A new procedure for the determination of molar mass distribution from sedimentation equilibrium runs in an analytical ultracentrifuge (AUC), 1. Measurements with interference optics

A new procedure for the determination of the number-, weight- and z-average molar masses M?_n, M?_w and M?_z and a model molar mass distribution W(M) has been developed from measurements of the sedimentation equilibrium. The method is based on the determination of the experimentally measured reduced concentration profile by extrapolation to zero concentration and direct calculation of the molar mass distribution W(M) by nonlinear regression of the integral equation according to the Simplex procedure. Results obtained in different solvents for the well characterized polystyrene NBS 706 with broad distribution are reasonable. Generally, it can be concluded that the suggested procedure is a suitable possibility for the determination of molar mass average values and the molar mass distribution of polymers if the latter is a log-normal, Schulz-Flory or Poisson distribution.     

Efficacy of Amodiaquine in uncomplicated falciparum malaria in Nigeria in an area with high-level resistance to Chloroquine and Sulphadoxine/Pyrimethamine

Falciparum Malaria is hyperendemic in southern Nigeria and chloroquine resistance is an increasing problem. Therefore, the parasitological and haematological response to treatment with amodiaquine was studied in children under 5 years during a 14-day follow-up. Of 105 children who accomplished the study (out of 114 who were enrolled), 95.3% were parasite-negative on thick blood film on day 7, which decreased to 89.5% on day 14. The haemoglobin levels increased on average by 1.3% on day 14 (±1.9) and more pronounced in children with anaemia < 10 g/dl on enrolment. The number of patients with adverse events (mainly pruritus and nausea) was few. This study shows that amodiaquine is effective, safe and affordable in an area with high resistance to chloroquine.     

Influence of selected wound dressings on PMN elastase in chronic wound fluid and their antioxidative potential in vitro

Exudates from non-healing wounds contain elevated levels of proteolytic enzymes, like elastase from polymorphonuclear granulocytes (PMN elastase), reactive oxygen species (ROS) and reactive nitrogen species (RNS). The overproduction of proteolytic enzymes leads to reduced concentrations of growth factors and proteinase inhibitors, resulting in an imbalance between degradation and remodelling processes. Thus, the reduction of protein-degrading enzymes and scavenging of ROS and RNS seem to be suitable ways to support the healing process of chronic stagnating wounds. The aim of this study was to test selected wound dressings from different biomaterials (collagen, oxidized regenerated cellulose (ORC) and ORC/collagen mixture), regarding their antioxidative potential in vitro and their influence on the concentration and activity of PMN elastase in chronic wound fluid. Antioxidant capacity of the investigated wound dressing was determined by a pholasin-based chemiluminescent assay. PMN elastase concentration was determined by means of ELISA. Enzyme activities could be measured by a fluorescence assay. As the presented data demonstrates, all tested materials showed antioxidant capacity. In addition, the investigated materials were able to reduce the concentration and activity of PMN elastase. Beside other aspects, such as biocompatibility, biodegradability, fluid absorption and clinical effects (e.g. angiogenesis and microcirculation), the understanding of these properties may help to support the further refinement of wound dressings for improved wound healing.     

Classification of anti-FcepsilonRI and anti-IgE autoantibodies in chronic idiopathic urticaria and correlation with disease severity

BACKGROUND: Circulating autoantibodies against FcepsilonRI, IgE, or both occur in approximately one third of patients with chronic idiopathic urticaria (CIU), but not all autoantibodies initiate histamine release. OBJECTIVE: We sought to classify patients with CIU into subsets on the basis of serum bioactivity and immunoreactivity and to examine the relationship between newly defined subtype and disease severity. METHODS: Sera from patients with CIU (n = 78), dermog-raphism (n = 15), and cholinergic urticaria (n = 10) and sera from healthy subjects (n = 39) were analyzed by means of Western blot analysis for anti-FcepsilonRI autoantibodies and for histamine release from basophils and dermal mast cells. In vivo reactivity of autologous serum was tested by means of intradermal injection, and CIU severity was determined on the basis of clinical interview. RESULTS: We classified sera from patients with CIU into 5 subsets: immunoreactive histamine-releasing anti-FcepsilonRI autoantibodies (n = 20 [26%]); immunoreactive anti-FcepsilonRI autoantibodies without histamine-releasing activity (n = 12 [15%]); anti-IgE-like autoantibodies (n = 7 [9%]); serum containing a mast cell-specific histamine-releasing factor (n = 7 [9%]); and sera with no identifiable factor (n = 32 [41%]). Patients with serum histamine-releasing activity had more severe urticaria than patients without such activity. Positive skin test responses to autologous sera were associated with histamine-releasing anti-FcepsilonRI autoantibodies but not with non-histamine-releasing anti-FcepsilonRI autoantibodies. Neither healthy subjects nor patients with dermographism or cholinergic urticaria had his-tamine-releasing anti-FcepsilonRI autoantibodies. CONCLUSION: These data support the specificity of functional anti-FcepsilonRI autoantibodies to CIU. The identification of distinctive subsets of patients suggests that other pathogenic mechanisms occur in CIU in addition to direct ligation of FcepsilonRI by autoantibodies causing dermal mast cell degranulation. Elucidating these mechanisms might lead to new treatments for CIU.     

Ultrastructural changes in hepatocytes of precision-cut rat liver slices after incubation for 24 and 48 hours

Hepatocytes of precision-cut rat liver slices were studied by means of transmission electron microscopy after long-term incubation (24–48 h) in comparison with freshly prepared slices, indicating reversible and irreversible intracellular alterations of the cells. After 24 h incubation the morphological image in transversal sections of slices is characterised by a central zone of damaged and necrotic cells flanked by two to several superficial layers of viable cells. This is typical of a diffusion gradient of oxygen tension and nutrient content from the surface to the centre of the slices. In adapted cells on the surface of the slices we observed an organelle-free layer of fine granular material in the apical cytoplasm followed by parallel oriented stacks of rough endoplasmic reticulum near by. Mitochondria of essentially normal appearance in adapted cells did not contain flocculent densities, which were observed in damaged cells only. The cytoplasm of parenchymal cells consisted of defined areas of clear cytoplasmic material containing numerous branching tubular profiles of smooth endoplasmic reticulum, presumably in the regions with depleted glycogen aggregates. Subcellular signs of necrosis are destroyed mitochondria, dilated endoplasmic reticulum free of ribosomes and clumping of chromatin in the nucleus of hepatocytes. No appreciable differences of the cell organelles were observed between 24 and 48 h of incubation, but the incidence and intensity of signs of necrosis increased with the duration of incubation and the thickness of the slices. The process of these changes may reflect the phenomenon of cellular adaptation and of hypoxic cellular injury in the periphery and the centre of the slices, respectively.     

Visualizing metabolic changes in breast-cancer tissue using 1H-NMR spectroscopy and self-organizing maps

In-vitro NMR spectroscopic examinations of tissue extracts can be combined with appropriate pattern-recognition and visualization techniques in order to monitor characteristic metabolic differences between tissue classes. In the present study, such techniques are applied to a set of 88 breast-tissue samples with the intention of identifying typical differences between various tissue classes. The set contains 49 breast-tumor samples of various tumor grades and 39 samples of healthy tissue. The metabolite compositions of the tissue extracts were investigated using a dual extraction technique and high-resolution (1)H-NMR spectroscopy. The spectra of the hydrophilic and the lipophilic compounds were assigned to three groups according to different malignancy grades of the respective tissue samples. The group characteristics were analyzed using the k-nearest-neighbor method and self-organizing-map visualizations. The results show an increase of UDP-hexose, phosphocholine and phosphoethanolamine concentrations according to the tumor grade. Higher concentrations of taurine were detected in the malignant samples. Myo-inositol and glucose content were elevated in control samples compared with malignant tissue. Both compounds also characterized different subgroups in the pool of unaffected tissue samples depending upon fat content or fibrosis. Several lipid metabolites showed a characteristic elevation with high malignancy.     

DNA end-independent activation of DNA-PK mediated via association with the DNA-binding protein C1D

DNA-dependent protein kinase (DNA-PK), which is involved in DNA double-strand break repair and V(D)J recombination, is comprised of a DNA-targeting component termed Ku and an ~465-kD catalytic subunit, DNA-PK_cs. Although DNA-PK phosphorylates proteins in the presence of DSBs or other discontinuities in the DNA double helix in vitro, the possibility exists that it is also activated in other circumstances via its association with additional proteins. Here, through use of the yeast two-hybrid screen, we discover that the recently identified high affinity DNA binding protein C1D interacts with the putative leucine zipper region of DNA-PK_cs. Furthermore, we show that C1D can interact with DNA-PK in mammalian cells and that C1D is a very effective DNA-PK substrate in vitro. Finally, we establish that C1D directs the activation of DNA-PK in a manner that does not require DNA termini. Therefore, these studies provide a function for C1D and suggest novel mechanisms for DNA-PK activation in vivo.     

Mineralization behaviour of collagen type I immobilized on different substrates

Collagen type I as a robust fibre protein and main component of the extracellular matrix of most tissues is increasingly utilized for surface engineering of biomaterials using different immobilization methods. In the present work we studied the mineralization behaviour of fibrillar collagen type I in simulated body fluid as a measure for conformational changes caused by adsorptive immobilization or immobilization by partial incorporation into the anodic oxide layer on c.p.-titanium using microscopic and vibration spectroscopic methods. Adsorptive immobilization on highly oriented pyrolytic graphite (HOPG) and c.p.-titanium without collagen were used as references. In the initial phase (1-24 h) the kinetics of formation and the morphology of calcium phosphate phases (CPP) are strongly influenced both by the substrate and the immobilization method. Compared to HOPG both types of immobilization on titanium increasingly inhibit the formation of CPP. For longer times (30 d) these initial differences disappear-mineralization product on titanium, irrespective of the presence of collagen, is a mixture of amorphous calcium phosphate and octacalcium phosphate. Contrary to this the mineralization of HOPG substrates results in hydroxy apatite. This is discussed with respect to the conditions during the immobilization as well as the resulting interactions between substrate and immobilized collagen. It is shown that the mineralization process exhibits a high sensitivity with respect to conformational changes caused by these interactions. Possible cell biological relevance of these conformational changes is discussed.     

Molecular and cytogenetic characterization of a non-mosaic isodicentric Y chromosome in a patient with Klinefelter syndrome

We report on an adult male with Klinefelter phenotype and an isodicentric Y chromosome (47,XX,+idic(Y)(q12)), a combination which has to the best of our knowledge not been reported before. The patient was hospitalized in forensic psychiatry because of repeated delinquency, aggressive, aberrant and inappropriate behavior, and borderline intelligence. Molecular cytogenetic studies (FISH) showed that the SRY gene was present on both ends of the idicY, while there was only one signal for the Yq subtelomere probe. Molecular investigations by multiplex PCR, using STS markers covering the short and long arm of the Y chromosome did not indicate a deletion of Y chromosomal material. Molecular investigations of STR markers located on Xp22.3 and Xq28 indicated paternal origin of the additional X chromosome and an error in paternal meiosis I. Results of FISH analysis and molecular investigations are compatible with a phenotype as described for individuals with a 48,XXYY karyotype and support the findings that isodicentric Y chromosomes are frequently accompanied by other sex chromosomal abnormalities.