Showing papers by "University of Graz published in 1994"

Rapid isolation of lipoproteins and assessment of their peroxidation by high-performance liquid chromatography postcolumn chemiluminescence.

Abstract: Publisher Summary Peroxidation of polyunsaturated lipids proceeds via a free radical chain reaction with consumption of molecular oxygen and concomitant formation of lipid hydroperoxides as the primary reaction products. These lipid hydroperoxides may undergo a variety of secondary reactions that result in the formation of reactive carbonyl products. Lipid peroxidation is commonly assessed by measurement of lipid hydroperoxides, diene conjugates, oxygen consumption, or secondary reaction products (example, malonylaldehyde and 4-hydroxynonenal. The different methods available vary greatly in their requirement for specialized (and expensive) equipment and, at least for biological systems, in their limitations and number of potential interferences and/or artifacts. This chapter discusses developments in the use of the high-performance liquid chromatography (HPLC) postcolumn chemiluminescence (CL) method 5 for the assessment of lipoprotein lipid peroxidation. Lipoprotein oxidation, particularly that of low-density lipoproteins (LDL), as oxidative LDL modification is thought to represent an early and important step in the development of atherosclerosis. In the HPLC postcolumn CL method, the different classes of unoxidized and oxidized lipids are extracted, separated (from one another and from lipophilic antioxidants) by HPLC, and detected sequentially by UV absorption and CL, respectively.

Endomysium antibodies in coeliac disease: an improved method.

Abstract: The ultra structural binding sites of endomysium antibodies have been studied on human umbilical cord tissue. The sensitivity and specificity of IgA endomysium antibodies were compared with recently described methods using basement membrane of smooth muscle of monkey oesophagus. Thirty adults affected by coeliac disease (10 in remission) and 75 healthy adult controls with normal intestinal mucosa (35 false antigliadin positive) were investigated. Sensitivity and correlation of endomysium antibodies with total villous atrophy in untreated coeliac disease patients were 100% on the human umbilical cord smooth muscles, and only 90% on the muscular layer of primate oesophagus. Indirect immunofluorescence was superior to peroxidase staining in detecting these IgA antibodies. The easy availability and enhanced testing sensitivity of the umbilical cord is an advance towards a better diagnostic tool for coeliac disease.

Methods to determine oxidation of low-density lipoproteins.

Abstract: Publisher Summary Low-density lipoprotein (LDL) is an important mediator in the pathogenesis of atherosclerosis. This chapter introduces methods to determine oxidation of low-density lipoproteins. Evaluation of lipoprotein oxidation in vivo, however, is difficult, and most of the investigations deal with in vitro oxidized LDL. During oxidation of LDL by cells or in cell-free systems the composition of the lipoprotein particle progressively changes with time; thus, continuous kinetic measurements or multiple analyses at different time intervals are necessary to assess the kinetics of the oxidation process. Oxidation of LDL in vitro is accompanied by characteristic changes of chemical, physicochemical, and biological properties, and a variety of methods may therefore be used for determining the extent and/or rate of oxidation of LDL. They include measurement of the increase of thiobarbituric acid-reactive substances (TBARS), total lipid hydroperoxides, defined lipid hydroperoxides, hydroxy and hydroperoxy fatty acids, conjugated dienes, oxysterols, lysophosphatides, aldehydes, and fluorescent chromophores, and measurements of the disappearance of endogenous antioxidants and polyunsaturated fatty acids, and oxygen uptake.

Characterization of lipid particles of the yeast, Saccharomyces cerevisiae.

Abstract: Lipid particles of the yeast, Saccharomyces cerevisiae, were isolated to high purity and their components were analysed. The hydrophobic core of this organelle consists of triacylglycerols and steryl esters, which are almost exclusively located to that compartment. Lipid particles are stabilized by a surface membrane consisting of phospholipids and proteins. Electron microscopy confirmed the purity of the preparations and the proposed structure deduced from biochemical experiments. Major proteins of lipid particles have molecular weights of 72, 52, 43 and 34 kDa, respectively. The 43 kDa protein reacts with an antiserum against human apolipoprotein AII. In lipid particles of the yeast mutant strain S. cerevisiae erg6, which is deficient in sterol delta 24-methyltransferase, this protein is missing thereby identifying the protein and confirming our previous finding (Zinser et al., 1993) that sterol delta 24-methylation is associated with lipid particles. A possible involvement of surface proteins of lipid particles in the interaction with other organelles is discussed with respect to sterol translocation in yeast.

Cellulose hydrolysis by the cellulases from Trichoderma reesei: a new model for synergistic interaction.

Abstract: The hydrolysis of Whatman no. 1 filter paper by purified cellulolytic components from Trichoderma reesei and the synergistic action of binary combinations of these enzymes on the same substrate were investigated. At 20 milligrams filter paper, enzyme concentrations needed to obtain half-maximal hydrolysis rates (KE values) were in the 3-4 microM range for the cellobiohydrolases (CBHs) and 0.05-0.10 microM for the endoglucanases (EGs). Catalytic-core proteins of CBH I and EG III, lacking the cellulose-binding domain, exhibit KE values 2.3 and 5.1 times higher than those of the intact enzymes. In synergistic combinations of two cellulases, the KE value of at least one enzyme was 3-10-fold reduced. CBH I/CBH II and CBH I/EG III combinations showed the most powerful synergism, and optimal ratios were a function of the total protein concentration. Results obtained in activity and adsorption assays using filter paper pretreated with one component, followed by inactivation and subsequent hydrolysis with the same or another cellulase component, point to a sequential enzymic attack of the cellulose and seems consistent with the mathematical model presented.

The Mattis Dementia Rating Scale: Normative data from 1,001 healthy volunteers.

Abstract: We administered the Mattis Dementia Rating Scale (MDRS) to 1,001 healthy volunteers, aged 50 to 80 years, randomly selected from our community. Multivariate regression analysis revealed educational level (p = 0.000004) and age (p = 0.00001), but no other sociodemographic or risk factors for stroke, to be significantly associated with the MDRS score. The age- and education-specific lowest quintile cutoff scores ranged from 140 in subjects aged 50 to 59 years with at least college experience to 130 in subjects aged 70 to 80 years with only 4 to 9 years of schooling. These percentile distributions obtained for decades of age and different levels of education should be useful reference values for clinicians and investigators when applying the MDRS to assess cognitive functioning.

A simple hypoplastic constitutive model for sand

Abstract: The paper presents a hypoplastic constitutive model for the three-dimensional non-linear stress-strain and dilatant volume change behaviour of sand. The model is developed without recourse to the concept in elastoplasticity theory such as yield surface, plastic potential and decomposition into elastic and plastic parts. Benefited from the non-linear tensorial functions available from the representation theorem the model possesses simple mathematical formulation and contains only four material parameters, which can be easily identified with triaxial compression tests. Comparison of the predictions with the experimental results shows that the model is capable of capturing the salient behaviour of sand under monotonic loading and is applicable to both drained and undrained conditions.

Proliferation centres in B‐cell malignant lymphoma, lymphocytic (B‐CLL): an immunophenotypic study

Abstract: Proliferation centres, also known as pseudofollicles, are present in approximately 90% of lymphocytic lymphomas (B-CLL). They consist of loosely arranged larger cells that often contain prominent nucleoli. In contrast to true B-cell follicles, which may be found entrapped within the small lymphocytic infiltrate in sections of B-CLL, proliferation centres are said not to contain follicular dendritic cells, although their presence has been occasionally recorded. We have carried out an immunohistochemical study of proliferation centres from 30 lymph node specimens of known cases of B-CLL, classified according to the Kiel classification, in six of which fresh frozen in addition to paraffin embedded tissue was available. We have compared proliferation centres with the surrounding small lymphocytic infiltrate and reactive B-cell follicles with respect to the presence of follicular dendritic cells and their associated network of IgM and complement, the cell proliferation fraction, the concentration of T-cells and the expression of bcl-2 protein. Most proliferation centres contained a delicate follicular dendritic cell network which was associated with IgM and complement, and showed a higher proliferation fraction than the surrounding small lymphocytic infiltrate. The proliferation centres contained more T-cells than the surrounding infiltrate and showed decreased expression of bcl-2 protein. Entrapped reactive B-cell follicles contained a much denser network of follicular dendritic cells, associated with much stronger expression of IgM and complement, and exhibited a very high proliferation fraction; they contained many T-cells and expressed very little bcl-2 protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Cellulose hydrolysis by the cellulases from Trichoderma reesei: adsorptions of two cellobiohydrolases, two endocellulases and their core proteins on filter paper and their relation to hydrolysis.

Abstract: Separate binding of several purified cellulolytic components of Trichoderma reesei on to filter paper was studied and concomitant hydrolysis rates evaluated. Enhancement of mass transfer from the bulk liquid to the solid substrate by agitation has two different effects on adsorption depending on the type of enzyme: (i) the fraction of cellobiohydrolase II (CBH II) and endoglucanase III (EG III) bound at equilibrium is increased, whereas (ii) the rate but not the extent of cellobiohydrolase I (CBH I) and endoglucanase I (EG I) adsorption is affected. The adsorption of CBH I core, a component lacking the cellulose-binding domain (CBD), is, however, not significantly influenced by mass transfer. The CBH I interdomain peptide (present in CBH I core b) does not participate in adsorption but enhances stability. The adsorption of CBH I core proteins is a fully reversible process whereas that of the intact CBH I is not. Thus, the interaction of the CBD with filter paper apparently accounts for the mass-transfer-limited binding rate and also for the irreversible adsorption of intact CBH I. Adsorption isotherms at 50 degrees C indicate very similar relative association constants for the intact cellulases (0.24-0.30 l/g of cellulose), but drastically reduced values for CBH I core proteins (0.03 l/g of cellulose). The specific activities of adsorbed CBH I and of its core proteins are identical and a linear relationship between adsorption and rates of hydrolysis is found only for these enzymes. Thus, non-productive binding on to cellulose seems evident in the case of CBH II and EG III but not CBH I.

DNA of Mycobacterium tuberculosis in Formalin-fixed, Paraffin-embedded Tissue in Tuberculosis and Sarcoidosis Detected by Polymerase Chain Reaction

Abstract: Infection with Mycobacterium tuberculosis is a major cause of death worldwide. Identification of mycobacteria in tissue sections is usually easily achieved by acid-fast stains, but this method sometimes gives unsatisfactory results. The authors therefore compared conventional staining techniques and polymerase chain reaction (PCR) for mycobacterial DNA sequences in 24 selected tissue samples from patients with tuberculosis. In all samples, either positive or negative with acid-fast stain, mycobacterial DNA fragments were detected. In addition, tissue samples from patients with clinically proven sarcoidosis were included as controls. Surprisingly, strong signals for mycobacterial DNA were found in 2 of 15 cases. Polymerase chain reaction is a useful technique in the demonstration of mycobacterial DNA fragments in patients with clinically suspected tuberculosis who have acid fast stain-negative histology. An epithelioid granulomatous reaction in the lung, negative by acid-fast stain and positive for mycobacterial DNA by PCR, however, does not permit a diagnosis of tuberculosis, because a positive result can also be obtained in cases of sarcoidosis. In some cases of sarcoidosis, the causal agent might be either cell wall defective mycobacteria or persistent intracellular DNA from mycobacteria.

Flow and stress characteristics in rigid walled and compliant carotid artery bifurcation models.

Abstract: Computer simulation of pulsatile non-Newtonian blood flow has been carried out in different human carotid artery bifurcation models In the first part of the investigation, two rigid walled models are analysed, differing in the bifurcation angle (wide angle and acute angle bifurcation) and in the shape of both the sinus (narrow and larger sinus width) and the bifurcation region (small and larger rounding of the flow divider), in order to contribute to the study of the geometric factor in atherosclerosis The results show a significant difference in the wall shear stress and in the flow separation Flow recirculation in the sinus is much more pronounced in the acute angle carotid An important factor in flow separation is the sinus width In the second part of the study, flow velocity and wall shear stress distribution have been analysed in a compliant carotid artery bifurcation model In the mathematical model, the non-Newtonian flow field and the idealized elastic wall displacement are coupled and calculated iteratively at each time step Maximum displacement of approximately 6% of the diastolic vessel diameter occurs at the side wall of the bifurcation region The investigation demonstrates that the wall distensibility alters the flow field and the wall shear stress during the systolic phase Comparison with corresponding rigid wall results shows that flow separation and wall shear stress are reduced in the distensible wall model

Nitric oxide synthase immunoreactivity in the enteric nervous system of the developing human digestive tract

Abstract: We have investigated indirectly the presence of nitric oxide in the enteric nervous system of the digestive tract of human fetuses and newborns by nitric oxide synthase (NOS) immunocytochemistry and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry. In the stomach, NOS immunoactivity was confined to the myenteric plexus and nerve fibres in the outer smooth musculature; few immunoreactive nerve cell bodies were found in ganglia of the outer submucous plexus. In the pyloric region, a few nitrergic perikarya were seen in the inner submucous plexus and some immunoreactive fibres were found in the muscularis mucosae. In the small intestine, nitrergic neurons clustered just underneath or above the topographical plane formed by the primary nerve strands of the myenteric plexus up to the 26th week of gestation, after which stage, they occurred throughout the ganglia. Many of their processes contributed to the dense fine-meshed tertiary nerve network of the myenteric plexus and the circular smooth muscle layer. NOS-immunoreactive fibres directed to the circular smooth muscle layer originated from a few NOS-containing perikarya located in the outer submucous plexus. In the colon, caecum and rectum, labelled nerve cells and fibres were numerous in the myenteric plexus; they were also found in the outer submucous plexus. The circular muscle layer had a much denser NOS-immunoreactive innervation than the longitudinally oriented taenia. The marked morphological differences observed between nitrergic neurons within the developing human gastrointestinal tract, together with the typical innervation pattern in the ganglionic and aganglionic nerve networks, support the existenc of distinct subpopulations of NOS-containing enterice neurons acting as interneurons or (inhibitory) motor neurons.

Sensitivity in the clinical diagnosis of malignant melanoma.

Abstract: Accurate clinical diagnosis of malignant melanoma is of great importance for early detection and further treatment. The purpose of this retrospective study was to evaluate the accuracy of clinical diagnosis by using different clinical and histopathological parameters. Calculations are based on a total of 44,258 histopathologically examined skin neoplasms, including 529 melanomas, which were recorded in the histological database of the Department of Dermatology, University of Graz during a 2 year period. The clinical diagnosis of the referring physicians was compared statistically with the final histopathological interpretation. Clinical diagnosis of melanoma showed a sensitivity of 70.1%, a specificity of 99.4%, and a positive predictive value of 60.7%. One hundred and fifty eight melanomas (29.9%) could not be diagnosed clinically. The age-dependent sensitivity was 47.6% in patients 80 years the value was 90.2%. Remarkably, the sensitivity in melanomas >4 mm thickness (64.8%) was lower than in 'melanoma in situ' (72.6%). Our findings underline the importance of further development of clinical examination techniques such as dermoscopy and digital epiluminescence microscopy.

Generation of glycerophospholipid molecular species in the yeast Saccharomyces cerevisiae. Fatty acid pattern of phospholipid classes and selective acyl turnover at sn‐1 and sn‐2 positions

Abstract: Acyl chains linked to phospholipids of the yeast, Saccharomyces cerevisiae, are mainly C16:1 and C18:1 accompanied by minor amounts of C14:0, C16:0 and C18:0. In view of this rather simple fatty acid composition, the question arose whether in yeast, as in higher eukaryotes, fatty acyl groups were characteristically distributed among the sn-1 and sn-2 positions of distinct phospholipid classes. Analysis of fatty acids linked to the sn-1 and sn-2 positions of the major phospholipids showed that indeed saturated fatty acyl groups predominated in the sn-1 positions. While the percentage of saturated fatty acids was low (10%) in phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) from cells grown on rich medium, it was higher in phosphatidylserine (PtdSer) (25%) and highest in phosphatidylinositol (PtdIns) (41%). Oleate was mainly linked to position sn-2, while palmitoleate predominated in position sn-1. Striking differences in the fatty acid distribution of phospholipids that are metabolically closely related (e.g. PtdSer and PtdEtn, PtdEtn and PtdCho, and PtdIns and PtdSer) suggest that pathways must exist for the generation of distinct phospholipid molecular species within the different phospholipid classes. The highly selective incorporation of exogenous [14C]palmitic acid (90%) and [3H]oleic acid (99%) into the sn-2 position of PtdCho, and the preferential incorporation of these fatty acids into the sn-2 position of PtdEtn (70 and 90%, respectively, for palmitic and oleic acid) are compatible with the postulate that phospholipase A2-mediated deacylation followed by reacylation of the lysophospholipids is involved in the generation of phospholipid species in yeast.

Calculation of the partial volume of organic compounds and polymers

Abstract: A novel, universal and easy-to-use approach is presented which allows the ab initio calculation of partial volumes of organic compounds, valid for aqueous solutions at 25°C The method is based on Traube’s additivity principle and concept of volume increments for atoms His concept, however, was improved considerably by manifold adaptations, corrections, and completions Major improvements were performed in context with the increments for nitrogen, and the decrements for ring formation Moreover, a procedure was developed for linking tabulated volumes of inorganic ions to calculated volumes of organic residues The validity of our approach was confirmed by a systematic comparison of calculated and experimental volumes of different classes of organic and biochemical compounds, including small molecules and polymers of nonionic and ionic nature The vast majority of calculated volumes is within a range of ± 2%, if compared with the experimental values A detailed table summarizing calculated and experimental partial volumes of diverse organic and biochemical molecules is presented and allows estimation of errors to be expected when performing calculations of substances of unknown volume The table may also be used as a powerful database for many purposes The prediction of partial volumes of compounds of quite different structure and composition is of great importance for many fields of research: correct interpretation of the results from ultracentrifugal and solution scattering studies, statements on various solute-solvent interactions, etc