Frequency of individual udder quarters with elevated CMT scores in cows' milk samples with low somatic cell counts.
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Animals↗
Biomedical subjects
G Pettersson
Publications and source records attributed to G Pettersson.
Family 7 cellobiohydrolases from Phanerochaete chrysosporium: crystal structure of the catalytic module of Cel7D (CBH58) at 1.32 A resolution and homology models of the isozymes.
Cellobiohydrolase 58 (Cel7D) is the major cellulase produced by the white-rot fungus Phanerochaete chrysosporium, constituting approximately 10 % of the total secreted protein in liquid culture on cellulose. The enzyme is classified into family 7 of the glycosyl hydrolases, together with cellobiohydrolase I (Cel7A) and endoglucanase I (Cel7B) from Trichoderma reesei. Like those enzymes, it catalyses cellulose hydrolysis with net retention of the anomeric carbon configuration. The structure of the catalytic module (431 residues) of Cel7D was determined at 3.0 A resolution using the structure of Cel7A from T. reesei as a search model in molecular replacement, and ultimately refined at 1.32 A resolution. The core structure is a beta-sandwich composed of two large and mainly antiparallel beta-sheets packed onto each other. A long cellulose-binding groove is formed by loops on one face of the sandwich. The catalytic residues are conserved and the mechanism is expected to be the same as for other family members. The Phanerochaete Cel7D binding site is more open than that of the T. reesei cellobiohydrolase, as a result of deletions and other changes in the loop regions, which may explain observed differences in catalytic properties. The binding site is not, however, as open as the groove of the corresponding endoglucanase. A tyrosine residue at the entrance of the tunnel may be part of an additional subsite not present in the T. reesei cellobiohydrolase. The Cel7D structure was used to model the products of the five other family 7 genes found in P. chrysosporium. The results suggest that at least two of these will have differences in specificity and possibly catalytic mechanism, thus offering some explanation for the presence of Cel7 isozymes in this species, which are differentially expressed in response to various growth conditions.
Amino Acid Sequence↗
Kinetics of the coupled reaction catalysed by a fusion protein of beta-galactosidase and galactose dehydrogenase.
The mechanistic implications of the kinetic behaviour of a fusion protein of beta-galactosidase and galactose dehydrogenase have been analysed in view of predictions based on experimentally determined kinetic parameter values for the galactosidase and dehydrogenase activities of the protein. The results show that the time course of galactonolactone formation from lactose in the coupled reaction catalysed by the fusion protein can be most satisfactorily accounted for in terms of a free-diffusion mechanism when consideration is given to the mutarotation of the reaction intermediate galactose. It is concluded that no tenable kinetic evidence is available to support the proposal that the fusion protein catalyses galactonolactone formation from lactose by a mechanism involving channelling of galactose.
Diffusion↗
Structural basis for enantiomer binding and separation of a common beta-blocker: crystal structure of cellobiohydrolase Cel7A with bound (S)-propranolol at 1.9 A resolution.
Cellobiohydrolase Cel7A (previously called CBH 1), the major cellulase produced by the mould fungus Trichoderma reesei, has been successfully exploited as a chiral selector for separation of stereo-isomers of some important pharmaceutical compounds, e.g. adrenergic beta-blockers. Previous investigations, including experiments with catalytically deficient mutants of Cel7A, point unanimously to the active site as being responsible for discrimination of enantiomers. In this work the structural basis for enantioselectivity of basic drugs by Cel7A has been studied by X-ray crystallography. The catalytic domain of Cel7A was co-crystallised with the (S)-enantiomer of a common beta-blocker, propranolol, at pH 7, and the structure of the complex was determined and refined at 1. 9 A resolution. Indeed, (S)-propranolol binds at the active site, in glucosyl-binding subsites -1/+1. The catalytic residues Glu212 and Glu217 make tight salt links with the secondary amino group of (S)-propranolol. The oxygen atom attached to the chiral centre of (S)-propranolol forms hydrogen bonds to the nucleophile Glu212 O(epsilon1) and to Gln175 N(epsilon2), whereas the aromatic naphthyl moiety stacks with the indole ring of Trp376 in site +1. The bidentate charge interaction with the catalytic glutamate residues is apparently crucial, since no enantioselectivity has been obtained with the catalytically deficient mutants E212Q and E217Q. Activity inhibition experiments with wild-type Cel7A were performed in conditions close to those used for crystallisation. Competitive inhibition constants for (R)- and (S)-propranolol were determined at 220 microM and 44 microM, respectively, corresponding to binding free energies of 20 kJ/mol and 24 kJ/mol, respectively. The K(i) value for (R)-propranolol was 57-fold lower than the highest concentration, 12.5 mM, used in co-crystallisation experiments. Still several attempts to obtain a complex with the (R)-enantiomer have failed. By using cellobiose as a selective competing ligand, the retention of the enantiomers of propranolol on the chiral stationary phase (CSP) based on Cel7A mutant D214N were resolved into enantioselective and non- selective binding. The enantioselective binding was weaker for both enantiomers on D214N-CSP than on wild-type-CSP.
Adrenergic beta-Antagonists↗
An amperometric cellobiose dehydrogenase-based biosensor can be used for measurement of cellulase activity.
The hemoflavoenzyme cellobiose dehydrogenase (CDH, EC 1.1.99.18) from Phanerochaete chrysosporium has been used in an amperometric redox polymer-based biosensor. Used in conjugation with a FIA system this biosensor can replace colorimetric assays for measuring cellobiose liberated from cellulose in a series of cellulase-containing samples. The biosensor gave the same result as the Somogyi-Nelson method in a less time-consuming and laborious manner. The two methods showed about the same precision.
Biosensing Techniques↗
Tracheal cancer in Denmark: a nationwide study.
OBJECTIVE: Most published series on tracheal cancer reflect single institution experiences. We used the nationwide Danish Cancer Registry to report on characteristics and treatment of tracheal cancers in Denmark. METHODS: One hundred and nine cases of primary tracheal cancers were extracted from the registry in the period 1978-1995. The clinical data, histological distribution and treatment modalities were analyzed. The cancers were staged in four groups (stage I-IV) according to size, location and spread. RESULTS: Seventeen cases were diagnosed at autopsy. Ninety-two cases were diagnosed in vivo and 84% of these within 3 months after the first consultation. Sixty-three percent of the cancers were squamous cell carcinomas and only 7% were adenoid cystic carcinomas. The disease was at stage I in 21%, stage II in 23%, stage III in 6% and stage IV in 50%. The majority of the patients received radiotherapy as single treatment. Only nine patients were offered surgery (six were resected and three were found inoperable). The overall survival rates for cases diagnosed in vivo were 1-year 32%, 2-year 20% and 5-year 13%. For the resected patients the 5- and 15-year survival rates were 50%. CONCLUSIONS: Tracheal cancers were rare and adenoid cystic carcinomas not as frequent as generally believed. Surgery was rarely offered. A resectability rate of only 10% is not adequately explained by selection bias and indicates a nihilistic attitude based on ignorance about surgical treatment of tracheal cancers. A more dedicated and aggressive approach with centralized workup and radical treatment is strongly recommended.
Adult↗
Mechanism of substrate inhibition in cellulose synergistic degradation.
A comprehensive experimental study of substrate inhibition in cellulose hydrolysis based on a well defined system is presented. The hydrolysis of bacterial cellulose by synergistically operating binary mixtures of cellobiohydrolase I from Trichoderma reesei and five different endoglucanases as well as their catalytic domains displays a characteristic substrate inhibition. This inhibition phenomenon is shown to require the two-domain structure of an intact cellobiohydrolase. The experimental data were in accordance with a mechanism where cellobiohydrolases previously bound to the cellulose by means of their cellulose binding domains are able to find chain ends by lateral diffusion. An increased substrate concentration at a fixed enzyme load will also increase the average diffusion distance/time needed for cellobiohydrolases to reach new chain ends created by endoglucanases, resulting in an apparent substrate inhibition of the synergistic action. The connection between the binding properties and the substrate inhibition is encouraging with respect to molecular engineering of the binding domain for optimal performance in biotechnological processes.
Cellulase↗
Esophageal injury during radiofrequency ablation for atrial fibrillation.
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Atrial Fibrillation↗
Cellobiohydrolase 58 (P.c. Cel 7D) is complementary to the homologous CBH I (T.r. Cel 7A) in enantioseparations.
Cellobiohydrolase 58 (EC 3.2.1.91, P.c. Cel 7D) from Phanerochaete chrysosporium was immobilized on silica and the resulting material, CBH 58-silica, was then used as a chiral stationary phase (CSP) in liquid chromatographic separations of enantiomers. The enantioselectivities obtained on CBH 58-silica were compared with those on CBH I-silica (a phase based on a corresponding cellulase from Trichoderma reesei). CBH 58-silica displayed higher selectivity than CBH I-silica for the more hydrophilic compounds, such as atenolol and metoprolol, although great similarities in chiral separation of beta-adrenergic antagonists were found between the two phases. None of the acidic compounds tested could be resolved on the CBH 58 phase. Moreover, the solutes were retained more on the CBH 58 phase in general, indicating an improved application potential in bioanalysis. Addition of cellobiose or lactose, both of which are inhibitors of cellulases, to the mobile phase impaired the enantioselectivity, indicating an overlap of the enantioselective and catalytic sites. The chiral analytes also functioned as competitive inhibitors and their inhibition constants were determined.
Cellulase↗
Do the extracellular enzymes cellobiose dehydrogenase and manganese peroxidase form a pathway in lignin biodegradation?
The extracellular enzyme manganese peroxidase is believed to degrade lignin by a hydrogen peroxide-dependent oxidation of Mn(II) to the reactive species Mn(III) that attacks the lignin. However, Mn(III) is not able to directly oxidise the non-phenolic lignin structures that predominate in native lignin. We show here that pretreatment of a non-phenolic lignin model compound with another extracellular fungal enzyme, cellobiose dehydrogenase, allows the manganese peroxidase system to oxidise this molecule. The mechanism behind this effect is demethoxylation and/or hydroxylation, i.e. conversion of a non-phenolic structure to a phenolic one, mediated by hydroxyl radicals generated by cellobiose dehydrogenase. This suggests that cellobiose dehydrogenase and manganese peroxidase may act in an extracellular pathway in fungal lignin biodegradation. Analytical techniques used in this paper are reverse-phase high-pressure liquid chromatography, gas chromatography connected to mass spectroscopy and UV-visible spectroscopy.
Anisoles↗
Is cellobiose dehydrogenase from Phanerochaete chrysosporium a lignin degrading enzyme?
Cellobiose dehydrogenase (CDH) is an extracellular redox enzyme of ping-pong type, i.e. it has separate oxidative and reductive half reactions. Several wood degrading fungi produce CDH, but the biological function of the enzyme is not known with certainty. It can, however, indirectly generate hydroxyl radicals by reducing Fe(3+) to Fe(2+) and O2 to H2O2. Hydroxyl radicals are then generated by a Fenton type reaction and they can react with various wood compounds, including lignin. In this work we study the effect of CDH on a non-phenolic lignin model compound (3,4-dimethoxyphenyl glycol). The results indicate that CDH can affect lignins in three important ways. (1) It breaks beta-ethers; (2) it demethoxylates aromatic structures in lignins; (3) it introduces hydroxyl groups in non-phenolic lignins. The gamma-irradiated model compound gave a similar pattern of products as the CDH treated model compound, when the samples were analyzed by HPLC, suggesting that hydroxyl radicals are the active component of the CDH system.
Carbohydrate Dehydrogenases↗
A critical review of cellobiose dehydrogenases.
Cellobiose dehydrogenase (CDH) is an extracellular enzyme produced by various wood-degrading fungi. It oxidizes soluble cellodextrins, mannodextrins and lactose efficiently to their corresponding lactones by a ping-pong mechanism using a wide spectrum of electron acceptors including quinones, phenoxyradicals, Fe(3+), Cu(2+) and triiodide ion. Monosaccharides, maltose and molecular oxygen are poor substrates. CDH that adsorbs strongly and specifically to cellulose carries two prosthetic groups; namely, an FAD and a heme in two different domains that can be separated after limited proteolysis. The FAD-containing fragment carries all known catalytic and cellulose binding properties. One-electron acceptors, like ferricyanide, cytochrome c and phenoxy radicals, are, however, reduced more slowly by the FAD-fragment than by the intact enzyme, suggesting that the function of the heme group is to facilitate one-electron transfer. Non-heme forms of CDH have been found in the culture filtrate of some fungi (probably due to the action of fungal proteases) and were for a long time believed to represent a separate enzyme (cellobiose:quinone oxidoreductase, CBQ). The amino acid sequence of CDH has been determined and no significant homology with other proteins was detected for the heme domain. The FAD-domain sequence belongs to the GMC oxidoreductase family that includes, among others, Aspergillus niger glucose oxidase. The homology is most distinct in regions that correspond to the FAD-binding domain in glucose oxidase. A cellulose-binding domain of the fungal type is present in CDH from Myceliophtore thermophila (Sporotrichum thermophile), but in others an internal sequence rich in aromatic amino acid residues has been suggested to be responsible for the cellulose binding. The biological function of CDH is not fully understood, but recent results support a hydroxyl radical-generating mechanism whereby the radical can degrade and modify cellulose, hemicellulose and lignin. CDH has found technical use in highly selective amperometric biosensors and several other applications have been suggested.
Carbohydrate Dehydrogenases↗
A new scaffold for binding haem in the cytochrome domain of the extracellular flavocytochrome cellobiose dehydrogenase.
BACKGROUND: The fungal oxidoreductase cellobiose dehydrogenase (CDH) degrades both lignin and cellulose, and is the only known extracellular flavocytochrome. This haemoflavoenzyme has a multidomain organisation with a b-type cytochrome domain linked to a large flavodehydrogenase domain. The two domains can be separated proteolytically to yield a functional cytochrome and a flavodehydrogenase. Here, we report the crystal structure of the cytochrome domain of CDH. RESULTS: The crystal structure of the b-type cytochrome domain of CDH from the wood-degrading fungus Phanerochaete chrysosporium has been determined at 1.9 A resolution using multiple isomorphous replacement including anomalous scattering information. Three models of the cytochrome have been refined: the in vitro prepared cytochrome in its redox-inactive state (pH 7.5) and redox-active state (pH 4.6), as well as the naturally occurring cytochrome fragment. CONCLUSIONS: The 190-residue long cytochrome domain of CDH folds as a beta sandwich with the topology of the antibody Fab V(H) domain. The haem iron is ligated by Met65 and His163, which confirms previous results from spectroscopic studies. This is only the second example of a b-type cytochrome with this ligation, the first being cytochrome b(562). The haem-propionate groups are surface exposed and, therefore, might play a role in the association between the cytochrome and flavoprotein domain, and in interdomain electron transfer. There are no large differences in overall structure of the cytochrome at redox-active pH as compared with the inactive form, which excludes the possibility that pH-dependent redox inactivation results from partial denaturation. From the electron-density map of the naturally occurring cytochrome, we conclude that it corresponds to the proteolytically prepared cytochrome domain.
Binding Sites↗
Kinetics of the coupled reaction catalysed by a fusion protein of yeast mitochondrial malate dehydrogenase and citrate synthase.
The mechanistic implications of the kinetic behaviour of a fusion protein of mitochondrial malate dehydrogenase and citrate synthase have been reanalysed in view of predictions based on experimentally determined kinetic parameter values for the dehydrogenase and synthase activities of the protein. The results show that the time-course of citrate formation from malate in the coupled reaction catalysed by the fusion protein can be most satisfactorily accounted for in terms of a free-diffusion mechanism when consideration is taken to the inhibitory effects of NADH and oxaloacetate on the malate dehydrogenase activity. The effect of aspartate aminotransferase on the coupled reaction is likewise fully consistent with that expected for a free-diffusion mechanism. It is concluded that no tenable kinetic evidence is available to support the proposal that the fusion protein catalyses citrate formation from malate by a mechanism involving channelling of the intermediate oxaloacetate.
Citrate (si)-Synthase↗
The control of haemolysis during transurethral resection of the prostate when water is used for irrigation: monitoring absorption by the ethanol method.
OBJECTIVE: To determine whether the addition of ethanol to water for irrigation during transurethral resection of the prostate (TURP) and monitoring breath ethanol could be used to detect irrigant absorption and to limit free plasma haemoglobin in cases of absorption. PATIENTS AND METHODS: One hundred patients (46 in Piteå, Sweden and 54 in Uong bi, Vietnam) underwent surgery for benign prostatic hyperplasia (BPH) under an intermittent irrigation technique using water containing 2% ethanol. An expired breath alcohol meter was used to monitor ethanol in the patients' breath every 5 min. Blood samples taken after TURP were assessed for free haemoglobin in 99 patients, and other markers of haemolysis were also evaluated in the Swedish group. RESULTS: Thirty-two patients had detectable ethanol in their breath. There was a close correlation between the maximum ethanol reading during surgery and the level of free plasma haemoglobin after TURP (r = 0.90, P < 0.001). There was no correlation between the duration of TURP and the free haemoglobin level. CONCLUSION: Monitoring breath ethanol during TURP assesses absorption and so can help to keep control of haemolysis. It is suggested that the value on the alcohol meter should not be allowed to exceed 0.15 (corresponding to a blood ethanol level of 0.15 per thousand), which should maintain the free plasma haemoglobin level at < 1.0 g/L after TURP. Restricting the operative duration per se is not a reliable safety measure.
Absorption↗
Outcome of septal myectomy in patients with hypertrophic obstructive cardiomyopathy.
OBJECTIVES: To study the outcome of septal myectomy in patients with hypertrophic obstructive cardiomyopathy. DESIGN: Septal myectomy in patients with hypertrophic cardiomyopathy with obstruction of the left ventricular outflow tract (HOCM) is symptomatically effective, and complication rates have been found to be low in large centres performing the procedure routinely. Representing a small centre we studied the outcome after septal myectomy in 11 consecutive patients, aged 44 +/- 21 (mean +/- SD) years with HOCM myectomized at our institution from 1991 to 1998. The patients were evaluated preoperatively using echocardiography and left-sided heart catheterization. RESULTS: Eight patients were operated on after medical treatment had failed and three after sudden deterioration of cardiac function. A Morrow myectomy was performed in 10 patients and a modified Konno procedure in one. Significant reductions were observed in left ventricular outflow tract gradients (77 +/- 29 to 10 +/- 7 mmHg, p < 0.01; n = 11), the degree of mitral valve regurgitation (grades 0-3) (1.7 +/- 1.0 to 0.8 +/- 0.7, p < 0.01; n = 11), NYHA functional classification score (2.4 +/- 1.0 to 1.5 +/- 0.7, p < 0.01; n = 11) and all five patients with angina preoperatively had an improved CCS angina classification score. There were no operative or early postoperative (30 days) deaths. One patient operated on with the modified Konno procedure was reoperated for a septal patch suture leak. During follow-up (43 +/- 24 months, range 11-83), the linearized mortality rate was 3.6% per year. One patient died from a pancreas cancer, one probably from coronary artery disease and one suddenly of unknown cause. CONCLUSION: We conclude that septal myectomy efficiently relieves symptoms in HOCM patients, possibly reflecting the direct as well as secondary effects of left ventricular outflow tract gradient reduction. The present results, obtained at a smaller centre for this procedure, should be considered when choosing from available therapeutic alternatives when medical therapy fails: dual chamber pacemaker implantation, percutaneous transluminal septal myocardial ablation or myectomy.
Adolescent↗
Glycogen content in rat liver. Importance for CT and MR imaging.
PURPOSE: CT and MR imaging are appropriate modalities for imaging of the liver. Contrast media are used to obtain a greater difference in attenuation and signal intensity, respectively, between normal liver tissue and focal lesions. However, no studies have attempted to determine whether physiological nutritional status of the liver during fasting is of importance for the native signal of normal liver tissue. MATERIAL AND METHODS: Using normal and fasting rats, we performed hepatic CT and MR imaging and glycogen analyses from excised tissue. RESULTS: A significantly higher liver attenuation in normal rats compared to fasting rats was found in CT. In MR images, there was a small but significantly lower liver signal-to-noise ratio in normal rats compared to fasting rats in T1-weighted and proton density-weighted images. Glycogen analyses showed depleted glycogen deposits in fasting rats and a mean glycogen content of 50.1 mg glucose equivalent/g liver tissue in normal rats. CONCLUSION: In CT, a normal nutritional status increases the native attenuation in normal liver tissue. The changes in attenuation in normal liver tissue correlate well with the additional attenuation of glycogen storage in the hepatocyte. The results indicate that the nutritional status is of less importance in MR imaging.
Animals↗
Discrimination between enantioselective and non-selective binding sites on cellobiohydrolase-based stationary phases by site specific competing ligands.
A systematic study was performed to investigate the influence of cellobiose or lactose on the enantioselective retention behaviour of some beta-blockers in liquid chromatography using Cellobiohydrolase (CHB) I from Trichoderma reesei or Cellobiohydrolase 58 from Phanerochaete chrysosporium immobilized on silica as stationary phases. The results revealed that the retention could be described by the function [equation; see text] where the observed capacity factor corresponds to the sum of an enantioselective mode being influenced by a site specific competing ligand (competitor) and a non-selective mode unaffected by the competitor. A non-constrained non-linear least-square regression gave in all cases virtually identical nondisplacable capacity factors (k'ns) for both enantiomers of the same drug. The experimental capacity factors (k'(x,C)) of the enantiomers all show a close fit to the adapted function. The Kd values calculated for the competitor were also virtually identical for each pair of enantiomers and were in accordance with Ki data determined for the competitors in classical enzyme kinetics experiments, demonstrating that one unique site; namely, the catalytic site, was responsible for the enantioselective binding. Similar results were obtained with the resolution of rac-alprenolol and rac-metoprolol on CBH I phase.
Binding Sites↗