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Biomedical subjects

A Lappalainen

Publications and source records attributed to A Lappalainen.

10 recordsLinked to original sources
PubMed2001

Calcification of the intervertebral discs and curvature of the radius and ulna: a radiographic survey of Finnish miniature dachshunds.

The vertebral column of 124 randomly selected miniature dachshunds, representing 4.5% of the population registered by the Finnish Kennel Club during the years 1988 to 1996, were radiographed. The front legs were also radiographed in order to evaluate the curvature of the radius and ulna. Calcified discs were found in 75.9% of the longhaired miniature dachshunds and in 86.7% of the wirehaired ones. The occurrence of signs associated with IDD was 16.5% in longhaired and 15.6% in wirehaired miniature dachshunds. The occurrence of signs of IDD in dogs with calcified discs was 20.0% and 17.9% in longhaired and wirehaired miniature dachshunds, respectively. In dogs without calcifications only one dog showed signs of IDD. The curvature of the radius and the ulna did not differ between the dogs with signs of IDD and the healthy ones, or between the dogs with and without intervertebral calcifications. Our results indicate that radiographic eradication based on the presence of intervertebral calcifications is not suitable for breeding purposes for the Finnish miniature dachshund population because the percentage of dogs without calcifications is small.

Animals↗
PubMed2000

Endoxylanase II from Trichoderma reesei has several isoforms with different isoelectric points.

Two minor xylanases present in Trichoderma reesei Rut C30 cultivation broth were purified as a mixture using ion-exchange, hydrophobic-interaction and gel chromatography. The purified enzyme preparation contained two active xylanases with pI values of 7.1 and 8.1. Both components had a molecular mass of 20 kDa. The purified xylanase preparation exhibited properties very similar to those of the previously isolated XYL II (pI 9.0) of T. reesei Rut C30. The activity and stability properties, apparent kinetic parameters as well as the titration curve forms were similar. The major difference in enzymic properties was the significantly lower specific activity of the pI-7.1+8.1 xylanase mixture (3350 nkat/mg) compared with the specific activity of XYL II (13500 nkat/mg). Amino acid sequences of tryptic peptides (34% of the total amino acid sequence was determined) were identical to the amino acid sequence of XYL II. Furthermore, in vitro modification of the pI-9.0 form of XYL II to pI-8.1 and pI-7.1 forms was demonstrated. Thus the purified xylanase preparation most probably contained two modified forms of XYL II. The primary amino acid sequence of XYL II contains 28 glutamine and asparagine residues and theoretically deamination of one of them lowers the pI to 8.06 and deamination of two amino acids lowers the pI to 7.02.

Amino Acid Sequence↗
PubMed1996

Immunoaffinity Chromatographic Purification of Cellobiohydrolase II Mutants from Recombinant Trichoderma reesei Strains Devoid of Major Endoglucanase Genes

Efficient purification of Trichoderma reesei cellobiohydrolase II (CBHII) requires the use of affinity chromatography based on a substrate analogue. Due to altered substrate binding, the purification of many active-site mutants of CBHII from the complex fungal culture media represents a considerable challenge. Here we describe a combination of two approaches to facilitate the purification: the first is based on the construction of novel engineered T. reesei strains devoid of the major contaminating endoglucanases, and the second uses immunoaffinity chromatography as the final purification step. Two different procedures for the preparation of the antibody matrix were tested. Crosslinking of the monoclonal antibody to Protein G matrix instead of the conventional immobilization via cyanogen bromide increased the binding efficiency. Three different active-site mutants of CBHII bound to the immunoaffinity column in neutral pH and were eluted in pH 2.7. The purity of the CBHII mutant preparations was tested using small chromophoric substrates and hydroxyethyl cellulose, which are hydrolyzed by many other cellulases but not by CBHII. The immunoaffinity column purified the CBHII mutants over 800-fold in a single step and resulted in homogeneous protein preparations free of proteolytically cleaved forms of CBHII. The use of the double replacement T. reesei production strains, especially the one lacking the genes coding for both the endogeneous CBHII and the endoglucanase II (EGII), helped to reduce the total endoglucanase activity in the preparations.

Journal Article↗
PubMed1996

Immunoaffinity chromatographic purification of cellobiohydrolase II mutants from recombinant trichoderma reesei strains devoid of major endoglucanase genes.

Efficient purification of Trichoderma reesei cellobiohydrolase II (CBHII) requires the use of affinity chromatography based on a substrate analogue. Due to altered substrate binding, the purification of many active-site mutants of CBHII from the complex fungal culture media represents a considerable challenge. Here we describe a combination of two approaches to facilitate the purification: the first is based on the construction of novel engineered T. reesei strains devoid of the major contaminating endoglucanases, and the second uses immunoaffinity chromatography as the final purification step. Two different procedures for the preparation of the antibody matrix were tested. Crosslinking of the monoclonal antibody to Protein G matrix instead of the conventional immobilization via cyanogen bromide increased the binding efficiency. Three different active-site mutants of CBHII bound to the immunoaffinity column in neutral pH and were eluted in pH 2.7. The purity of the CBHII mutant preparations was tested using small chromophoric substrates and hydroxyethyl cellulose, which are hydrolyzed by many other cellulases but not by CBHII. The immunoaffinity column purified the CBHII mutants over 800-fold in a single step and resulted in homogeneous protein preparations free of proteolytically cleaved forms of CBHII. The use of the double replacement T. reesei production strains, especially the one lacking the genes coding for both the endogeneous CBHII and the endoglucanase II (EGII), helped to reduce the total endoglucanase activity in the preparations.

Binding Sites↗
PubMed1992

Reproductive status, blood chemistry, gill histology and growth of perch (Perca fluviatilis) in three acidic lakes.

Perch (Perca fluviatilis L.) were sampled soon after spawning in three small acidic lakes (pH 4.3-6.1, Al(lab) 5-106 microg litre(-1), Ca2+ 0.01-0.08 mmol litre(-1)) and in one circumneutral lake (pH 5.9-6.4, Al(lab) 4-12 microg litre(-1), Ca2+ 0.06-0.07 mmol litre(-1)) in southern Finland. Due to the delayed spawning of perch in the acidic lakes, sampling in those lakes was performed later than in the reference lake. In spite of that, the gonadosomatic index (GSI) of males in all the acidic lakes was significantly greater than in the reference lake. Of the two lakes with similar low water pH, the effects on reproduction were more prominent in the lake with higher water Al content. The plasma Ca2+ concentrations of females in the acidic lakes were significantly smaller than in the females of the reference lake. The low female:male plasma Ca2+ ratio (1.0-1.32) depicted delay of spawning. Stress in perch in acidic water was also seen in elevated blood haematocrit values, especially in females. On the other hand, a low plasma Cl- level, a common response to acidic water in salmonids, was not detected in perch in the most acidic lakes. The amount of Al accumulated in the gill epithelium was highest in the most acidified lake with high Al concentration, but was also pronounced in a lake with low pH and low Al concentration.

Journal Article↗
PubMed1985

A new appraisal of the endoglucanases of the fungus Trichoderma reesei.

The properties and enzymic activity of endoglucanases (EC 3.2.1.4) of the fungus Trichoderma reesei were studied by means of immunological methods and by using polyglycosidic substrates. Endoglucanases exist in the culture liquid as a series of immunologically related components. The most active endoglucanase component has an Mr of 43 000 and pI value of 4.0. The most abundant components have a value of pI about 5.0, an Mr of 56 000-67 000 and specific activity only one-fifth of that of the pI-4.0 component. During purification and storage the endoglucanases are spontaneously modified; the relative proportion of components having greater Mr values, more alkaline pI values and lower specific activities is increased. The hexose content of the endoglucanase components is 2-7%. Endoglucanases hydrolyse soluble beta-1,4 glycans. The enzymes described here differ from endoglucanase preparations described previously in not showing activity towards insoluble substrates. The role of endoglucanases in wood hydrolysis is consequently limited to the stage where wood constituents are already in soluble form.

Cellulase↗
PubMed1985

Combination of three techniques for the study of complex enzyme mixtures: polyacrylamide gel electrophoresis, immunodiffusion and detection of enzymatic activity.

A technique is presented in which two different separation methods are combined. The first separation is carried out in polyacrylamide gel electrophoresis, which is then followed by immunodiffusion in agarose against appropriate antisera. Soluble and insoluble macromolecular carbohydrates were used as substrates in the detection of the various enzymatic activities. These three methods in combination can provide new information on enzymatically active proteins in mixtures, such as isoenzymes, and multiple forms varying in molecular size.

Electrophoresis, Polyacrylamide Gel↗
PubMed1983

Cellobiohydrolase from Trichoderma reesei.

A 1,4-beta-D-glucan cellobiohydrolase (EC 3.2.1.91) was purified from the culture liquid of Trichoderma reesei by using biospecific sorption on amorphous cellulose and immunoaffinity chromatography. A single protein band in polyacrylamide-gel electrophoresis and one arc in immunoelectrophoresis corresponded to the enzyme activity. The Mr was 65 000. The pI was 4.2-3.6. The purified enzyme contained about 10% hexose. The enzyme differs from previously described cellobiohydrolases in being more effective in the hydrolysis of cellulose.

Cellulose 1,4-beta-Cellobiosidase↗
PubMed1980

The amino acid sequence of pea (Pisum sativum) leghemoglobin.

The amino acid sequence has been determined for leghemoglobin component I from root nodules of pea, Pisum sativum. Pea leghemoglobin is one polypeptide chain composed of 147 amino acids, it contains one methionine residue at position 144, and three histidines, which are at positions 60, 92 and 101. The sequence has at least seven polymorphic residues, but it was not possible to separate the polymorphic protein forms which had identical electric charge. The approximate molecular weight of pea leghemoglobin component I is 16,350. The other major leghemoglobin component (II) from pea has an amino acid composition very similar to that of leghemoglobin component I, suggesting that the gene has duplicated relatively recently. P. sativum leghemoglobin differs from that of Vicia faba by 22--23%, depending on the polymorphic form. The leghemoglobins from Phaseolus vulgaris and Glycine max differ from pea leghemoglobin by 35--44%, and Lupinus luteus leghemoglobins differ from it by 45--48%. The seven leghemoglobins so far sequenced have 50 residues (33%) which are common to all.

Amino Acid Sequence↗