Biomedical subjects

B Svensson

Publications and source records attributed to B Svensson.

At least 73 records · Page 4Linked to original sources

PubMed1999

Thermodynamics of reversible and irreversible unfolding and domain interactions of glucoamylase from Aspergillus niger studied by differential scanning and isothermal titration calorimetry.

The stability of three forms of glucoamylase from Aspergillus niger has been investigated by differential scanning and isothermal titration calorimetry: Glucoamylase 1 (GA1), which consists of a catalytic domain and a starch-binding domain (SBD) connected by a heavily O-glycosylated linker region; glucoamylase 2 (GA2), which lacks SBD; and a proteolytically cleaved glucoamylase (GACD), which contains the catalytic domain and part of the linker region. The structures of the catalytic domain with part of the linker region and of SBD are known from crystallography and NMR, respectively, but the precise spatial arrangement of the two domains in GA1 is unknown. To investigate the stability of the three glucoamylase forms, we unfolded the enzymes thermally by differential scanning calorimetry (DSC). Aggregation occurs upon heating GA1 and GA2 at pH values between 2.5 and 5.0, whereas no aggregation is observed at higher pH (5.5-7.5). At all pH values, the catalytic domain of GA1 and GA2 unfolds irreversibly, while SBD unfolds reversibly in the pH range 5. 5-7.5 where aggregation does not occur. The unfolding of the catalytic domain of all glucoamylase forms seems to follow an irreversible one-step mechanism with no observable reversible intermediates on the experimental time scale. SBD of GA1 unfolds reversibly, and the ratio between the van't Hoff and calorimetric enthalpies is 1.4 +/- 0.1. Assignment of peaks of the DSC profile to the domains at pH 7.5 is achieved by using two different ligands: Acarbose, a very strong inhibitor that binds exclusively to the catalytic domain, and beta-cyclodextrin, a small starch analogue of which 2 molecules bind solely to the two binding sites present in SBD. Differences are seen in the unfolding processes of GA1 and GA2 since the former unfolds with one peak at all pH values, while the calorimetric trace of the latter can be resolved into more peaks depending on pH and the chemical composition of the buffers. In general, peaks corresponding to unfolding of GA2 are more complex than the peaks of GA1 and GACD. Some part of GA2 unfolds before the rest of the molecule which may correspond to the linker region or a particular early unfolding part of the catalytic domain. This leads to the conclusion that the structure of the GA2 molecule has a larger cooperative unfolding unit and is less stable than the structures of GA1 and GACD and that the C-terminal part of the linker region has a destabilizing effect on the catalytic domain.

Acarbose↗

PubMed1999

Comparison between automatic ribotyping and random amplified polymorphic DNA analysis of Bacillus cereus isolates from the dairy industry.

Discrimination by automatic ribotyping and random amplified polymorphic DNA PCR, RAPD, was compared for 40 different B. cereus dairy isolates, 4 different B. mycoides isolates and 6 culture collection strains. RAPD-PCR has previously shown to be useful for tracing contamination routes of B. cereus to milk. Automatic ribotyping using EcoRI and PvuII separated the B. cereus and B. mycoides isolates/strains into 36 different ribotypes. RAPD-typing with primers generated 40 different RAPD-profiles. However, 17 isolates clustered into eight groups, irrespective of the primer and restriction enzyme used, and in all but one case, the isolates with the same pattern were isolated from the same dairy. Automatic ribotyping proved to be a useful, standardized and quick method to discriminate between B. cereus strains, only slightly less discriminatory than RAPD-typing.

Animals↗

PubMed1999

Mass spectrometric identification of a stable catalytic cysteinesulfinic acid residue in an enzymatically active chemically modified glucoamylase mutant.

Mass spectrometric identification of cysteinsulfinic acid resulting in restoration of activity of chemically modified Glu400 Cys catalytic-base glucoamylase (GA) mutants is described. This oxidation unexpectedly occurred during attempts to carboxyalkylate the Cys400 GA mutant using three different alkylation reagents. However, mass spectrometric peptide mapping did not show the presence of carboxyalkylation of the Cys400 residue but suggested an oxidation to cysteinsulfinic acid based on the observed mass increment. The presence of cysteinsulfinic acid was confirmed by employing matrix-assisted laser desorption/ionization mass spectrometry combined with post-source decay analysis. Furthermore, strong enhancement of metastable fragmentation was observed for peptides containing oxidized Cys in comparison with non-oxidized peptide.

Amino Acid Sequence↗

PubMed1999

Rehabilitation of schizophrenic and other long-term mentally ill patients. Results from a prospective study of a comprehensive inpatient treatment program based on cognitive therapy.

A total of 28 long-term mentally ill patients, the majority schizophrenic, treated with cognitive therapy in the context of milieu-therapy and group therapy, were investigated at admission and discharge with regard to changes in symptomatology, quality of life, global functioning, need for medication, and perceived target complaints. The results showed a significant relief in perceived burden of illness and an improvement in quality of life. A better pre-admission functioning with regard to social functioning, occupational function, and symptoms predicted a more favourable outcome.

Adult↗

PubMed1999

Structure-function relationships in glucoamylases encoded by variant Saccharomycopsis fibuligera genes.

The mutation Gly467-->Ser in Glu glucoamylase was designed to investigate differences between two highly homologous wild-type Saccharomycopsis fibuligera Gla and Glu glucoamylases. Gly467, localized in the conserved active site region, S5, is replaced by Ser in the Gla glucoamylase. These amino acid residues are the only two known to occupy this position in the elucidated glucoamylase sequences. The data from the kinetic analysis revealed that replacement of Gly467 with Ser in Glu glucoamylase decreased the kcat towards all substrates tested to values comparable with those of the Gla enzyme. Moreover, the mutant glucoamylase appeared to be less stable compared to the wild-type Glu glucoamylase with respect to thermal unfolding. Microcalorimetric titration studies of the interaction with the inhibitor acarbose indicated differences in the binding between Gla and Glu enzymes. The Gla glucoamylase, although less active, binds acarbose stronger (Ka congruent with 10(13).M(-1)) than the Glu enzyme (Ka congruent with 10(12).M(-1)). In all enzymes studied, the binding of acarbose was clearly driven by enthalpy, with a slightly favorable entropic contribution. The binding of another glucoamylase inhibitor, 1-deoxynojirimycin, was about 8-9 orders of magnitude weaker (Ka congruent with 10(4).M(-1)) than that of acarbose. From comparison of kinetic parameters for the nonglycosylated and glycosylated enzymes it can be deduced that the glycosylation does not play a critical role in enzymatic activity. However, results from differential scanning calorimetry demonstrate an important role of the carbohydrate moiety in the thermal stability of glucoamylase.

Acarbose↗

PubMed1999

The prevalence of rheumatoid arthritis in Sweden.

The aim of this study was to ascertain the prevalence of rheumatoid arthritis (RA) in a Swedish general adult population. A questionnaire about chronic pain was mailed to a total of 3928 subjects who were chosen as a random sample of the population in two communities in the county of Halland. All persons answering affirmatively to questions intended to identify patients with RA were invited to a clinical examination. X-rays of hands and feet, and analyses of rheumatoid factor and C reactive protein were performed provided that the patients fulfilled two or more of the five clinical items of the 1987 ARA criteria. Furthermore, non-participants were searched for in a patient register and in medical records from the local rheumatology unit in an attempt to identify further cases. Using the modified 1987 ARA criteria for population studies the prevalence rate of RA was calculated to 0.51% (95%, CI = 0.31-0.79).

Adult↗

PubMed1999

Relationships among patient and therapist ratings of therapeutic alliance and patient assessments of therapeutic process: a study of cognitive therapy with long-term mentally ill patients.

The therapeutic alliance as rated by therapists and patients was assessed every 5 weeks throughout the treatment period in an in-patient treatment program for schizophrenic and other long-term mentally ill patients. The patients (N = 26) also assessed perceived curative factors (Curative Factors Questionnaire) and made therapy session evaluations (Session Evaluation Questionnaire). The most important patient rated factors showing a relationship with therapist-rated alliance were in the initial phase of treatment depth in the therapy sessions, in the working phase the experience of involvement in the treatment, and in the discharge phase perceived helpfulness of encouragement and reassurance. The investigation of curative factors, session evaluations, and alliance as rated by patients showed a relationship in the initial phase between alliance and encouragement, reassurance and awareness, in the working phase between alliance and depth in sessions and "talking to someone who understands," and in the discharge phase between alliance and self-understanding and problem solution.

Attitude of Health Personnel↗

PubMed1999

Therapeutic alliance in cognitive therapy for schizophrenic and other long-term mentally ill patients: development and relationship to outcome in an in-patient treatment programme.

A total of 26 schizophrenic and other long-term mentally ill patients receiving cognitive therapy sessions twice a week in the context of a comprehensive in-patient treatment programme were assessed every 5 weeks with regard to therapeutic alliance. The therapists rated the alliance according to the Psychotherapy Status Report and the patients rated it according to the Patient Collaboration Scale. The results showed either good or fair and stable therapeutic alliance for the majority of the patients throughout the treatment period. Few correlations were found between patient characteristics and initial alliance. The therapists' ratings showed a positive relationship between initial alliance and a favourable outcome of treatment at discharge.

Adolescent↗

PubMed1999

Cervical spine fractures in the elderly: factors influencing survival in 65 cases.

To evaluate whether a cervical spine fracture increases the death risk in elderly patients, and to define risk factors, we studied the survival of 65 patients (26 women) with a mean age of 77 (66-99) years. 8 of the patients were tetraparetic. In 35 patients, the upper cervical spine was fractured. 7 patients suffered from ankylosing spondylitis. Severe co-morbidity was present in 16. Survival status and the date of death were retrieved from the government official personal registry. The expected survival was calculated from data retrieved from the Swedish National Board of Health and Welfare. Variables having a possible relation with survival (i.e., a p-value < 0.10 when entered into a Kaplan-Meier survival analysis) were used in a Cox multiple regression survival analysis. 53 (24-105) months after injury, 25 of the 65 patients had died. The survival was significantly lower than the expected values. Severe co-morbidity (risk ratio: 5,6), neurological injury (6,4), high age (1,1), and ankylosing spondylitis (5,5) proved to be significant risk factors for death. Thus, a cervical spine fracture may lead to earlier death in a patient with a severe co-morbidity. A neurological complication constitutes a risk also for a previously healthy individual. Patients having ankylosing spondylitis (with increased death risk) run a higher than normal risk of sustaining a cervical spine fracture.

Age Distribution↗

PubMed1999

Bacillus cereus spores in raw milk: factors affecting the contamination of milk during the grazing period.

Psychrotrophic Bacillus cereus is a limiting factor for the shelf-life of pasteurized milk, particularly during the grazing season. Potential sources of contamination and factors that might affect the spore content of milk were studied in detail for a group of eight cows during three 2-wk study periods from June to September over 2 yr. The spore content of milk was strongly associated with the degree of contamination of the teats with soil. High water content of soil, low evaporation of water and dirty access alloys were the most important factors correlating with high spore concentrations. The spore content of soil varied from < 50 to 380,000/g, depending on time and sampling site. The milking equipment did not contribute significantly to the contamination. The spore contents in air during milking (< 100 cfu/m3) and in feed (silage, hay, fresh grass, and concentrates) were too low to be of importance for contamination. The spore content in dung was also low. Further support that soil was the major contamination source was found by comparison of genetic fingerprints by random amplified polymorphic DNA polymerase chain reaction of isolates of B. cereus from soil and milk and by teat cleansing experiments, which resulted in reduced contamination levels in milk.

Animals↗

PubMed1999

The role of the nurse in community psychiatry.

The transformation of psychiatry during the last decades has also resulted in changed and more independent roles for mental health workers of all categories. The nurse has traditionally had a central role in psychiatric care. In the community oriented care the nurse still plays an important role which can be summarised in four points: 1. focus on the rehabilitation of long term mentally ill patients, 2. the case manager function, 3. to provide individual support in the daily living and 4. to provide support to the relatives of the patients. Those different roles are described. The research being done on these different approaches is presented and it is concluded that case management, social skills training and family intervention programmes have all proved to be effective. In these programmes nurses often play a central role.

Community Mental Health Services↗

PubMed1998

Thermodynamics of binding of heterobidentate ligands consisting of spacer-connected acarbose and beta-cyclodextrin to the catalytic and starch-binding domains of glucoamylase from Aspergillus niger shows that the catalytic and starch-binding sites are in close proximity in space.

The binding to glucoamylase 1 from Aspergillus niger (GA1) of a series of four synthetic heterobidentate ligands of acarbose and beta-cyclodextrin (beta-CD) linked together has been studied by isothermal titration calorimetry. GA1 consists of a catalytic and a starch-binding domain (SBD) connected by a heavily O-glycosylated linker region. Acarbose is a strong inhibitor of glucoamylase and binds exclusively in the catalytic site, while the cyclic starch mimic beta-CD binds exclusively to the two sites of SBD. No spacer or spacer arms of 14, 36, and 73 A in their extended conformations connect acarbose and beta-CD. These compounds were used as probes for bidentate ligand binding to both domains in order to estimate the distance between the catalytic site and the SBD binding site in solution. DeltaH of binding of the four heterobidentate ligands is within experimental uncertainty equal to the sum of DeltaH of binding of free acarbose and beta-CD, indicating ligand binding to both domains. However, the binding constants are 4-5 orders of magnitude smaller than for the binding of acarbose (K approximately 10(12) M-1), increasing with spacer length from 2 x 10(7) M-1 for no spacer to 1 x 10(8) M-1 for the 73 A spacer. Subsequent titrations with beta-CD of the glucoamylase-bidentate ligand complexes revealed that only one of the two binding sites of SBD was vacant. Further titrations with acarbose to these mixtures showed complete displacement of the acarbose moiety of the bidentate ligands from the catalytic sites. These experiments show that the bidentate ligands bind to both the catalytic domain and SBD. The weakening of the bidentate ligand binding compared to acarbose is a purely entropic effect point to steric hindrance between SBD and the beta-CD moiety. To test this, titrations of glucoamylase 2, a form containing the catalytic domain and the linker region but lacking SBD, with the bidentate ligands were carried out. The results were indistinguishable from the binding of free acarbose. Thus, the reduced affinity of the bidentate ligands observed with GA1 stems from interactions due to SBD. The results show that the catalytic and starch-binding sites are in close proximity in solution and thus indicate considerable flexibility of the linker region.

Acarbose↗

PubMed1998

Mitogen activated protein (MAP) kinase activity is increased in adult mouse superior cervical ganglia during culturing.

We have used adult mouse superior cervical ganglia (SCG) to study the role of mitogen activated protein (MAP) kinase activity during axonal outgrowth in vitro. An initial peak in activity within the first hours of culture was followed by a substantially higher activity after 1 to 2 days, a time when axons were actively growing. The latter peak is probably a result of both higher levels of protein and increased activity. The addition of nerve growth factor stimulated both outgrowth and kinase activity, whereas treating the cultures with the kinase inhibitor PD98059 had an opposite effect. Taken together, the results suggest that activation of the MAP kinase pathway could be involved in the initiation as well as regulation of axonal outgrowth from adult SCG.

Animals↗

PubMed1998

Barley alpha-amylase bound to its endogenous protein inhibitor BASI: crystal structure of the complex at 1.9 A resolution.

BACKGROUND: Barley alpha-amylase is a 45 kDa enzyme which is involved in starch degradation during barley seed germination. The released sugars provide the plant embryo with energy for growth. The major barley alpha-amylase isozyme (AMY2) binds with high affinity to the endogenous inhibitor BASI (barley alpha-amylase/subtilisin inhibitor) whereas the minor isozyme (AMY1) is not inhibited. BASI is a 19.6 kDa bifunctional protein that can simultaneously inhibit AMY2 and serine proteases of the subtilisin family. This inhibitor may therefore prevent degradation of the endosperm starch during premature sprouting and protect the seed from attack by pathogens secreting proteases. RESULTS: The crystal structure of AMY2 in complex with BASI was determined and refined at 1.9 A resolution. BASI consists of a 12-stranded beta-barrel structure which belongs to the beta-trefoil fold family and inhibits AMY2 by sterically occluding access of the substrate to the active site of the enzyme. The AMY2-BASI complex is characterized by an unusual completely solvated calcium ion located at the protein-protein interface. CONCLUSIONS: The AMY2-BASI complex represents the first reported structure of an endogenous protein-protein complex from a higher plant. The structure of the complex throws light on the strict specificity of BASI for AMY2, and shows that domain B of AMY2 contributes greatly to the specificity of enzyme-inhibitor recognition. In contrast to the three-dimensional structures of porcine pancreatic alpha-amylase in complex with proteinaceous inhibitors, the AMY2-BASI structure reveals that the catalytically essential amino acid residues of the enzyme are not directly bound to the inhibitor. Binding of BASI to AMY2 creates a cavity, exposed to the external medium, that is ideally shaped to accommodate an extra calcium ion. This feature may contribute to the inhibitory effect, as the key amino acid sidechains of the active site are in direct contact with water molecules which are in turn ligated to the calcium ion.

Amino Acid Sequence↗

PubMed1998

Molecular structure of a barley alpha-amylase-inhibitor complex: implications for starch binding and catalysis.

alpha-Amylases are widely occurring, multidomain proteins with a catalytic (beta/alpha)8-barrel. In barley alpha-amylase, insight into the catalytic mechanism is gained from the X-ray crystal structure of its molecular complex with acarbose, a pseudotetrasaccharide that acts like a transition-state analogue and which is shown to bind at two specific regions of the enzyme. The structure of the complex has been refined to an R-factor of 15.1% for all observations with Fo>sigma(Fo) between 10 and 2.8 A resolution. A difference Fourier map produced after refinement of the native structure against the data of the acarbose complex clearly revealed density corresponding to two oligosaccharide-binding sites. One of these is defined as the surface-located starch granule-binding site characteristic of cereal alpha-amylases. It involves stacking of two acarbose rings on Trp276 and Trp277. The other binding region is the active site covering subsites -1, +1 and +2. Here, Glu204 is positioned to act in general acid/base catalysis protonating the glucosidic oxygen atom assisted by Asp289. A water molecule that bridges Glu204 and Asp289 is found at the entrance cavity containing a total of five water molecules. This water molecule is proposed to reprotonate Glu204 and supply the hydroxyl ion for nucleophilic attack on the glucosyl C1 atom. Asp 179 acts as the nucleophile that can bind covalently to the substrate intermediate after bond cleavage. The present complex structure together with the conservation of active-site residues among alpha-amylases and related enzymes, are consistent with a common catalytic mechanism for this class of retaining carbohydrases.

Acarbose↗

PubMed1998

Restoration of catalytic activity beyond wild-type level in glucoamylase from Aspergillus awamori by oxidation of the Glu400-->Cys catalytic-base mutant to cysteinesulfinic acid.

Glucoamylase catalyzes the hydrolysis of glucosidic bonds with inversion of the anomeric configuration. Site-directed mutagenesis and three-dimensional structure determination of the glucoamylase from Aspergillus awamori previously identified Glu179 and Glu400 as the general acid and base catalyst, respectively. The average distance between the two carboxyl groups was measured to be 9.2 A, which is typical for inverting glycosyl hydrolases. In the present study, this distance was increased by replacing the catalytic base Glu400 with cysteine which was then oxidized to cysteinesulfinic acid. Initially, this oxidation occurred during attempts to carboxyalkylate the Cys400 residue with iodoacetic acid, 3-iodopropionic acid, or 4-bromobutyric acid. However, endoproteinase Lys-C digestion of modified glucoamylase followed by high-pressure liquid chromatography in combination with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry on purified peptide fragments demonstrated that all enzyme derivatives contained the cysteinesulfinic acid oxidation product of Cys400. Subsequently, it was demonstrated that treatment of Glu400-->Cys glucoamylase with potassium iodide in the presence of bromine resulted in complete conversion to the cysteinesulfinic acid product. As expected, the catalytic base mutant Glu400-->Cys glucoamylase had very low activity, i.e., 0.2% compared to wild-type. The oxidation of Cys400 to cysteinesulfinic acid, however, restored activity (kcat) on alpha-1,4-linked substrates to levels up to 160% of the wild-type glucoamylase which corresponded to approximately a 700-fold increase in the kcat of the Glu400-->Cys mutant glucoamylase. Whereas Glu400-->Cys glucoamylase was much less thermostable and more sensitive to guanidinium chloride than the wild-type enzyme, the oxidation to cysteinesulfinic acid was accompanied by partial recovery of the stability.

Alkylation↗

PubMed1998

Enzymatic properties of the cysteinesulfinic acid derivative of the catalytic-base mutant Glu400-->Cys of glucoamylase from Aspergillus awamori.

The pKa of the catalytic base was lowered and its distance to the general acid catalyst, Glu179, was increased in the glucoamylase from Aspergillus awamori by replacing the catalytic base Glu400 with cysteine followed by oxidation to cysteinesulfinic acid [Fierobe, H.-P., Mirgorodskaya, E., McGuire, K. A., Roepstorff, P., Svensson, B. and Clarke, A. J. (1998) Biochemistry 37, 3743-3752. 1H NMR spectroscopy demonstrated that the oxidized mutant Glu400-->Cys-SO2H glucoamylase, like the wild-type, catalyzed hydrolysis with inversion of the anomeric configuration of the product. Relative to the catalytic base mutant Glu400-->Cys, the Cys400-SO2H glucoamylase had 700 times higher kcat toward maltose, while K(m) was unchanged. Compared to wild-type glucoamylase, the Cys400-SO2H derivative had kcat values of 150-190% and 85-320% on malto- and isomaltooligosaccharides, respectively, while K(m) values were similar to those of wild-type with the two disaccharides and 3.5-5.5- and 1.8-2.5-fold higher for the longer malto- and isomaltooligosaccharides substrates, respectively. The pH-activity dependence at saturating concentration of maltose indicated that the pKa of the catalytic base Cys400-SO2H was about 0.5 pH unit lower than that of wild-type Glu400. The Ki of Cys400-SO2H glucoamylase for the pseudotetrasaccharide and potent inhibitor acarbose increased more than 10(4)-fold, but Ki values of the mono- and disaccharide analogues 1-deoxynojirimycin and beta-O-methylacarviosinide were unchanged, suggesting perturbation at binding subsites beyond the catalytic center. A distinct property of Cys400-SO2H glucoamylase was the catalysis of the condensation of beta-D-glucopyranosyl fluoride and subsequent hydrolysis of the product to beta-glucose, under conditions where this was not detected for the wild-type enzyme.

1-Deoxynojirimycin↗

PubMed1998

Secretion, purification, and characterisation of barley alpha-amylase produced by heterologous gene expression in Aspergillus niger.

Efficient production of recombinant barley alpha-amylase has been achieved in Aspergillus niger. The cDNA encoding alpha-amylase isozyme 1 (AMY1) and its signal peptide was placed under the control of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter and the A. nidulans trpC gene terminator. Secretion yields up to 60 mg/l were obtained in media optimised for alpha-amylase activity and low protease activity. The recombinant AMY1 (reAMY1) was purified to homogeneity and found to be identical to native barley AMY1 with respect to size, pI, and immunoreactivity. N-terminal sequence analysis of the recombinant protein indicated that the endogenous plant signal peptide is correctly processed in A. niger. Electrospray ionisation/mass spectrometry gave a molecular mass for the dominant form of 44,960 Da, in accordance with the loss of the LQRS C-terminal residues; glycosylation apparently did not occur. The activities of recombinant and native barley alpha-amylases are very similar towards insoluble and soluble starch as well as 2-chloro-4-nitrophenol beta-D-maltoheptaoside and amylose (degree of polymerisation = 17). Barley alpha-amylase is the first plant protein efficiently secreted and correctly processed by A. niger using its own signal sequence.

Aspergillus niger↗