Endogenous Benzodiazepine Receptor Ligands in Human and Animal Hepatic Encephalopathy

The role of endogenous benzodiazepine receptor ligands in the pathogenesis of hepatic encephalopathy was studied in humans and in rat models of hepatic encephalopathy. Endogenous benzodiazepine ligands were extracted from rat brain and human CSF by acid treatment and purification by HPLC. Detection and partial characterization of these endogenous benzodiazepine ligands were carried out using both radioreceptor binding assays and radioimmunoassays with anti-benzodiazepine antibodies. Four different benzodiazepine receptor ligands were identified in human and rat tissue, two of which may be diazepam and desmethyldiazepam, based on elution profiles and anti-benzo-diazepine antibody reactivity. Human CSF and serum from patients with hepatic encephalopathy contained approximately 10 times more endogenous benzodiazepine receptor ligand than CSF from controls or nonencephalopathic patients with liver disease. The levels of brain benzodiazepine receptor ligand compounds were also increased approximately 10-fold in rats suffering from fulminant hepatic failure, but not in rats with portacaval shunts, a model of chronic hepatic disease. The increased concentrations of these substances could be behaviorally significant and may contribute to the pathogenesis of hepatic encephalopathy.     

Effect of nucleosome distortion on the linking deficiency in relaxed minichromosomes

The wrapping of closed circular DNA on a protein surface, followed by relaxation with a topoisomerase and removal of proteins, produces a characteristic DNA linking deficiency, delta Lk. We show that the magnitude of delta Lk depends upon the surface shape, and we calculate changes in delta Lk caused by particular distortions of the protein wrapping surface. If the DNA remains attached to the surface during distortion, the DNA winding number, phi, is not altered. The change in delta Lk is then equal to the change in the surface linking number, SLk, which is a straightforward measure of the wrapping of the DNA around the surface. For left-handed wrapping, as in a nucleosome, SLk = -n, the number of times that the DNA axis winds around the axis of the protein complex. We calculate values of SLk for the helical wrapping of a constant length of DNA on protein surfaces having the shapes of cylinders and of ellipsoids and hyperboloids of revolution. If the equatorial radius of the protein is fixed, change in shape from a cylinder to a hyperboloid increases SLk, while the corresponding change to an ellipsoid reduces SLk. We apply the general results to the interpretation of experiments in which minichromosomes are relaxed with topoisomerase at various temperatures and delta Lk is determined. The result is that a distortion of the nucleosome core by at most 5% (the change in the radius at the axial extremity relative to the equator) is sufficient to explain the observed delta Lk changes.     

Comparison of different cationic probes for electron microscopic visualization of bacterial polyanionic capsular material

We have isolated from the ovine rumen eight bacterial strains belonging to the speciesButyrivibrio fibrisolvens. DNA hybridization studies showed that the eight strains could be divided into four homology groups, of which none was closely related to the type strain ATCC 19171. Measurement of cross-hybridization between selected pairs of bacterial strains showed that DNA types which produced low, but significant, cross-hybridization on dot-blots were able to form heteroduplexes with between 8.4% and 32.9% of the efficiency of homoduplex formation. Thermal denaturation of the same heteroduplexes resulted in Tm values 6.4–7.5°C lower than those of the homologous duplexes formed under the same conditions. In some cases, hybridization between strains was below the level of reliable measurement. Similar experiments with ten recently isolated strains ofBacteroides ruminicola sub-sp.brevis revealed a similar degree of genetic divergence between isolates.     

Rhizobium meliloti exopolysaccharide Mutants Elicit Feedback Regulation of Nodule Formation in Alfalfa

Nodule formation by wild-type Rhizobium meliloti is strongly suppressed in younger parts of alfalfa (Medicago sativum L.) root systems as a feedback response to development of the first nodules (G Caetano-Anollés, WD Bauer [1988] Planta 175: 546-557). Mutants of R. meliloti deficient in exopolysaccharide synthesis can induce the formation of organized nodular structures (pseudonodules) on alfalfa roots but are defective in their ability to invade and multiply within host tissues. The formation of empty pseudonodules by exo mutants was found to elicit a feedback suppression of nodule formation similar to that elicited by the wild-type bacteria. Inoculation of an exo mutant onto one side of a split-root system 24 hours before inoculation of the second side with wild-type cells suppressed wild-type nodule formation on the second side in proportion to the extent of pseudonodule formation by the exo mutants. The formation of pseudonodules is thus sufficient to elicit systemic feedback control of nodulation in the host root system: infection thread development and internal proliferation of the bacteria are not required for elicitation of feedback. Pseudonodule formation by the exo mutants was found to be strongly suppressed in split-root systems by prior inoculation on the opposite side with the wild type. Thus, feedback control elicited by the wild-type inhibits Rhizobium-induced redifferentiation of host root cells.     

Chemotaxis,induced gene expression and competitiveness in the rhizosphere

Rhizobia are soil bacteria which symbiotically infect legume roots and generate nodules in which they fix atmospheric nitrogen for the plant in exchange for photosynthetically fixed carbon. A crucial aspect of signal exchange between these symbionts is the secretion of phenolic compounds by the host root which induce nodulation gene expression in the bacteria. Stimulation of nod gene expression by host phenolics is required for nodule formation, is biochemically specific at 10^-6 M, and is mediated by nodD. We and others have shown that rhizobia display chemotaxis to 10^-9 M of the same phenolic compounds. Chemotaxis to inducer phenolics is selectively reduced or abolished by mutations in certain nod genes governing nodulation efficiency or host specificity. Conversely, mutations in rhizobia that affect general motility or chemotaxis have substantial effects on nodulation efficiency and competitiveness. These findings suggest that microbes entering the rhizosphere environment may utilize minor, non-nutrient components in root exudates as signals to guide their movement towards the root surface and elicit changes in gene expression appropriate to this environment.     

Comparison of gene expression of extracellular matrix molecules in brain microvascular endothelial cells and astrocytes.

By use of random-primed cDNA probes the expression of extracellular matrix molecules in cerebral microvascular endothelial cells (cEC) and in astrocytes from mouse brain was examined. Two phenotypically different batches of cloned cEC were used. Expression of major adhesive ECM molecules, constituting the endothelial basement membrane (i.e., fibronectin, laminin A, B and collagen IV) and of other attachment factors, such as SPARC (osteonectin), tenascin and thrombospondin 1, was examined. We have demonstrated that cEC of different morphology display variations in the expression of fibronectin (FN), thrombospondin 1 (TSP1) and collagen IV (C IV). Astrocytes were shown to contain FN, TSP1, TN and SPARC mRNA. Unexpectedly, SPARC mRNA could not be detected in any of the capillary endothelial cells examined. Therefore, we suggest that astrocytes are likely to be involved in endothelial differentiation and function in the central nervous system via ECM molecule secretion.     

Nicotinic Autoreceptors Mediating Enhancement of Acetylcholine Release Become Operative in Conditions of "Impaired" Cholinergic Presynaptic Function

Abstract: The existence in the mammalian CNS of release-inhibiting muscarinic autoreceptors is well established. In contrast, few reports have focused on nicotinic autoreceptors mediating enhancement of acetylcholine (ACh) release. Moreover, it is unclear under what conditions the function of one type of autoreceptor prevails over that of the other. Rat cerebrocortex slices, prelabeled with [³H]choline, were stimulated electrically at 3 or 0.1 Hz. The release of [³H]ACh evoked at both frequencies was inhibited by oxotremorine, a muscarinic receptor agonist, and stimulated by atropine, a muscarinic antagonist. Nicotine, ineffective at 3 Hz, enhanced [³H]ACh release at 0.1 Hz; mecamylamine, a nicotinic antagonist, had no effect at 3 Hz but inhibited [³H]ACh release at 0.1 Hz. The cholinesterase inhibitor neostigmine decreased [³H]ACh release at 3 Hz but not at 0.1 Hz; in the presence of atropine, neostigmine potentiated [³H]ACh release, an effect blocked by mecamylamine. In synaptosomes depolarized with 15 mM KCI, ACh inhibited [³H]ACh release; this inhibition was reversed to an enhancement when the external [Ca²⁺] was lowered. The same occurred when, at 1.2 mM Ca²⁺, external [K⁺] was decreased. Oxotremorine still inhibited [³H]ACh release at 0.1 mM Ca²⁺. When muscarinic receptors were inactivated with atropine, the K⁺ (15 mM)-evoked release of [³H]ACh (at 0.1 mM Ca²⁺) was potently enhanced by ACh acting at nicotinic receptors (EC₅₀? 0.6 µM). In conclusion, synaptic ACh concentration does not seem to determine whether muscarinic or nicotinic autoreceptors are activated. Although muscarinic autoreceptors prevail under normal conditions, nicotinic autoreceptors appear to become responsive to endogenous ACh and to exogenous nicotinic agents under conditions mimicking impairment of ACh release. Our data may explain in part the reported efficacy of cholinesterase inhibitors (and nicotinic agonists) in Alzheimer's disease.     

Cloning of Choriocarcinoma Cells Shows That Invasion Correlates with Expression and Activation of Gelatinase A

Implantation and placental development are dependent upon trophoblast invasion of the endometrium. While the villous trophoblast does not display invasive behavior, the extravillous cytotrophoblast is highly invasive. By cloning BeWo choriocarcinoma cells, we have isolated two distinct clones that share similarities with villous and extravillous cytotrophoblasts. When cultured at the surface of a type I collagen gel, BeWo MC-1 cells were not invasive, whereas BeWo MC-2 cells rapidly invaded this matrix. When injected subcutaneously in nude mice, BeWo MC-1 cells developed a localized tumor and BeWo MC-2 cells developed larger tumors with micrometastases. Gelatinase A expression and minute amounts of gelatinase B were detected in the parental cell line and in both clones. However, the parental and the BeWo MC-2 cells secreted 5- to 10-fold more gelatinase A than the BeWo MC-1 cells. Laminin and matrigel stimulated the production of gelatinase A in BeWo MC-2 cells. Type I collagen promoted the conversion of the 72-kDa progelatinase A in an active enzyme only in parental BeWo and in BeWo MC-2 cells. These clones provide an interesting model for studying the complex mechanisms regulating implantation as well as the controlled invasiveness during implantation compared to tumor invasion.