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.     

Heparin-like molecules regulate the number of epidermal growth factor receptors on vascular smooth muscle cells

The effect of heparin on the binding of epidermal growth factor (EGF) to vascular smooth muscle cells (SMC) was examined. Heparin pretreatment of SMC obtained from bovine aortic explant tissue resulted in significant reductions in the amount of EGF bound. Decreases in mitogen binding were observed with both growth arrested as well as exponentially growing cultures. The heparin concentrations (10-100 micrograms/ml) and pretreatment times (48-72 h) necessary for suppression of EGF binding correlated with the concentrations and temporal requirements necessary for growth inhibition. Chondroitin sulfate, which has negligible antiproliferative activity, had no effect on EGF binding. However, a highly inhibitory heparan sulfate species obtained from postconfluent SMC suppressed EGF binding by 45%. Platelet-derived growth factor and insulin-like growth factor-1 binding were unaffected by heparin. Scatchard analysis revealed that heparin induced 50 to 60% reductions in the numbers of high and low affinity EGF receptors without detectable changes in the binding affinity or ratio of high to low receptors. Experiments were also performed with enzymatically dispersed SMC. These cultures were inhibited by heparin in a time dependent manner which was partially reversible in the presence of EGF. Subsequent studies revealed that heparin suppressed EGF binding in these cultures by 20 to 40%. In summary, heparin reduces the number of EGF receptors on both explant and enzyme dispersed SMC by a mechanism which closely parallels the antiproliferative effects of this glycosaminoglycan.     

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.     

Heparin inhibition of smooth muscle cell proliferation: a cellular site of action

The potential of a given amount of heparin to inhibit smooth muscle cell (SMC) proliferation can be increased more than 13 fold if quiescent cultures are pretreated with this mucopolysaccharide for 48 h. The large increase in antiproliferative activity was attributable to a 74% inhibition of the first cell cycle traverse of SMC after serum addition. If the mucopolysaccharide was added to SMC coincident with serum, the initial cell cycle traverse was only suppressed by 27%. In both heparin pretreated and nonpretreated SMC cultures, 48 to 72 h elapsed before substantial inhibition was observed. The inhibitory effects of heparin were reversible and inversely proportional to the starting cell density of the cultures. The effects of known heparin binding proteins on the inhibitory capability of heparin were examined. Neither platelet-derived growth factor (PDGF), low density lipoprotein (LDL), nor platelet factor 4 (PF4) were able to reduce the antiproliferative effects. Heparin retained full biological activity in medium containing serum depleted of all heparin binding proteins by heparin-Sepharose chromatography. These results indicate that heparin does not inhibit growth by preventing serum mitogens or nutrients from interacting with SMC. Rather, our data suggest that heparin is slowly internalized by SMC following binding to specific, non-PF4 dissociable sites. Heparin may accumulate intracellularly and block a crucial point in the proliferative machinery of SMC.     

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.     

DETERMINATION BY NEUTRON ACTIVATION OF COPPER, MANGANESE, AND ZINC IN THE PINEAL BODY AND OTHER AREAS OF BRAIN TISSUE

Abstract— Neutron activation analysis was used for the simultaneous determination of copper, manganese and zinc in the brains of calves, cows and pigs. Measurements of these three elements in as many as 11 different regions of the brain showed that the highest content is always found in the pineal body. Typical values for calf brains are: Cu, ∼20 μg/g; Mn, ∼3 μg/g; and Zn, ∼90 μg/g dry pineal tissue. As a first approximation, the ratio of Cu:Mn:Zn is roughly constant for all regions.     

Inheritance of mitochondrial aldehyde dehydrogenase: genotyping in Chinese,Japanese and South Korean families reveals dominance of the mutant allele

Summary Genotyping of mitochondrial aldehyde dehydrogenase (ALDH I) was performed in enzymatically amplified DNA of 20 Chinese, Japanese and South Korean families (85 individuals) and in 113 unrelated persons by employing allele-specific oligonucleotide probes and dot blot hybridization. Genotyping individuals with phenotypic deficiency of ALDH I activity always showed the presence of at least one mutant allele. The data are compatible with a model assuming dominant inheritance of the mutant allele, which we have previously suggested on the basis of a population study.     

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.     

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.