Tumor promoter and fibronectin induce actin stress fibers and focal adhesion sites in spreading human erythroleukemia (HEL) cells

The effects of plasma fibronectin (pFn) and the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate (TPA) on adhesion and cytoskeletal organization of human erythroleukemia (HEL) cells were studied. HEL cells, that normally grow in suspension, attached rapidly on pFn-coated growth substratum and some cells showed spreading. Upon exposure to TPA most of the cells adhered and showed some degree of spreading also when plated on plastic. The spread cells showed mostly peripheral accumulations of F-actin in addition to actin fibers seen in some of the cells. When the cells were plated in the presence of TPA on pFn or on pFn-fragments, containing the cell binding site, all the cells adhered rapidly, spread extensively, organized prominent F-actin stress fibers and typical ventral plaques of vinculin and alpha-actinin. Both proteins were revealed also in the suspended cells by Western blot analysis. When plated on substratum coated with other pFn-fragments or laminin, the HEL cells did not adhere or spread. Both adhesion on pFn as well as formation of stress fibers in the presence of TPA could be prevented by the synthetic peptide Arg-Gly-Asp-Ser (RGDS). HEL cells were also able to organize typical ventral fibrillar arrays of Fn. Immunostaining and metabolic labeling experiments showed that the cells did not contain or synthesize Fn, indicating that the plaques were formed from exogenous pFn by the cells. The results suggest that Fn and TPA synergistically induce the organization of the actomyosin system in HEL cells by promoting the formation of prominent actin stress fibers and focal adhesion sites.     

Subcellular Location and Neuronal Release of Diazepam Binding Inhibitor

Diazepam binding inhibitor (DBI), a peptide located in CNS neurons, blocks the binding of benzodiazepines and beta-carbolines to the allosteric modulatory sites of gamma-aminobutyric acid (GABAA) receptors. Subcellular fractionation studies of rat brain indicate that DBI is compartmentalized. DBI-like immunoreactivity is highly enriched in synaptosomes obtained by differential centrifugation in isotonic sucrose followed by a Percoll gradient. In synaptosomal lysate, DBI-like immunoreactivity is primarily associated with synaptic vesicles partially purified by differential centrifugation and continuous sucrose gradient. Depolarization induced by high K+ levels (50 mM) or veratridine (50 microM) released DBI stored in neurons of superfused slices of hypothalamus, hippocampus, striatum, and cerebral cortex. The high K+ level-induced release is Ca2+ dependent, and the release induced by veratridine is blocked by 1.7 microM tetrodotoxin. Depolarization released GABA and Met5-enkephalin-Arg6-Phe7 together with DBI. DBI is also released by veratridine depolarization, in a tetrodotoxin-sensitive fashion, from primary cultures of cerebral cortical neurons, but not from cortical astrocytes. Depolarization fails to release DBI from slices of liver and other peripheral organs. These data support the view that DBI may be released as a putative neuromodulatory substance from rat brain neurons.     

Characterization of two lipoproteins containing apolipoproteins B and E from lesion-free human aortic intima

Lesion-free areas of aortic intimas from seven men, 30 to 49 years old, were extracted with aqueous buffer within a few hours after an accidental or sudden death. Two lipoprotein fractions could be isolated by density gradient ultracentrifugation from all cases. The mean composition of fraction I (d less than 1.012 g/ml) resembled that reported for the cholesteryl ester-rich, beta-migrating very low density lipoprotein (beta-VLDL); the composition of fraction II (d 1.021-1.046 g/ml) resembled that of plasma low density lipoprotein (LDL). Mean diameter of the particles was 35 +/- 8 nm in fraction I and 25 +/- 5 nm in fraction II (22 +/- 2 nm in plasma LDL). Both fractions contained apolipoproteins B (apoB) and E (apoE), and had increased electrophoretic mobilities and reduced contents of linoleic acid. The immunoreactivity of apoB to a polyclonal and two monoclonal antibodies in both fractions was not different from that of plasma lipoproteins. The apoE isoform patterns in both fractions were similar to those obtained from the respective postmortem plasmas. When incubated with mouse peritoneal macrophages, fractions I and II enhanced the incorporation of radioactive oleate into cholesteryl esters by 10- to 20-fold and 3- to 4-fold, respectively, in comparison to plasma LDL. In conclusion, our results indicate that lesion-free human aortic intima contains two types of apoB- and apoE-containing lipoprotein particles, both of which might be potentially atherogenic.     

Construction of linear GlcNAcβ1-6Galβ1-OR type oligosaccharides by partial cleavage of GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-OR sequences with jack bean β-N-acetylhexosaminidase

Radiolabelled GlcNAc beta 1-3(GlcNAc beta 1-6)Gal (1), GlcNAc beta 1-3)GlcNAc beta 1-6)Gal beta 1-OCH3 (4), GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4Glc (7), and GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc (10) were cleaved partially with jack bean beta-N-acetylhexosaminidase (EC 3.2.1.30), and the digests were analysed chromatographically. All four oligosaccharides were hydrolysed faster at the (1-6) branch, than at the (1-3) branch, but a high branch specificity was observed only with the glycan 4. The saccharides 1 and 7 resembled each other in the kinetics of the enzyme-catalysed release of their two non-reducing N-acetylglucosamine units, but the glycan 10 was rather different. The partial digestions made it possible to obtain radiolabelled GlcNAc beta 1-6Gal, GlcNAc beta 1-6Gal beta 1-OCH3, GlcNAc beta 1-6Gal beta 1-4Glc, and, in particular, GlcNAc beta 1-6Gal beta 1-4GlcNAc.     

Pancreatic Duct Obstruction in the Pig: Light Microscopy of Chronic Pancreatitis

Morphological changes of pancreatic tissue in young pigs caused by surgical ligation of the main pancreatic duct are described. Nineteen animals from 6 to 7 weeks in age were operated on and necropsied 3 or 6 to 8 weeks later. Twelve pigs developed a pronounced chronic pancreatitis with complete exocrine insufficiency. Of the 7 animals failing to develop ectasia of pancreatic ducts, 2 died due to surgical complications. In addition, 3 pigs were sham-operated and served as controls. In macroscopical studies it was observed that in the pronounced pancreatitis cases the ligated duct was greatly dilated by a clear watery fluid. Only remnants of pale and firm grandular tissues were seen around the ectatic ducts. Microscopically, typical changes of chronic pancreatitis were noted. Complete disappearance of acini was followed by ductular cell proliferations. Glandular tissues were divided into lobuli by fibrotic tissues and fat cells. The wall of the main pancreatic duct was greatly thickened and fibrotic, presenting intensely proliferating ductular cells and round cell infiltrates. Furthermore, enlarged endocrine islets surrounded by connective tissue fibres were seen.     

Laminin chains in the basement membranes of human thymus

Summary In recent studies, the α2 chain of laminin (Ln) has been suggested to be the only laminin α chain expressed in mouse and human thymus. We have now used chain-specific monoclonal antibodies and indirect immunofluorescence microscopy to study the expression of laminin chains in samples of foetal and 6-year-old human thymus. The subepithelial basement membrane of the capsule of foetal 16- to 18-week thymus presented a bright immunoreactivity for Ln α1, α3, β1, β3 and γ1 chains but not for α2 chain, suggesting the expression of laminins-1 and-5. Most cortical and medullary epithelial cells, including Hassall's corpuscles, however, lacked laminin immunoreactivity. Immunoreactivity for Ln β2 chain was only seen in basal laminae of larger blood vessels. In thymic specimens from 6-year-old children, immunoreactivity for the laminin α1, α3, β1, β3 and γ1 chains was invariably found in subepithelial basement membrane of the capsule and that for laminin α2 chain was now also distinct but more heterogeneous. Furthermore, the thymic subepithelial basement membrane of the capsule at all stages showed immunore-activity for collagen type VII, forming the anchoring fibres in epithelial basement membranes. The subcapsular thymic epithelium also showed immunoreactivity for the BP 230 antigen and β₄ integrin subunit, both components of hemidesmosomes. The present results show that the thymic subepithelial basement membrane of the capsule presents properties which are commonly seen in stratified and combined epithelia, and are compatible with suggestions of the antigenic similarity of thymic epithelial cells and keratinocytes.     

Behaviour of cyclic bank voles under risk of mustelid predation: do females avoid copulations?

Mustelid odours have been shown to suppress breeding in captive bank voles (Clethrionomys glareolus) from cyclic populations (Ylönen 1989; Ylönen and Ronkainen 1994). The mechanism behind the suppression is unknown. Based on a series of behavioural trials and breeding experiments with pairs of bank voles in breeding condition, we suggest that the primary cause for breeding suppression is a change in female mating behaviour. Experimental female-male pairs (n=34) exposed to mustelid odour decreased their general activity compared to control pairs (n=34). When encountering males in behavioural trials, females exposed to stoat odour were more aggressive and actively avoided precopulatory behaviours of males. No copulations were observed in experimental pairs compared to five in control pairs during the behavioural trials. Males actively approached females in general but male behaviour did not change under exposure to mustelid odours. We suggest that females are more vulnerable to mustelid predators than males and therefore actively avoid copulations in the (indirect) presence of mustelids. As well as this behavioural response, internal abortive mechanisms (cf. Bruce 1959) could play a role in the observed breeding suppression.     

Microfungal species composition and fungal biomass in a coniferous forest soil polluted by alkaline deposition

Isolations of soil microfungi from the humus (F/H-layer) of a coniferous forest soil which was either unpolluted (pH 4.1) or polluted (pH 6.6) for 25 years by deposition of alkaline dust, were made by soil washing and spore plating. Both techniques revealed similar changes in species composition. Alkaline dust exposure caused a reduction in overall species numbers, but led to higher relative isolation frequencies of Mortierella alpina, Oidiodendron tenuissimum, Penicillium montanese, Sagenomella verticillata, and Trichosporiella sporotrichioides. The incidence of M. isabellina, O. cf. clamydosporium, P. spinulosum, Penicillium sp. 1, P. sclerotiorum, Trichoderma viride, and Verticillium bulbillosum was reduced on polluted sites. The amount of the mainly fungal-derived phospholipid fatty acid 18 : 26 decreased by 23%, while the amount of ergosterol increased by 9% in the polluted soil. Offprint requests to: H. Fritze.     

Factors affecting species number and density of dabbling duck guilds in North Europe

We addressed how species number and pair density in guilds of co-existing species is related to habitat structure, and to the abundance and diversity of food resources using the assemblage of seven species of dabbling ducks (genus Anas ) breeding in 60 lakes distributed over six regions in temperate north Europe
Partial correlation and multiple regression revealed that species richness was best predicted by habitat structural diversity as indexed by a principal component analysis based on 18 vegetation and lake characteristics, and by the abundance of aquatic and emergent prey We found no effect of lake size or prey size diversity on species richness Pair density was correlated with the percentage of shoreline with horsetails ( Equisetum ), by habitat structural diversity and by the abundance of emergent invertebrate prey Neither prey size diversity nor abundance of aquatic prey correlated with pair density Species richness and pair density in North European duck guilds vary both with habitat structure and prey availability