Andrzej Gindzieński

Comparative studies of osteoblast and fibroblast type I collagen in a patient with osteogenesis imperfecta type IV

The expression of type I collagen has been compared in fibroblast and osteoblast cultures of a patient with moderately severe osteogenesis imperfecta (OI) type IV, with respect to control cells. Electrophoretic analysis of type I collagen showed that both OI osteoblasts and fibroblasts synthesized normal chains and chains with delayed migration. However, the osteoblasts contained a higher proportion of abnormal chains than fibroblasts from the proband. Pulse‐chase experiments showed that the trimers containing abnormal chains were cleared more rapidly from osteoblasts than fibroblasts. Moreover, the collagen secreted by OI osteoblasts had thermal stability 1°C higher than collagen secreted by OI fibroblasts. These results suggest that the abnormal collagen in osteoblasts may be more resistant to intra‐ and extracellular degradation and may thus have better survival than in fibroblasts. This finding could have implications for understanding the clinical phenotype of OI. Copyright

MUC 1 mucin content in gastric juice of duodenal ulcer patients: effect of Helicobacter pylori eradication therapy

It has been suggested that Helicobacter pylori can interact via carbohydrate structures with gastric mucins. Particularly, the Lewis b structures of the secretory MUC 5AC mucin are considered to be putative receptors for bacterial adhesins. Also the epithelial MUC 1 mucin is implicated by some authors to have a major role in the mechanism of infection. The main objective of our study was to evaluate MUC 1 mucin levels in human gastric juice before and at the end of eradication therapy. Any possible changes could suggest the participation of MUC 1 in H. pylori infection. We assume that the amount of the soluble form of MUC 1 exfoliated to the juice correlates with MUC 1 expressed on epithelial cells. Gastric juice samples of 14 duodenal ulcer patients infected with H. pylori were assayed before and at the end of eradication. In all samples, DNA content was determined. Mucin fractions were isolated by gel exclusion chromatography. High molecular mass material containing MUC 1 was subjected to 4%–12% polyacrylamide gradient gels, electrotransfer to Immobilon P and immunodetection. In 12 infected patients (86%), the initial low level of MUC 1 mucin in gastric juice increased at the end of eradication. In comparison to the infected patients, neutral carbohydrate and DNA content in gastric juice diminished after treatment. Our results indicate that the bacterium affects the soluble form of MUC 1 mucin, thus suggesting a likely role of this mucin in the course of H. pylori infection.

Phenotype variability in a daughter and father with mild osteogenesis imperfecta correlated with collagen and prolidase levels in cultured skin fibroblasts.

Studies of collagen biosynthesis and prolidase activity were performed on cultured skin fibroblasts obtained from a female patient and her father, who displayed variable phenotypes of mild osteogenesis imperfecta (OI). For comparison, the same studies were also performed on age-matched controls. Biosynthesis of collagen in fibroblasts of the less affected father was reduced to approximately 50% of control levels, whereas in cells of the more severely affected daughter, it was decreased to about 20% of control levels. Furthermore, the decrease in collagen synthesis in OI fibroblasts was accompanied by a parallel decrease in prolidase activity and expression of beta1 integrin and insulin-like growth factor-I (IGF-I) receptors recovered from the cells. Therefore, prolidase, as well as IGF-I and beta1 integrin receptors involved in collagen metabolism regulation, may represent important factors influencing OI phenotype.

Characterisation of glycoforms of ascitic fluids in benign and malignant diseases.

OBJECTIVES To evaluate the differences in glycoforms of ascitic fluids which can be considered as useful in discrimination between benign and malignant patients. DESIGN AND METHODS The investigations were carried out on 17 benign and 13 malignant patients. To obtain high molecular weight material, gel exclusion chromatography was applied. To evaluate the relative amounts of different glycoforms, ELISA tests with biotinylated lectins were used. RESULTS The fucosylation pattern was found to be similar in malignant and benign group. Fucose linked by alpha 1-6 bond was the most abundant structure. Sialylation was found to be more typical for malignant patients. Alpha 2-6 bond was observed on higher level than alpha 2-3 linkage. T and Tn antigens were present on rather low level with a slight prevalence of T antigen in malignant group. The glycoforms recognized by DSA lectin were more numerous in benign patients. CONCLUSIONS The evaluation of the level of some ascitic fluids glycoforms can be useful in differentiation between malignant and benign diseases.

Elongation factor 2 as a target for selective inhibition of protein synthesis in vitro by the novel aromatic bisamidine.

The effect of the novel aromatic bisamidine 1 on protein synthesis in cell-free translational system isolated from rat livers was studied. The bisamidine 1 caused inhibition of [14C]leucine incorporation into proteins proportionally to its concentration. To establish a precise mechanism of inhibition, we evaluated the effect of the bisamidine 1 on the isolated ribosomes and purified to homogeneity elongation factors. Preincubation of the bisamidine 1 with ribosomes resulted in partial inhibition of their activity in whole elongation system. The eucaryotic elongation factor 1 (eEF-1) was not significantly affected by the bisamidine 1. In contrast to eEF-1, the bisamidine 1 preincubated with the eucaryotic elongation factor 2 (eEF-2) caused total inhibition of its activity in the translocation process. The inhibitory effect of the bisamidine 1 on eEF-2 activity was confirmed in diphtheria toxin-dependent ADP-ribosylation reaction. The results suggest a high action specificity of the bisamidine 1 as potential anticancer drug, since the primary target seems to be highly conserved protein-elongation factor 2.

Application of the 50% Hydrazine Solution Method for O-Glycans Release, their Chemical Labeling, and HPLC Separation

ABSTRACT Mucins are high molecular mass glycoproteins with oligosaccharides O-bonded to the protein core. β-elimination is the most popular method used for releasing of O-glycans. However to such released glycoforms it is difficult to introduce a label to amplify a signal for oligosaccharide detection. In our study we used a combination of the β-elimination and hydrazinolysis methods. Released glycoforms were labeled with para-amino benzooic acid ethyl ester (ABBE) and fractionated on HPLC column. This combined procedure seems to be a good tool for O-glycans analysis.