Neuropathological changes in scrapie and Alzheimer's disease are associated with increased expression of apolipoprotein E and cathepsin D in astrocytes.

With the rationale that the neuropathological similarities between scrapie and Alzheimer's disease reflect convergent pathological mechanisms involving altered gene expression, we set out to identify molecular events involved in both processes, using scrapie as a model to study the time course of these changes. We differentially screened a cDNA library constructed from scrapie-infected mice to identify mRNAs that increase or decrease during disease and discovered in this way two mRNAs that are increased in scrapie and Alzheimer's disease. These mRNAs were subsequently shown by sequence analysis to encode apolipoprotein E and cathepsin D (EC 3.4.23.5). Using in situ hybridization and immunocytochemistry to define the cellular and anatomic pathology of altered gene expression, we found that in both diseases the increase in apolipoprotein E and cathepsin D mRNAs and proteins occurred in activated astrocytes. In scrapie, the increase in gene expression occurred soon after the amyloid-forming abnormal isoform of the prion protein has been shown to accumulate in astrocytes. In Alzheimer's disease, the increased expression of cathepsin D also occurred in association with beta-amyloid. These studies reveal some of the molecular antecedents of neuropathological changes in scrapie and Alzheimer's disease and accord new prominence to the role of astrocytes in neurodegenerative conditions.     

A new method for the reconstitution of the anion transport system of the human erythrocyte membrane

Summary The anion transport protein of the human erythrocyte membrane, band 3, was solubilized and purified in solutions of the non-ionic detergent Triton X-100. It was incorporated into spherical lipid bilayers by the following procedure: (1) Dry phosphatidylcholine was suspended in the protein solution. Octylglucopyranoside was added until the milky suspension became clear. (2) The sample was dialyzed overnight against detergentfree buffer. (3) Residual Triton X-100 was removed from the opalescent vesicle suspension by sucrose density gradient centrifugation and subsequent dialysis. Sulfate efflux from the vesicles was studied, under exchange conditions, using a filtration method. Three vesicle subpopulations could be distinguished by analyzing the time course of the efflux. One was nearly impermeable to sulfate, and efflux from another was due to leaks. The largest subpopulation, however, showed transport characteristics very similar to those of the anion transport system of the intact erythrocyte membrane: transport numbers (at 30°C) close to 20 sulfate molecules per band 3 and min, an activation energy of approx. 140 kJ/mol, a pH maximum at pH 6.2, saturation of the sulfate flux at sulfate concentrations around 100mm, inhibition of the flux by H₂DIDS and flufenamate (approx.K _l-values at 30°C: 0.1 and 0.7 m, respectively), and right-side-out orientation of the transport protein (as judged from the inhibition of sulfate efflux by up to 98% by externally added H₂DIDS). Thus, the system represents, for the first time, a reconstitution of all the major properties of the sulfate transport across the erythrocyte membrane.     

Na+/Ca2+ countertransport in plasma membrane of rat pancreatic acinar cells

Summary The presence of a coupled Na⁺/Ca²⁺ exchange system has been demonstrated in plasma membrane vesicles from rat pancreatic acinar cells. Na⁺/Ca²⁺ exchange was investigated by measuring⁴⁵Ca²⁺ uptake and⁴⁵Ca²⁺ efflux in the presence of sodium gradients and at different electrical potential differences across the membrane (=) in the presence of sodium. Plasma membranes were prepared by a MgCl₂ precipitation method and characterized by marker enzyme distribution. When compared to the total homogenate, the typical marker for the plasma membrane, (Na⁺+K⁺)-ATPase was enriched by 23-fold. Markers for the endoplasmic reticulum, such as RNA and NADPH cytochromec reductase, as well as for mitochondria, the cytochromec oxidase, were reduced by twofold, threefold and 10-fold, respectively. For the Na⁺/Ca²⁺ countertransport system, the Ca²⁺ uptake after 1 min of incubation was half-maximal at 0.62 mol/liter Ca²⁺ and at 20 mmol/liter Na⁺ concentration and maximal at 10 mol/liter Ca²⁺ and 150 mmol/liter Na⁺ concentration, respecitively. When Na⁺ was replaced by Li⁺, maximal Ca²⁺ uptake was 75% as compared to that in the presence of Na⁺. Amiloride (10^–3 mol/liter) at 200 mmol/liter Na⁺ did not inhibit Na⁺/Ca²⁺ countertransport, whereas at low Na⁺ concentration (25 mmol/liter) amiloride exhibited dose-dependent inhibition to be 62% at 10^–2 mol/liter. CFCCP (10^–5 mol/liter) did not influence Na⁺/Ca²⁺ countertransport. Monensin inhibited dose dependently; at a concentration of 5×10^–6 mol/liter inhibition was 80%. A SCN^– or K⁺ diffusion potential (=), being positive at the vesicle inside, stimulated calcium uptake in the presence of sodium suggesting that Na⁺/Ca²⁺ countertransport operates electrogenically, i.e. with a stoichiometry higher than 2 Na⁺ for 1 Ca²⁺. In the absence of Na⁺, did not promote Ca²⁺ uptake. We conclude that in addition to ATP-dependent Ca²⁺ outward transport as characterized previously (E. Bayerdörffer, L. Eckhardt, W. Haase & 1. Schulz, 1985,J. Membrane Biol. 84:45–60) the Na⁺/Ca²⁺ countertransport system, as characterized in this study, represents a second transport system for the extrusion of calcium from the cell. Furthermore, the high affinity for calcium suggests that this system might participate in the regulation of the cytosolic free Ca²⁺ level.     

Characterization of the ugp region containing the genes for the phoB dependent sn-glycerol-3-phosphate transport system of Escherichia coli

Summary The ugp structural genes, coding for the pho regulon dependent sn-glycerol-3-phosphate transport system, were cloned in pBR322 and characterized. The expression of the cloned ugp system was phoB dependent. Cells containing the ugp plasmid overproduced the G3P binding protein upon phosphate starvation. Tn5 mutagenesis of the cloned DNA revealed that the ugp genes are organized in two separate operons which comprise at least four genes: ugpB and ugpD constitute one operon, ugpA and ugpC constitute the other. The structural gene for the G3P binding protein (G3PBP) is ugpB.The ugpC gene product was also synthesized in minicells as a polypeptide, with an apparent molecular weight of 40,000. No gene products could be assigned to the ugpA and ugpD genes. Hybridization experiments allowed the physical characterization of 20 kb of DNA adjacent to the ugp genes on the E. coli chromosome including the liv genes.     

Light-temperature interactions in the control of photosynthesis in Antarctic phytoplankton

Summary During October/November 1983 photosynthetic responses of natural phytoplankton from the Scotia Sea and Bransfield strait to light and temperature were examined in incubators. Both assimilation numbers at saturating light levels and the slopes of the light-limited portions of the photosynthesis versus irradiance curves were smaller than in algae from lower latitudes. However, both parameters increased significantly with rising temperatures. Light-saturated photosynthesis on the average exhibited a Q₁₀-value of ca. 4.2 between-1.5°C and +2°C. Light-limited photosynthesis between-1.5°C and +5°C rose at a rate corresponding to a Q₁₀-value of roughly 2.6. Above +5°C, temperature enhancement of both light-saturated and light-limited photosynthetic rates was minimal or absent. Our results suggest that under extremely low temperatures light-limited photosynthetic rates become temperature-dependent due to changes in maximum quantum yields.     

Mengovirus-Induced Cytopathic Effect in L-Cells: Protective Effect of Interferon

The effect of interferon on mengovirus-induced cytopathic effect (CPE) in L cells, the cut-off of host-cell protein synthesis, and production of mature virus were found to be dependent on the concentration of interferon. CPE and inhibition of host protein synthesis were not affected until the concentration of interferon was increased 100-fold over that required to reduce viral yields by 90%.     

Acetylcholinesterase in der Pars distalis von Zonotrichia leucophrys gambelii (Aves)

Zusammenfassung In beiden Lobi der Pars distalis photosensitiver Zonotrichia leucophrys gambelii-Männchen bewirkt die photoperiodische Stimulation unter Langtagbedingungen das Auftreten von Acetylcholinesterase, die, solange die Vögel unter Kurztagbedingungen gehalten wurden, nicht oder nur in geringer Menge vorhanden war. Das Enzym ist auf PAS-positive Zellen beschränkt, doch nicht in allen von ihnen anzutreffen. Nach dem Erreichen eines Maximums zwischen Tag 31 und Tag 52 sinkt der AChE-Gehalt wieder und nähert sich nach 80 Tagen dem Ausgangsstadium. Der Parallelismus zwischen den Testesgewichten und der AChE-Aktivität läßt vermuten, daß die AChE-Zellen gonadotropes Hormon bilden.

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Acetylcholinesterase in the Pars distalis of Zonotrichia leucophrys gambelii (Aves)

Summary Photoperiodic stimulation of photosensitive male white-crowned sparrows, Zonotrichia leucophrys gambelii, causes the appearance of acetylcholinesterase activity in the cephalic and caudal lobes of the pars distalis. In photosensitive birds held on short daily photoperiods little or no activity can he demonstrated histochemically. The enzyme is restricted to PAS-positive cells. After attaining a maximum between 31 and 52 days after the beginning of photoperiodic stimulation (20 hours of light per day), AChE activity declines and after 80 days returns approximately to the initial level of short-day birds (eight hours of light per day). The correlation between the increase in weight of the testes and AChE activity, and other considerations, suggest that these AChE cells must produce gonadotropic hormones. Our observations are generally consistent with the results of the biochemical studies of Russell (1968) on the AChE activity in the pars distalis of the same species.

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Mit Unterstützung durch National Institutes of Health (Grant 5R01 NB 06187, Prof. Farner, Principal Investigator).

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Institut für Haustierkunde der Universität Kiel (Direktor: Prof. Dr. Dr. h. c. Wolf herre).     

Monoclonal Antibodies for Detection and Serotyping of Cucumber Mosaic Virus

Two monoclonal antibodies (MAbs) to cucumber mosaic virus (CMV) were selected from a panel of MAbs for use in the direct DAS (double antibody sandwich)-ELISA. Two different test procedures were developed: an ELISA with polyclonal and monoclonal antibodies (mixed ELISA) for the routine detection of CMV and a MAb-ELISA with two MAbs directed against different epitopes for the specific detection of the N serotype which is prevalent in GDR. The conventional two-step incubation of plates precoated with IgG was compared with simultaneous incubation of test sample and labelled antibody (one-step incubation). The mixed ELISA proved to be more sensitive than the direct DAS-ELISA with polyclonal antisera in detecting CMV in crude sap of infected plants. On the other hand, the MAb-ELISA could be used for serotyping of CMV isolates which is important in epidemiological investigations and in resistance breeding. Both the two-step and the one-step procedures gave similar results with some advantages of the latter procedure. One-step incubation is not only time-saving but seems to be also more sensitive with regard to the detection limit. However, care must be taken to circumvent the hook-effect occurring at high virus concentrations.     

Microtubules are involved in the secretion of proteins at the apical cell surface of the polarized epithelial cell, Madin-Darby canine kidney

Microtubule-disrupting drugs (nocodazole, colchicine) and cytochalasin D, which inhibits the polymerization of the actin microfilaments, were used to study the role of the cytoskeleton in protein secretion in the polarized Madin-Darby canine kidney (MDCK) epithelial cells. Two proteins were analyzed. The gp 80 glycoprotein complex, which in untreated cells is sorted into the apical pathway and lysozyme, which is released randomly at both cell surfaces in transfected MDCK cells. Our results show that cytochalasin D has no influence on the transport of the gp 80 complex and lysozyme to either cell surface. However, in the presence of nocodazole or colchicine the secretion of both proteins at the apical cell surface is reduced by 50% with a concomitant increase in the basolateral release. These data suggest that microtubules are necessary for an efficient secretion of proteins at the apical cell surface of MDCK cells. In regard to the yet unresolved discrepancy concerning the involvement of microtubules in the transport of membrane proteins to the apical surface of MDCK cells, our results are consistent with the data of Rindler et al. (Rindler, M. J., Ivanov, I. E., and Sabatini, D. D. (1987) J. Cell. Biol. 104, 231-241) who observed a nonpolarized delivery of the influenza virus hemagglutinin in the presence of nocodazole or colchicine.     

Community composition and soil properties in northern Bolivian savanna vegetation

Abstract. Direct gradient analysis (Canonical Correspondence Analysis) of northern Bolivian savanna vegetation revealed correlations between the composition of plant communities and physical and chemical soil properties. Cover/abundance values for 193 species from 27 sites were related to data on eight soil factors. A water-regime variable and the percentage of sand and silt were correlated with the first axis of the species-environment biplot and explained most of the variation in community composition. Along this axis, species and sites of flood-plain vegetation were separated from sites not affected by flooding rivers. The second axis of the biplot was correlated with soil-chemical variables, namely extract-able phosphate, base saturation, organic carbon, pH, and effective cation exchange capacity. Part of the variation in community composition can be explained by these soil nutrient variables. Grassland communities were separated from woody vegetation along the soil nutrient gradient, and floodplain communities of white-water rivers from those of clear-water rivers. The results of the gradient analysis indicate that the soil texture-moisture gradient is the prime factor determining the variation in the floristic composition of the savanna communities examined, and that, in addition, the soil nutrient gradient accounts for some of the variation.