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Biomedical subjects

M Penttilä

Publications and source records attributed to M Penttilä.

At least 55 records · Page 3Linked to original sources
PubMed1997

Functional vs. radical maxillary surgery. Failures after functional endoscopic sinus surgery.

Randomized FES or Caldwell-Luc (C-L) operations were performed in 150 patients suffering from chronic maxillary sinusitis during 1987-1989. Follow-up examination was done for 143 patients (95%) 1 year postoperatively. Although 15 patients had already deceased, questionnaires were obtained from 128 patients (85%) 5-9 years after operation. One year after surgery 51% of the C-L patients and 77% of the FES patients reported no symptoms or distinct improvement in their global symptoms, whereas 5-9 years postoperatively 82% of the C-L and 76% of the FES patients reported this outcome, respectively. Thirteen C-L operated patients (18%) and 14 FES patients (20%) have been reoperated during 7-9 years follow-up. In most cases revision surgery was done for polyposis or maxillary sinusitis. In the long term, altogether 21 patients (28%), including 14 reoperated patients, were failures after FES. These included also 4 patients with global symptoms unchanged at late inquiry and 3 other patients, though getting better, reported unwillingness for FES operation if they had known the result beforehand.

Chronic Disease↗
PubMed1997

Bacterial findings in acute maxillary sinusitis--European study.

Bacteriology of acute maxillary sinusitis was studied in 569 patients in 16 centers of 6 countries located throughout Europe during 1992-1994 by ENT specialists. Patients with symptoms of acute sinusitis lasting less than 3 weeks with ongoing purulent nasal discharge were included. Diagnosis was verified by sinus x-ray or ultrasonography and a positive aspiration finding in maxillary sinus puncture. One or more pathogens were isolated from the maxillary sinus aspirates of 375 (66%) patients. Fifty-six percent of patients harboured 1 pathogen and 10% multiple pathogenic organisms, respectively. Haemophilus influenzae was the most common pathogen isolated (148 isolates), occurring as a single pathogen in 14% of the patients. The occurrence of H. influenzae was highest in Finnish military hospital patients (43-48%), as compared with the non-military Finnish patients (9-11%) or to patients from other European centers (mean 13%). H. influenzae was more frequently beta-lactamase positive in other European centers (22%) than in Finnish centers (7%). Streptococcus pneumoniae was the most common pathogen isolated in other European centers (20%) but second most common in Finnish centers (13%). Moraxella catarrhalis occurred at quite similar frequency among Finnish centers (9-14%), but clearly less often in other centers (mean 4%). S. aureus, which in acute maxillary sinusitis is regarded as a contaminant from the nasal cavity, was more prevalent in other European centers (12%) than in Finnish centers (4%). In patients with acute maxillary sinusitis reliable bacteriological samples should be taken by antral aspiration directly from the diseased sinus.

Acute Disease↗
PubMed1996

Functional analysis of the cellobiohydrolase I promoter of the filamentous fungus Trichoderma reesei.

Functional analysis of the cellulase promoter cbh1 of the filamentous fungus Trichoderma reesei was carried out using the Escherichia coli lacZ gene as a reporter. An assay based on cultivation on solid medium in microtiter plates was developed that allows rapid and reliable semiquantitative analysis of beta-galactosidase expression of a large number of transformants. A series of deletions and specifically designed alterations were made covering 2.2 kb of the cbh1 promoter. Removal of sequences upstream of nucleotide -500 in relation to the initiator ATG abolished glucose repression. Mutation of a single hexanucleotide sequence 5'GTGGGG at nucleotide -720 was sufficient for derepression. This site is similar to the binding sites of the glucose repressors MIG1 of Saccharomyces cerevisiae and CREA/CREI of filamentous fungi. Removal of the glucose repressor site did not affect sophorose induction. Sophorose induction of the promoter was retained even in deletion derivatives lacking sequences upstream of position -161, which retained about 70 bp upstream of the transcription start point and only 30 bp upstream of the TATA box.

Base Sequence↗
PubMed1996

Use of a modified alcohol dehydrogenase, ADH1, promoter in construction of diacetyl non-producing brewer's yeast.

The bacterial gene, encoding alpha-acetolactate decarboxylase (alpha-ALDC), was expressed in a bottom-fermenting brewer's yeast under the control of a modified Saccharomyces cerevisiae alcohol dehydrogenase (ADH1) promoter which lacks the upstream regions from -800 bp to -1500 bp. In pilot scale brewing conditions, the level of alpha-ALDC produced was high enough to reduce the concentration of diacetyl so that lagering was not required. alpha-ALDC active brewer's yeast strains were also shown to be suitable for high gravity brewing.

Alcohol Dehydrogenase↗
PubMed1996

Three alpha-galactosidase genes of Trichoderma reesei cloned by expression in yeast.

Three alpha-galactosidase genes, agl1, agl2 and agl3, were isolated from a cDNA expression library of Trichoderma reesei RutC-30 constructed in the yeast Saccharomyces cerevisiae by screening the library on plates containing the substrate 5-bromo-4-chloro-3-indolyl-alpha-D-galactopyranoside. The genes agl1, agl2 and agl3 encode 444, 746 and 624 amino acids, respectively, including the signal sequences. The deduced amino acid sequences of AGLI and AGLIII showed similarity with the alpha-galactosidases of plant, animal, yeast and filamentous fungal origin classified into family 27 of glycosyl hydrolases whereas the deduced amino acid sequence of AGLII showed similarity with the bacterial alpha-galactosidases of family 36. The enzymes produced by yeast were analysed for enzymatic activity against different substrates. AGLI, AGLII and AGLIII were able to hydrolyse the synthetic substrate p-nitrophenyl-alpha-D-galactopyranoside and the small galactose-containing oligosaccharides, melibiose and raffinose. They liberated galactose from polymeric galacto(gluco)mannan with different efficiencies. The action of AGLI towards polymeric substrates was enhanced by the presence of the endo-1,4-beta-mannanase of T. reesei. AGLII and AGLIII showed synergy in galacto(gluco)mannan hydrolysis with the endo-1,4-beta-mannanase of T. reesei and a beta-mannosidase of Aspergillus niger. The calculated molecular mass and the hydrolytic properties of AGLI indicate that it corresponds to the alpha-galactosidase previously purified from T. reesei.

Amino Acid Sequence↗
PubMed1996

The glucose repressor gene cre1 of Trichoderma: isolation and expression of a full-length and a truncated mutant form.

The cre1 genes of the filamentous fungi Trichoderma reesei and T. harzianum were isolated and characterized. The deduced CREI proteins are 46% identical to the product of the glucose repressor gene creA of Aspergillus nidulans, encoding a DNA-binding protein with zinc fingers of the C2H2 type. The cre1 promoters contain several sequence elements that are identical to the previously identified binding sites for A. nidulans CREA. Steady-state mRNA levels for cre1 of the T. reesei strain QM9414 varied depending on the carbon source, being low on glucose-containing media. These observations suggest that cre1 expression may be autoregulated. The T. reesei strain Rut-C30, a hyper-producer of cellulolytic enzymes, was found to express a truncated form of the cre1 gene (cre1-1) with an ORF corresponding to a protein of 95 amino acids with only one zinc finger. Unlike QM9414 the strain Rut-C30 produced cellulase mRNAs on glucose-containing medium and transformation of the full-length cre1 gene into this strain caused glucose repression of cbh1 expression, demonstrating that cre1 regulates cellulase expression.

Amino Acid Sequence↗
PubMed1996

The alpha-glucuronidase-encoding gene of Trichoderma reesei.

The Trichoderma reesei cDNA coding for alpha-glucuronidase (GLRI), which releases glucuronic acid attached to xylose units of xylan, was cloned and sequenced. The deduced N-terminal amino acid (aa) sequence of the protein was verified by sequencing of the purified GLRI. The aa sequence of the GLRI displayed no similarity with any aa sequence available in the data bases.

Amino Acid Sequence↗
PubMed1996

Acetyl xylan esterase from Trichoderma reesei contains an active-site serine residue and a cellulose-binding domain.

The axe1 gene encoding acetyl xylan esterase was isolated from an expression library of the filamentous fungus Trichoderma reesei using antibodies raised against the purified enzyme. Apparently axe1 codes for the two forms, pI 7 and pI 6.8, of acetyl xylan esterase previously characterized. The axe1 encodes 302 amino acids including a signal sequence and a putative propeptide. The catalytic domain has no amino acid similarity with the reported acetyl xylan esterases but has a clear similarity, especially in the active site, with fungal cutinases which are serine esterases. Similarly to serine esterases, the axe1 product was inactivated with phenylmethylsulfonyl fluoride. At its C-terminus it carries a cellulose binding domain of fungal type, which is separated from the catalytic domain by a region rich in serine, glycine, threonine and proline. The binding domain can be separated from the catalytic domain by limited proteolysis without affecting the activity of the enzyme towards acetylated xylan, but abolishing its capability to bind cellulose.

Acetylesterase↗
PubMed1996

Genetic and biochemical characterization of the Trichoderma reesei hydrophobin HFBI.

The hfb1 gene of the filamentous fungus Trichoderma reesei, previously cloned as a gene which was abundantly expressed when the fungus was grown on glucose-containing medium, was shown to encode a novel fungal hydrophobin. The encoded 97-amino-acid protein is cysteine-rich and has a typical signal sequence for secretion. Signal-sequence cleavage and putative proteolytic processing results in the mature HFBI protein of 75 amino acids. Antibodies raised against the HFBI protein expressed in Escherichia coli detected the T. reesei HFBI protein in the fungal cell wall and in the culture medium of submerged glucose-containing cultures. The identity of HFBI was verified by N-terminal and peptide sequencing of proteins purified both from the cell wall and culture medium. In the cell wall most of the HFBI formed SDS-insoluble complexes that could be extracted with trifluoroacetic acid. Bubbling or freezing of the culture medium caused HFBI to form aggregates that coprecipitated with a yellow pigment produced by the fungus.

Amino Acid Sequence↗
PubMed1996

Cloning of genes encoding alpha-L-arabinofuranosidase and beta-xylosidase from Trichoderma reesei by expression in Saccharomyces cerevisiae.

A cDNA expression library of Trichoderma reesei RutC-30 was constructed in the yeast Saccharomyces cerevisiae. Two genes, abf1 and bxl1, were isolated by screening the yeast library for extracellular alpha-L-arabinofuranosidase activity with the substrate p-nitrophenyl-alpha-L-arabinofuranoside. The genes abf1 and bxl1 encode 500 and 758 amino acids, respectively, including the signal sequences. The deduced amino acid sequence of ABFI displays high-level similarity to the alpha-L-arabinofuranosidase B of Aspergillus niger, and the two can form a new family of glycosyl hydrolases. The deduced amino acid sequence of BXLI shows similarities to the beta-glucosidases grouped in family 3. The yeast-produced enzymes were tested for enzymatic activities against different substrates. ABFI released L-arabinose from p-nitrophenyl-alpha-L-arabinofuranoside and arabinoxylans and showed some beta-xylosidase activity toward p-nitrophenyl-beta-D-xylopyranoside. BXLI did not release L-arabinose from arabinoxylan. It showed alpha-L-arabinofuranosidase, alpha-L-arabinopyranosidase, and beta-xylosidase activities against p-nitrophenyl-alpha-L-arabinofuranosidase, p-nitrophenyl-alpha-L-arabinopyranoside, and p-nitrophenyl-beta-D- xylopyranoside, respectively, with the last activity being the highest. It was also able to hydrolyze xylobiose and slowly release xylose from polymeric xylan. ABFI and BXLI correspond to a previously purified alpha-L-arabinofuranosidase and a beta-xylosidase from T. reesei, respectively, as confirmed by partial amino acid sequencing of the Trichoderma-produced enzymes. Both enzymes produced in yeasts displayed hydrolytic properties similar to those of the corresponding enzymes purified from T. reesei.

Amino Acid Sequence↗
PubMed1996

Improved production of Trichoderma harzianum endochitinase by expression in Trichoderma reesei.

The chromosomal endochitinase gene (ThEn-42) of the mycoparasite fungus Trichoderma harzianum P1 was isolated and overexpressed in the filamentous fungus Trichoderma reesei under the promoter of the major cellulase gene cbhl1. The host strain RutC-30 did not produce any endogenous endochitinase activity. The prepro region of the T harzianum endochitinase was correctly processed in T. reesei. No differences in expression were observed when the prepro region was replaced with the CBHI signal sequence. Shake flask cultivation yielded 130 mg of active enzyme per liter, which in terms of activity represents about a 20-fold increase over the endochitinase activity produced by T. harzianum. The presence of multiple copies of the expression cassette in the transformant resulted in limitation in transcription and/or regulation factors needed for full activity of the cbh1 promoter, although this was not the major limiting factor for higher expression of endochitinase. The endochitinase was very sensitive to an acidic protease at the late stages of T. reesei cultivation. T. reesei RutC-30 appeared to be tolerant of the endochitinase and can be used as a production host for this enzyme, which has antifungal activity toward plant pathogens.

Amino Acid Sequence↗
PubMed1995

A short-chain dehydrogenase gene from Pichia stipitis having D-arabinitol dehydrogenase activity.

An NAD(+)-dependent D-arabinitol dehydrogenase (polyol dehydrogenase) gene was isolated from Pichia stipitis CBS 6054 and cloned in Saccharomyces cerevisiae. The gene was isolated by screening of a lambda-cDNA library with a zymogram technique. D-Arabinitol, xylitol, D-glucitol and galactitol are substrates for the recombinant protein. With D-arabinitol as substrate the reaction product is D-ribulose. The molecular weight of the native tetramer enzyme is 110,000 Da and the monomer is 30,000 Da. The amino acid sequence is homologous to the short-chain dehydrogenase family. It is 85.5% identical to a D-arabinitol dehydrogenase from Candida albicans. The gene in P. stipitis was induced by D-arabinitol and P. stipitis was able to grow on D-arabinitol. The physiological role of D-arabinitol metabolism is discussed.

Amino Acid Sequence↗
PubMed1995

Production of recombinant proteins in the filamentous fungus Trichoderma reesei.

The potential of the filamentous fungus Trichoderma reesei for producing heterologous proteins has recently been demonstrated with a number of secreted proteins. Rate-limiting steps and ways of improving the production have been studied, especially using antibody Fab fragments. Major improvements have been achieved by producing the foreign protein fused to the fungal cellulase cellobiohydrolase I. In addition to the strong inducible cbh1 promoter, other promoters, which are regulated in a different manner, have been developed.

Genetic Engineering↗
PubMed1995

Production of Trichoderma reesei cellulases on glucose-containing media.

The filamentous fungus Trichoderma reesei was shown to secrete active cellobiohydrolase I and the endoglucanase I catalytic core domain into the culture medium when the fungus was grown on glucose-containing medium. The expression of the proteins was driven by the promoters of the elongation factor 1 alpha, tef1, and the unidentified gene for cDNA1. The cDNA1 promoter gave the best yields. The highest amounts of cellobiohydrolase I and the endoglucanase I core, being 50 to 100 mg/liter, accounted for more than half of the total protein secreted by the fungus. The levels obtained with the tef1 promoter were 20 to 50 times lower.

Base Sequence↗
PubMed1995

Xylose-metabolizing Saccharomyces cerevisiae strains overexpressing the TKL1 and TAL1 genes encoding the pentose phosphate pathway enzymes transketolase and transaldolase.

Saccharomyces cerevisiae was metabolically engineered for xylose utilization. The Pichia stipitis CBS 6054 genes XYL1 and XYL2 encoding xylose reductase and xylitol dehydrogenase were cloned into S. cerevisiae. The gene products catalyze the two initial steps in xylose utilization which S. cerevisiae lacks. In order to increase the flux through the pentose phosphate pathway, the S. cerevisiae TKL1 and TAL1 genes encoding transketolase and transaldolase were overexpressed. A XYL1- and XYL2-containing S. cerevisiae strain overexpressing TAL1 (S104-TAL) showed considerably enhanced growth on xylose compared with a strain containing only XYL1 and XYL2. Overexpression of only TKL1 did not influence growth. The results indicate that the transaldolase level in S. cerevisiae is insufficient for the efficient utilization of pentose phosphate pathway metabolites. Mixtures of xylose and glucose were simultaneously consumed with the recombinant strain S104-TAL. The rate of xylose consumption was higher in the presence of glucose. Xylose was used for growth and xylitol formation, but not for ethanol production. Decreased oxygenation resulted in impaired growth and increased xylitol formation. Fermentation with strain S103-TAL, having a xylose reductase/xylitol dehydrogenase ratio of 0.5:30 compared with 4.2:5.8 for S104-TAL, did not prevent xylitol formation.

Gene Expression Regulation, Enzymologic↗
PubMed1995

Cloning and expression in Saccharomyces cerevisiae of a Trichoderma reesei beta-mannanase gene containing a cellulose binding domain.

beta-Mannanase (endo-1,4-beta-mannanase; mannan endo-1,4-beta-mannosidase; EC 3.2.1.78) catalyzes endo-wise hydrolysis of the backbone of mannan and heteromannans, including hemicellulose polysaccharides, which are among the major components of plant cell walls. The gene man1, which encodes beta-mannanase, of the filamentous fungus Trichoderma reesei was isolated from an expression library by using antiserum raised towards the earlier-purified beta-mannanase protein. The deduced beta-mannanase consists of 410 amino acids. On the basis of hydrophobic cluster analysis, the beta-mannanase was assigned to family 5 of glycosyl hydrolases (cellulase family A). The C terminus of the beta-mannanase has strong amino acid sequence similarity to the cellulose binding domains of fungal cellulases and is preceded by a serine-, threonine-, and proline-rich region. Consequently, the beta-mannanase is probably organized similarly to the T. reesei cellulases, having a catalytic core domain separated from the substrate-binding domain by an O-glycosylated linker. Active beta-mannanase was expressed and secreted by using the yeast Saccharomyces cerevisiae as the host. The results indicate that the man1 gene encodes the two beta-mannanases with different isoelectric points (pIs 4.6 and 5.4) purified earlier from T. reesei.

Amino Acid Sequence↗