The Mr 95,000 gelatin-binding protein in differentiated human macrophages and granulocytes

Cultured adherent human macrophages and a promonocytic cell line, U 937, were previously shown to produce a Mr 95,000 gelatin-binding protein. The protein has no immunologic cross-reactivity with the well- characterized gelatin-binding protein fibronectin and the Mr 70,000 gelatin-binding protein produced by a variety of mesenchymal or epithelial cell types (T. Vartio et al, J Biol Chem 257:8862, 1982). In the present study the Mr 95,000 protein was found in Triton X-100 extracts of granulocytes purified from human blood buffy coat. The protein, as isolated by gelatin-agarose, was immunologically cross- reactive with the corresponding macrophage protein in immunoblotting assay. When peripheral blood and bone marrow cells were examined for the presence of the Mr 95,000 protein by indirect immunofluorescence, positive staining was detected only in differentiated granulocytes but not to any significant extent in metamyelocytes, myelocytes, promyelocytes, or in normal or leukemic blasts. In granulocytes the protein had a granular cytoplasmic distribution. In freshly prepared monocyte cultures, the Mr 95,000 protein was detected in low amounts in the cytoplasm, while along with differentiation of the cells into macrophages, the immunofluorescence increased in a reticular and vesicular cytoplasmic pattern and in a juxtanuclear cap, probably representing the Golgi complex. In conclusion, the Mr 95,000 gelatin- binding protein was specifically detected in macrophages and granulocytes and may thus serve as a differentiation marker for these phagocytic cells.     

Constrictive pericarditis following coronary bypass reoperation. Fibrotic pericardium and a foreign body reaction

A 65-year-old male patient underwent two bypass operations because of coronary artery disease. After the second operation he developed congestive heart failure with breathlessness, ankle swelling, hepatomegaly and poor exercise tolerance. Echocardiographic and haemodynamic findings were characteristic of constrictive pericarditis. Pericardiectomy was performed three months after the second operation. The retrosternal space was replaced with fibrotic, patchy hyalinic tissue, and the pericardium was thick and rigid. Histologically, the thickened pericardium showed dense fibrosis and foreign-body granulomas with large multinuclear giant cells and irregular crystals. This report indicates that foreign body reaction following coronary artery bypass operation may result in constrictive pericarditis with severe heart failure.     

Lack of fibronectin-binding plasma membrane proteins may explain defective pericellular matrix formation in transformed fibroblasts and fibrosarcoma cells

Affinity of iodinated fibronectin (Fn) and its defined proteolytic fragments to electrophoretically separated polypeptides of normal and malignant cells was studied in an overlay assay. Cellular 125I-Fn and a major 125I-Fn fragment (Mr 120,000-140,000), containing the cell-binding site, revealed in fibroblasts Mr 170,000, Mr 140,000, and Mr 47,000 Fn-binding polypeptides of which the first two could also be found in the plasma membrane preparations. Binding of 125I-Fns to Mr 170,000 and Mr 140,000 polypeptides was inhibited by the synthetic peptide Arg-Gly-Asp-Ser and to all 3 polypeptides by Fns and Mr 120,000-140,000 fibronectin fragment. Both fibrosarcoma cells and SV40-virus-transformed fibroblasts appeared to lack the Mr 140,000 Fn-binding polypeptide. Binding was similar when Fn from normal fibroblasts or fibrosarcoma cells was used in the assay, while plasma 125I-Fn had weaker affinity towards the Mr 140,000 polypeptide. Instead, proteolytic Fn-fragments, lacking the cell binding site, did not bind to any proteins in the assay. Radioactive cell-surface labelling showed differences in the corresponding surface polypeptide profiles of normal and malignant cells. The results suggest that the failure of pericellular matrix deposition in malignant cells could be due to either defective surface exposition or defective binding property of the Fn-receptor-like polypeptides.     

Preparative polyacrylamide gel electrophoresis in the isolation of the nephritogenic proteins of passive Heymann nephritis

To study kidney antigens involved in the formation of glomerular subepithelial immune deposits in passive Heymann nephritis polypeptides of 500, 130 and 105 kDa were isolated from rat kidney brush border (BB) membrane fraction using preparative polyacrylamide gel electrophoresis. Polyclonal antibodies raised against these proteins were specific for their respective antigens in immunoblotting. All three antisera bound to proximal tubular BB of kidney and to apical surfaces of several other epithelia as shown by indirect immunofluorescence on frozen sections of normal rat tissues. The anti-500 kDa and anti-105 kDa, but not the anti-130 kDa, antibodies also stained glomeruli and the anti-105 kDa antibodies also endothelial cells. After injection into rats the anti-500 kDa IgG bound to kidney glomeruli forming diffuse, granular deposits of rabbit IgG along the glomerular capillary walls, as shown by direct immunofluorescence. In electron microscopy the immune deposits were subepithelial and electron dense. The deposits remained in glomeruli for at least 60 days and increased with time. Deposits of C3 were not detected and proteinuria did not develop. The anti-130 kDa and the anti-105 kDa IgGs did not form glomerular deposits after in vivo injections. The results suggest that the 500 kDa and the 105 kDa proteins or related antigens are present in glomeruli and the 500 kDa protein is located on the epithelial side of the glomerular basement membrane. Circulating antibodies can bind to the 500 kDa protein forming immune complexes which rearrange and form electron dense deposits. The results further demonstrate that preparative gel electrophoresis is a useful technique for the isolation of kidney proteins of immunopathologic interest.     

Transformation-enhancing activity of gelatin-binding fragments of fibronectin.

Studies have established that cryoprecipitates of the plasma of tumor patients contain a biological activity enhancing morphological cell transformation (transformation-enhancing factor; TEF) in cultures of chicken embryo fibroblasts infected with temperature-sensitive mutants of Rous sarcoma virus. We report here that similar TEF activity is effected by defined fragments of human plasma fibronectin obtained by limited digestion with major humoral or tissue proteinases. TEF activity was obtained from plasminolytic fragments of fibronectin and from cathepsin G-treated fibronectin. No activity was recorded from intact dimeric fibronectin or its reduced and alkylated subunits, from fibrinogen or its plasminolytic fragments, or from plasmin (EC 3.4.21.7) or cathepsin G (EC 3.4.21.20) treated or untreated with proteinase inhibitors. All of the TEF activity of the proteolytic fragments of fibronectin was located on the gelatin-binding peptides. The minimum effective doses in the TEF assay were 750 ng/ml of plasmin-treated fibronectin, 100 ng/ml of gelatin-binding plasminolytic fibronectin (enriched in Mr 180,000--190,000 polypeptides), and 100 ng/ml of gelatin-binding fragments of cathepsin G-treated fibronectin (enriched in a Mr 30,000 fragment). TEF activity of proteinase-treated fibronectin was inhibited by gelatin and by intact dimeric fibronectin. The potent TEF activity of proteolytic fragments of fibronectin raises the possibility that they may have a role in malignant transformation.     

Adhesion protein YadA of Yersinia species mediates binding of bacteria to fibronectin.

The interaction between fibronectin and Yersinia strains was studied. Wild-type Y. enterocolitica strains expressing the virulence-plasmid-encoded adhesion protein YadA adhered strongly to fibronectin-coated coverslips, while their plasmid-cured variants expressed weaker binding. The cloned yadA gene of Y. enterocolitica or Y. pseudotuberculosis conferred fibronectin-binding ability both to Escherichia coli and to Y. psuedotuberculosis strains lacking the YadA protein. The YadA protein did not mediate binding to isolated fragments of fibronectin or to soluble fibronectin.     

Rhabdomyosarcoma of the oesophagus

Summary A 61-year-old man was operated for a large tumor, 12×4 cm in size, in the lower third of the oesophagus. The tumor had the appearance of a pleomorphic rhabdomyosarcoma showing cross striations by light microscopy and typical sarcomeres by electron microscopy. This is the fifth undisputed oesophageal rhabdomyosarcoma described in the literature and the first examined using electron microscope.     

Binding sites for streptococci and staphylococci in fibronectin

Purified cathepsin G fragments of fibronectin were used to locate the binding sites for streptococci and staphylococci in the fibronectin molecule. The iodinated, NH2-terminal, 30-kilodalton (kd) fragment bound to group A and G streptococci and to Staphylococcus aureus. The 125I-labeled, COOH-terminal, 120- to 140-kd fragment bound weakly to group A streptococcus strain and to S. aureus when tested in a buffer of low ionic strength. The 30- and 120- to 140-kd fragments inhibited the binding of iodinated fragments to bacteria. The two fragments were, on a molar basis, equally effective, and they were more potent inhibitors than intact fibronectin. The gelatin-binding 40-kd fragment neither bound to any of the bacterial strains nor inhibited the binding of 125I-labeled 30-kd or 125I-labeled 120- to 140-kd fragments to bacteria. The results indicate that fibronectin has at least two separate binding sites for streptococci and staphylococci, one in the NH2-terminal region and another in the COOH-terminal region of the molecule, both capable of specific interaction with a complementary structure exposed on streptococcal and staphylococcal cell surfaces.     

School Bullies’ Intention to Change Behavior Following Teacher Interventions: Effects of Empathy Arousal,Condemning of Bullying,and Blaming of the Perpetrator

This study examines how bullies’ perceptions of how they were treated by a teacher (or other school personnel) during discussions aimed at putting an end to bullying influenced their intention to change their behavior. After each discussion, which took place as part of the implementation of an anti-bullying program, bullies anonymously reported the extent to which they felt that the teacher aroused their empathy for the victim, condemned their behavior, or blamed them. Half of the schools implementing the program were instructed to handle these discussions in a confrontational way—telling the bully that his behavior is not tolerated—while the other half were instructed to use a non-confronting approach. Schools were randomly assigned to one of the two approaches. A total of 341 cases (188 in primary and 153 in secondary schools) handled in 28 Finnish schools were analyzed. Regression analyses showed that attempts at making bullies feel empathy for the victim and condemning their behavior both increased bullies’ intention to stop. Blaming the bully had no significant effect. Bullies’ intention to change was the lowest when both empathy-arousal and condemning behavior were low. The effects of empathy arousal were stronger when condemning the behavior was low (and vice versa), suggesting that teachers tackling bullying should make sure to use at least one of these strategies. When choosing not to raise the child’s empathy, clear reprobation of the behavior is key.     

Tissue type plasminogen activator,but not urokinase,exerts transformation-enhancing activity

Previous studies have established that plasma cryoprecipitates of tumor patients, culture media of transformed cells and defined proteolytic fragments of fibronectin enhance the morphological cell transformation (TEF activity) in cultures of chicken embryo fibroblasts infected with temperature-sensitive mutants of Rous sarcoma virus. We now report that purified human tissue type plasminogen activator (t-PA), but not urokinase (u-PA), has a similar TEF activity, at doses as low as 2 ng/ml (30 pM). Specific antibodies effectively neutralized the activity. No significant contamination (≤1%) between the preparations of t-PA and fibronectin (FN) or its fragments (FNdp) was detected. The results suggest that t-PA may have a direct role in the process of morphological cell transformation in vitro.