Pseudorabies virus glycoproteins gII and gp50 are essential for virus penetration.

Pseudorabies virus (PrV) glycoproteins gII and gp50 are major constituents of the viral envelope and targets of neutralizing monoclonal antibodies. Both are homologs of essential glycoproteins found in herpes simplex virus, gB (gII) and gD (gp50). We recently isolated a gII-negative PrV deletion mutant on complementing cell lines and established the essential character of gII for PrV replication (I. Rauh, F. Weiland, F. Fehler, G. Keil, and T.C. Mettenleiter, J. Virol. 65: 621-631, 1991). In this report, we describe the isolation of a gp50-negative PrV mutant after constructing cell lines that constitutively express gp50 and phenotypically complement the gp50 defect. Analysis of the gp50- mutant proved that gp50 is essential for PrV replication. Further studies showed that both gII and gp50 are required for viral penetration into target cells. The penetration defect in the gII and gp50 deletion mutants could be overcome by experimental polyethylene glycol-induced membrane fusion. Surprisingly, whereas gII proved to be essential for both penetration and cell-cell spread of the virus, gp50 was required only for penetration and appeared dispensable for direct cell-cell spread.     

Pharmacology of insect GABA receptors

A GABA-operated Cl^– channel that is bicuculline-insensitive is abundant in the nervous tissue of cockroach, in housefly head preparations and thorax/abdomen preparations, and in similar preparations from several insect species. Bicuculline-insensitive GABA-operated Cl^– channels, which are rare in vertebrates, possess sites of action of benzodiazepines, steroids and insecticides that are pharmacologically-distinct from corresponding sites on vertebrate GABA_A receptors. The pharmacological profile of the benzodiazepine-binding site linked to an insect CNS GABA-operated Cl^– channel resembles more closely that of vertebrate peripheral benzodiazepine-binding sites. Six pregnane steroids and certain polychlorocycloalkane insecticides, which are active att-butylbicy-clophosphorothionate (TBPS)-binding sites, also differ in their effectiveness on vertebrate and insect GABA receptors. Radioligand binding and physiological studies indicate that in insects there may be subtypes of the GABA receptor. Molecular biology offers experimental approaches to understanding the basis of this diversity.Special issue dedicated to Dr. Eugene Roberts     

Altered drug translocation mediated by the MDR protein: Direct,indirect, or both?

Overexpression of the MDR protein, or p-glycoprotein (p-GP), in cells leads to decreased initial rates of accumulation and altered intracellular retention of chemotherapeutic drugs and a variety of other compounds. Thus, increased expression of the protein is related to increased drug resistance. Since several homologues of the MDR protein (CRP, ltpGPA, PDR5, sapABCDF) are also involved in conferring drug resistance phenomena in microorganisms, elucidating the function of the MDR protein at a molecular level will have important general applications. Although MDR protein function has been studied for nearly 20 years, interpretation of most data is complicated by the drug-selection conditions used to create model MDR cell lines. Precisely what level of resistance to particular drugs is conferred by a given amount of MDR protein, as well as a variety of other critical issues, are not yet resolved. Data from a number of laboratories has been gathered in support of at least four different models for the MDR protein. One model is that the protein uses the energy released from ATP hydrolysis to directly translocate drugs out of cells in some fashion. Another is that MDR protein overexpression perturbs electrical membrane potential () and/or intracellular pH (pH_i) and therebyindirectly alters translocation and intracellular retention of hydrophobic drugs that are cationic, weakly basic, and/or that react with intracellular targets in a pH_i, or -dependent manner. A third model proposes that the protein alternates between drug pump and Cl^– channel (or channel regulator) conformations, implying that both direct and indirect mechanisms of altered drug translocation may be catalyzed by MDR protein. A fourth is that the protein acts as an ATP channel. Our recent work has tested predictions of these models via kinetic analysis of drug transport and single-cell photometry analysis of pH_i, , and volume regulation in novel MDR and CFTR transfectants that have not been exposed to chemotherapeutic drugs prior to analysis. This paper reviews these data and previous work from other laboratories, as well as relevant transport physiology concepts, and summarizes how they either support or contradict the different models for MDR protein function.     

Astrocytes alter their polarity in organotypic slice cultures of rat visual cortex

The ultrastructure of astrocytes in an organotypic slice culture of the rat visual cortex was investigated using ultrathin sections and freeze-fracture replicas. After a culture period of 9–15 days, a glial scaffold formed that separated the bulk of the slice neuropil from the medium and the underlying plasma clot. However, the glial cells and processes did not build a dense barrier but allowed the outgrowth of neurites. A basal lamina covering the medium-oriented surface of the astrocytes was not found. In freeze-fracture replicas, orthogonal arrays of particles (OAP) were characteristic components of astrocytic membranes. The OAP density in membranes bordering the medium was 35±13 OAP/m², corresponding to 2.5% of this membrane area; the OAP density in membranes within the slice neuropil was 22±12 OAP/², corresponding to 1.4% of this membrane area. Although the difference was significant, it was greatly reduced when comparing OAP densities in endfoot and non-endfoot membranes in vivo. Another mode of polarity was recognized in astrocytes of the organotypic slice culture. In membranes of astrocytes bordering upon the medium, the density of non-OAP intramembranous particles (IMP) was clearly higher (1130±136 IMP/ m²) than in membranes of astrocytes in the center of the slice (700±172 IMP/m²). This pronounced IMP-related polarity was observed neither in vivo nor in cultured astrocytes. The present study suggests, together with data from the literature, that the distribution of astrocytic OAP across the cell surface is influenced by the existence of a basal lamina and neuronal activity, and that astrocytes possess a more remarkable plasticity of membrane structure than previously suspected.     

Dynamic properties of cortical evoked (10 Hz) oscillations: theory and experiment

Experiments probed the dynamic properties of stimulus-evoked (10 Hz) oscillations in somatosensory cortex of anesthetized rats. Experimental paradigms and statistical time series analysis were based on theoretical ideas from a dynamic approach to temporal patterns of neuronal activity. From the results of a double-stimulus paradigm we conclude that the neuronal response contains two components with different dynamics and different coupling to the stimulus. Based on this result a quantitative dynamic model is derived, making use of normal form theory for bifurcating vector fields. The variables used are abstract, but measurable, dynamic components. The model parameters capture the dynamic properties of neuronal response and are related to experimental results. A structural interpretation of the model can be given in terms of the collective dynamics of neuronal groups, their mutual interaction, and their coupling to peripheral stimuli. The model predicts the stimulusdependent lifetime of the oscillations as observed in experiment. We show that this prediction relies on the basic concept of dynamic bistability and does not depend on the modeling details.     

l-Fucose residues on cellulose-based dialysis membranes: Quantification of membrane-associatedl-fucose and analysis of specific lectin binding

Contact of mononuclear human leukocytes with cellulose dialysis membranes may result in complement-independent cell activation, i.e. enhanced synthesis of cytokines, prostaglandins and an increase in 2-microglobulin synthesis. Cellular contact activation is specifically inhibited by the monosaccharidel-fucose suggesting that dialysis membrane associatedl-fucose residues are involved in leukocyte activation. In this study we have detected and quantitatedl-fucose on commercially-available cellulose dialysis membranes using two approaches. A sensitive enzymatic fluorescence assay detectedl-fucose after acid hydrolysis of flat sheet membranes. Values ranged from 79.3±3.6 to 90.2±5.0 pmol cm^–2 for Hemophan® or Cuprophan® respectively. Enzymatic cleavage of terminal -l-fucopyranoses with -l-fucosidase yielded 7.7±3.3 pmoll-fucose per cm² for Cuprophan. Enzymatic hydrolysis of the synthetic polymer membranes AN-69 and PC-PE did not yield detectable amounts ofl-fucose. In a second approach, binding of the fucose specific lectins ofLotus tetragonolobus andUlex europaeus (UEAI) demonstrated the presence of biologically accessiblel-fucose on the surface of cellulose membranes. Specific binding was observed with Cuprophan®, and up to 2.6±0.3 pmoll-fucose per cm² was calculated to be present from Langmuir-type adsorption isotherms. The data presented are in line with the hypothesis that surface-associatedl-fucose residues on cellulose dialysis membranes participate in leukocyte contact activation.     

Investigations on the cuticle of the polychaete elytra using energy dispersive X-ray analysis

Energy dispersive X-ray analysis of elements with Z>11 in a SEM (scanning electron microscope) was used to investigate the elytra ofLagisca extenuata, Lepidonotus clava, andHarmothoe areolata from Naples (Italy) and Banyuls (France). High concentrations of halogens and a few other elements were found in certain papillae in samples from both locations. Additional TEM-examinations and X-ray analysis of thin sections revealed that the halogen concentration is inversely related to the collagen content of the matrix. The halogens are presumably bound to tyrosines, which occur in these structures. In addition, accumulation of Mn²⁺ and possibly Fe³⁺ in the papillae might depend on environmental conditions. The results show that valuable information about the chemical composition of biological structures can be obtained by energy dispersive X-ray analysis. Moreover, the results indicate that this method may be useful for environmental investigations.This work is part of a doctoral thesis, written at the Zoological Institute of the University at Hamburg.     

Neue Algen aus den Grundwasserweihern bei Kienberg-Gaming,N.-Ö

Ohne Zusammenfassung     

Culture of a Gastric Non‐Helicobacter pylori Helicobacter from the Stomach of a 14‐Year‐Old Girl

Helicobacter felis belongs to the fastidious gastric non‐Helicobacter pylori helicobacter species that are typically found in the stomach of cats and dogs. These bacteria have the potential to colonize the human stomach and are then associated with gastritis, gastroduodenal ulcers, and MALT lymphoma. Strains cultured from the human stomach are rare. Here, we present the first isolation of H. felis from a gastric biopsy specimen of a 14‐year‐old girl who presented with persistent epigastric pain. The strain was cultured using our routine protocol for H. pylori and identified by phylogenetic analyses of partial urease AB and gyrB gene sequences.