperoxia for 5 days and then returned to room air (resulting in acute retinal ischaemia). RT-PCR demonstrated that all the members of the VEGF family are expressed in the retina and in situ hybridization (ISH) located their mRNAs primarily in ganglion cells. Similarly to VEGF itself, VEGF-C, PDGF-A, and PDGF-B were upregulated during retinal ischaemia (P < 0.05). Only PlGF gene expression increased during hyperoxia (P < 0.01). The expression pattern of these growth factors suggests a role in the normal retina and during vaso-obliterative and ischaemic phases.

Animals↗
PubMed1999

Expression, purification, and characterization of the recombinant calcium-binding equine lysozyme secreted by the filamentous fungus Aspergillus niger: comparisons with the production of hen and human lysozymes.

Equine lysozyme (EqL) has been expressed from a synthetic gene and secreted from a heterologous host, the filamentous fungus Aspergillus niger. By including 100 mM Ca2+ in the growth medium, secreted yields of more than 50 mg/liter could be achieved using polyvinylpyrrolidone (PVP) complete medium. In a soya medium yields of up to 150 mg/liter were achieved. The production of recombinant human lysozyme (HuL) from A. niger with yields of over 40 mg/liter was also achieved using PVP medium. Addition of Ca2+ to the growth medium reduced the yield of both HuL and hen egg white lysozyme (HEWL). Sequence differences between the three lysozymes, EqL, HuL, and HEWL, resulted in different susceptibilities to cleavage by A. niger proteases. An improved procedure for the purification of EqL and HuL from A. niger allowed separation of the proteins from pigments produced by the fungus. Detailed spectroscopic analysis, including 2D 1H NMR, for recombinant EqL and recombinant HuL confirm that both proteins possess their native structure and are purified to homogeneity.

Amino Acid Sequence↗
PubMed1999

Oxidative insult specifically decreases levels of a mitochondrial transcript.

The effects of oxidative insult, applied with hydrogen peroxide, on gene transcript levels in a human lymphocyte cell line (Molt-17) were investigated using mRNA differential display. Several cDNA fragments corresponding to putatively up- or down-regulated transcripts were isolated. One of these was found to hybridize to two discrete transcripts on Northern blots of Molt-17 cell RNA. The more abundant transcript, that has previously been demonstrated to correspond to the mRNA for mitochondrial ATPase subunits 8 and 6, was unaffected by the hydrogen peroxide treatment. In contrast, levels of the rarer, larger transcript were consistently reduced in a rapid, sustained, and dose-dependent manner following hydrogen peroxide treatment. Prior supplementation of the cells with beta carotene provided some protection against the reduction in levels of this transcript following hydrogen peroxide treatment. In contrast, vitamins C and E had no effect at the concentrations tested. We have now cloned the cDNA corresponding to this stress-responsive transcript and demonstrated that it is an incompletely processed product of the mitochondrial genome encompassing ATPase subunits 8 and 6 plus the adjacent gene for cytochrome c oxidase subunit 3. This decrease in one specific mitochondrial transcript may represent a novel mechanism for differential expression of mitochondrially-encoded genes.

Adenosine Triphosphatases↗
PubMed1999

Corneal guttata: a comparative clinical and specular micrographic study.

BACKGROUND: The relationship between corneal guttata and Fuchs' endothelial dystrophy is unclear, with the result that the clinical differentiation of the two conditions is often made on the basis of the presence or absence of symptoms. METHODS: In this study the authors compare the clinical and specular micrographic findings, as recorded from 20 patients noted to have biomicroscopic clinical findings consistent with corneal guttata or early Fuchs' endothelial dystrophy. RESULTS: Results confirm the increased prevalence of corneal findings in elderly women. The topographical distribution of guttata, across the posterior corneal surface, as observed clinically was confirmed specular micrographically (rS = 0.55, p = 0.01). Increased numbers of guttata correlated with a statistically significant reduction in the endothelial cell counts recorded from the midperipheral cornea (rS = 0.49, p = 0.02). The relationship between the presence of guttata and a reduction in cellular hexagonality or an increase in polymegethism failed to reach a statistically significant level. Pigment deposits adherent to the posterior endothelial surface were also noted in all but one of the eyes examined. CONCLUSIONS: The authors advocate the use of a grading scale, developed from specular micrographs taken during the course of the study, to assist in the clinical classification of corneal guttata and pre-clinical Fuchs' dystrophy. The authors also recommend specular microscopy as a tool capable of differentiating corneal guttata from pigment deposits in even the most severely affected cases.

Aged↗
PubMed1999

Plasma levels of endothelin-1 in diabetic retinopathy in pregnancy.

PURPOSE: Raised circulating levels of the potent vasoconstrictor endothelin-1 (ET-1) have been demonstrated in diabetes and pregnancy-induced hypertension. Pregnant women with diabetic retinopathy are known to be at a higher risk of developing pregnancy-induced hypertension than those without retinopathy. To examine the association between ET-1, pregnancy, diabetes and diabetic retinopathy, circulating ET-1 levels were measured in each trimester in a cohort of women with and without diabetes during pregnancy. METHODS: A cohort of women with diabetes (n = 13) and normal controls (n = 8) were followed throughout pregnancy. Detailed clinical and fundoscopic examinations were carried out according to ETDRS protocols. Plasma ET-1 levels were measured in each trimester using a sensitive radioimmunoassay. Those with diabetes were further divided into those with retinopathy (n = 7) and those without (n = 6). RESULTS: Plasma levels of ET-1 increased progressively during normal pregnancy and peaked in the third trimester. Women with diabetes had significantly higher levels of plasma ET-1 (14.0 vs 4.6 pg/ml in the first trimester, 14.0 vs 4.8 pg/ml in the second trimester and 15.8 vs 7.2 pg/ml in the third trimester) compared with those without diabetes. These were no significant differences in plasma ET-1 levels between women with diabetes who had pre-existing diabetic retinopathy and those without. CONCLUSION: This study has shown that ET-1 levels rise during normal pregnancy, and are higher in women with diabetes, which may reflect pre-existing endothelial damage. Although no association could be demonstrated between diabetic retinopathy and serum ET-1 levels, this may reflect the small sample size in this study.

Adult↗
PubMed1999

Two fatty acid delta9-desaturase genes, ole1 and ole2, from Mortierella alpina complement the yeast ole1 mutation.

Genes encoding two distinct fatty acid delta9-desaturases were isolated from strains of the oleaginous fungus Mortierella alpina. Two genomic sequences, delta9-1 and delta9-2, each containing a single intron, were cloned from strain CBS 528.72 while one cDNA clone, LM9, was isolated from strain CBS 210.32. The delta9-1 gene encoded a protein of 445 aa which shared 99% identity with the LM9 gene product. These proteins also showed 40-60% identity to the delta9-desaturases (Ole1p) of other fungi and contained the three conserved histidine boxes, C-terminal cytochrome b5 fusion and transmembrane domains characteristic of endoplasmic reticulum membrane-bound delta9-desaturases. LM9 and delta9-1 are therefore considered to represent the same gene (ole1). The ole1 gene was transcriptionally active in all M. alpina strains tested and its function was confirmed by complementation of the Saccharomyces cerevisiae ole1 mutation. Fatty acid analysis of yeast transformants expressing the CBS 210.32 ole1 gene showed an elevated level of oleic acid (18:1) compared to palmitoleic acid (16:1), the major fatty acid component of wild-type S. cerevisiae. This indicated that the M. alpina delta9-desaturase had a substrate preference for stearic acid (18:0) rather than palmitic acid (16:0). Genomic clone delta9-2 (ole2) also encoded a protein of 445 aa which had 86% identity to the delta9-1 and LM9 proteins and whose ORF also complemented the yeast ole1 mutation. The transcript from this gene could only be detected in one of the six M. alpina strains tested, suggesting that its expression may be strain-specific or induced under certain physiological conditions.

Fatty Acid Desaturases↗
PubMed1999

Ophthalmic and visual profile of guide dog owners in Scotland.

BACKGROUND/AIMS: Out of an estimated 90,000 visually impaired people in Scotland, 509 make use of a guide dog. Initial research in Northern Ireland suggests that the ophthalmic profile of guide dog owners (GDOs) is highly specific. The aim of this study was to compare the ophthalmic and visual characteristics of Scottish GDOs with other groups of visually impaired people. METHODS: A random sample of GDOs from central and northern Scotland (n = 82) underwent a detailed assessment of residual vision (distance and near acuity, visual fields, contrast, and glare sensitivity). Comparative data were obtained from two populations of visually impaired non-GDOs-one group attending hospital ophthalmic and low vision clinics (n = 50) the other social services rehabilitation clients (n = 35). All participants completed a questionnaire to elicit ophthalmic history, age, and registration details. RESULTS: GDOs were found to be significantly younger and more profoundly visually impaired than non-GDOs. The main causes of visual impairment were retinitis pigmentosa (23%), optic atrophy (15%), and retinopathy of prematurity (7%). Ninety nine per cent of GDOs were registered blind and had been visually impaired for an average of 39 years. Only 31% were totally blind. CONCLUSION: GDOs represent a unique minority of the visually impaired population. Epidemiological registration trends would suggest that the numbers of young profoundly visually impaired people are unlikely to increase relative to their elderly counterparts. This has implications on the future demand for guide dog ownership.

Adult↗
PubMed1999

Comet assay of UV-induced DNA damage in retinal pigment epithelial cells.

PURPOSE: The molecular mechanisms mediating photic injury to the retinal pigment epithelial (RPE) cell are not clearly understood. This study examined qualitative and quantitative aspects of DNA damage caused by broadband UVA and UVB radiation in RPE cells. METHODS: Cultured bovine RPE cells were exposed to doses of between 0 and 0.9 J/cm2 UVA or 0 and 0.09 J/cm2 UVB, as either a suspension or as an attached monolayer. The damage to DNA resulting in single-strand breaks was assessed by means of the comet assay in which the damaged DNA migrates out of the nucleus forming a tail, and this was quantified using image analysis. Two measurements were taken: the mean percentage of tail DNA, which reflects the overall level of DNA damage in the group of cells, and the Olive tail moment, which represents the extent of migration and thus the pattern of DNA damage in individual cells. Cells were processed by the comet assay immediately after UV exposure in acute experiments. To study the occurrence of DNA repair, RPE cells were first exposed to UVB and then incubated at 37 degrees C for either 1 or 24 hours before processing for the comet assay. RESULTS: UVA- and UVB-exposed cells showed a mean percentage of tail DNA that was significantly greater than in unexposed cells. Olive tail moment was higher in cells exposed to larger doses of UVB. This parameter also showed a bimodal distribution when assessed 24 hours after exposure to UVB indicating the presence of two distinct subpopulations of cells with small and large tail moments. Cells with very large tail moments were not seen with doses below 0.045 J/cm2. CONCLUSIONS: Relatively low doses of UVA and UVB induce the formation of DNA strand breaks in cultured RPE. The tail moment profiles for cells incubated for 24 hours after UVB irradiation are consistent with the occurrence of DNA repair in most cells exposed to low doses and apoptosis in a subpopulation of the cells exposed to high doses.

Animals↗
PubMed1998

Determinants of the fidelity of processing glucoamylase-lysozyme fusions by Aspergillus niger.

Fusion proteins are used to enhance the yields of heterologous proteins secreted from filamentous fungi. In Aspergillus niger, the target protein is normally fused downstream of the carrier protein glucoamylase with a Lys-Arg KEX2-like cleavage site at the junction. This is cleaved in vivo to release mature protein but the processing is not always accurate. We have used N-terminal mutant lysozymes to vary the sequence immediately downstream of the KEX site, and also varied the amino acid sequence upstream of the KEX processing site, to study the fidelity of processing. The sequences both upstream and downstream of the KEX2 site affected the fidelity of cleavage. With some constructs, a range of processing sites were apparent and the relative proportions were time dependent in batch cultures of A. niger. Aberrant processing was related to the secondary-structure preferences of the amino acids in and around the KEX site. Downstream of the processing site, the fidelity of processing decreased in proportion to the tendency for helix formation.

Amino Acid Sequence↗