Alzheimer-like paired helical filaments and antiparallel dimers formed from microtubule-associated protein tau in vitro

Recent evidence from several laboratories shows that the paired helical filaments of Alzheimer's disease brains consist mainly of the protein tau in an abnormally phosphorylated form, but the mode of assembly is not understood. Here we use EM to study several constructs derived from human brain tau and expressed in Escherichia coli. All constructs or tau isoforms are rodlike molecules with a high tendency to dimerize in an antiparallel fashion, as shown by antibody labeling and chemical crosslinking. The length of the rods is largely determined by the region of internal repeats that is also responsible for microtubule binding. One unit length of the repeat domain (three or four repeats) is around 22-25 nm, comparable to the cross-section of Alzheimer PHF cores. Constructs corresponding roughly to the repeat region of tau can form synthetic paired helical filaments resembling those from Alzheimer brain tissue. A similar self-assembly occurs with the chemically cross-linked dimers. In both cases there is no need for phosphorylation of the protein.     

ADP-ribosylation of gelsolin-actin complexes by clostridial toxins.

ADP-ribosylation of the 1:1 (G-A) and 1:2 (G-A-A) gelsolin-actin complexes by Clostridium perfringens iota toxin and Clostridium botulinum C2 toxin was studied. Iota toxin ADP-ribosylated actin in the G-A complex from human platelets as effectively as skeletal muscle actin. The Km for NAD (4 microM) was identical for both substrates. C2 toxin ADP-ribosylated actin in the G-A complex with lower efficacy than nonmuscle actin from platelet cytosol. In the G-A-A complex both actin molecules were ADP-ribosylated by iota toxin. The G-A complex bound ADP-ribosylated actin (Ar) to form the G-A-Ar complex in which the weakly bound actin is ADP-ribosylated. Vice versa, ADP-ribosylated 1:1 gelsolin-actin complex (G-Ar) was able to bind unmodified actin to yield the G-Ar-A complex. ADP-ribosylation did not change the nucleation activity of either the G-Ar complex or the G-Ar-A complex. When monomeric actin was added to the G-A-Ar complex, polymerization of actin was delayed by about 10 min. According to a quantitative kinetic analysis, the delay of polymerization corresponded to the rate of dissociation of ADP-ribosylated actin from the G-A-Ar complex. This suggests that the nucleation activity of the G-A-A complex is inhibited by ADP-ribosylation of the weakly bound actin and that the inhibition can be removed by dissociation of ADP-ribosylated actin from the G-A-Ar complex.     

Conformational analysis of a ribopentanucleoside tetraphosphate in aqueous solution. A two-dimensional NMR study at 500 MHz.

The 30 ribose proton resonances of the pentaribonucleoside tetraphosphate m6(2)AUm6(2)AUm6(2)A have been assigned unequivocally by means of spin-echo-correlated spectroscopy, 2D J-resolved spectroscopy and Nuclear Overhauser difference spectroscopy, carried out at 500 MHz. A detailed comparison of the conformational properties of the title compound with its constituent fragments m6(2)AUm6(2)AU, m6(2)AUm6(2)A, m6(2)AU and the relevant monomers is given. Chemical shift data indicate the existence of a doubly "bulged out" conformer, in which the two interior U-fragments are not involved in regular nearest neighbour stacking interactions. The coupling constants of the ribose-ring are interpreted in terms of the N/S equilibrium, and population distributions along the backbone angles beta and gamma are presented. The combined data suggest a strong similarity between the 5'-terminal triplets in m6(2)AUm6(2)AUm6(2)A, m6(2)AUm6(2)AU and m6(2)AUm6(2)A2.     

Histochemische und elektronenoptische Untersuchungen über Adenosintriphosphatasen des endometrialen Oberflächenund Drüsenepithels der Ziege (Capra hircus)

Zusammenfassung Im Oberflächen- und Drüsenepithel des nicht graviden Uterus der Ziege wurden licht- und elektronenoptische Untersuchungen über den Nachweis der Mg⁺⁺-, Ca⁺⁺- und der (Mg⁺⁺-Na⁺-K⁺)-aktivierbaren ATPasen durchgeführt.Spezifische Enzymaktivitäten lassen sich nur an Proben aus der Follikelphase nachweisen. In der Corpus luteum-Phase dagegen liegen diese Enzyme nicht in nachweisbarer Intensität vor.Die lichtmikroskopisch an den seitlichen Zellflächen nachgewiesenen Niederschläge der ATPasen finden sich elektronenoptisch als schwarze Granula an der Außenlamelle der lateralen Plasmalemm-Abschnitte. An apikalen und basalen Zellmembran-Bereichen in beiden Zyklusphasen zu beobachtende Reaktionsprodukte sind das Ergebnis einer Spontanhydrolyse des Substrats ATP bzw. von Spaltungen aufgrund der unspezifischen alkalischen Phosphatase. Haftkomplexe und Desmosomen sind regelmäßig frei von Bleisalz-Niederschlägen. Es können weder regionale Unterschiede in der Reaktionsintensität der Enzyme noch Differenzen im Hinblick auf die Verteilung und ultrastrukturelle Lokalisation von Niederschlägen der Mg⁺⁺-bzw. der Ca⁺⁺-aktivierbaren ATPase festgestellt werden.

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Histochemical and electron microscopic studies of the adenosine triphosphatases in the endometrial surface and glandular epithelium of the goat (Capra hircus)

Summary Light and electron microscopic studies were performed on the Mg⁺⁺-, Ca⁺⁺-, and the (Mg⁺⁺-Na⁺-K⁺)-activated ATPases in the surface and glandular epithelium of the non-pregnant uterus of the goat.Specific enzyme activities have been shown only in the epithelial samples collected during the follicular phase. In the luteal phase, none of these enzymes have been demonstrated with certainty. Precipitates of the ATPases which have been shown with the light microscope on the sides of the cell surface, can electron optically be observed as black granules on the outer lamella of the lateral plasmalemma. Reaction products on the apical and basal cell membrane regions observable in both cyclic phases, result from spontaneous hydrolysis of the substrate ATP, i.e. splitting caused by non-specific alkaline phosphatase. Junctional complexes and desmosomes are regularly free from lead salt precipitates. Neither regional differences in reaction intensity of the enzymes nor differences in the distribution and ultrastructural localization of either Mg⁺⁺-or Ca⁺⁺-activated ATPase precipitates can be observed.

Frau E. Merl danken wir für sorgfältige technische Mitarbeit.     

Biosynthesis of proteoglycans by proliferating and differentiating normal human keratinocytes cultured in serum-free medium

Normal human keratinocytes (NHK) were cultured in serum-free medium, containing low (0.1 mM) or high (2 mM) calcium, to obtain proliferating and differentiating cultures, respectively. Proteoglycan (PG) synthesis of proliferating and differentiating NHK was investigated. Cultures were labeled with 35S-sulfate, and the PGs were extracted from medium and cell layer. The newly synthesized PGs were isolated by ion-exchange chromatography on a column of DEAE-Sephacel. The molecular properties of the PGs and the size and composition of glycosaminoglycans (GAGs) were determined. In general, the PGs are relatively small size (Mr 70,000-120,000). The PGs of proliferating cultures are larger in molecular size than the PGs of differentiating cultures, and this is due to the degradation of the GAG chains. The molecular weight of the GAG chains of proliferating NHK ranged from 4,800 to 22,000, and the range for GAGs from differentiating cultures varied from 2,800 to 9,600. By compositional analysis, these PGs proved to contain heparan sulfate, chondroitin sulfate, and dermatan sulfate as determined by nitrous acid degradation, and chondroitinase ACII and ABC digestion. No significant differences were found in the overall GAG composition of the medium secreted PGs of proliferating and differentiating cultures. In contrast, cell-associated PGs of differentiating cells had higher levels of heparan sulfate than those of proliferating cells.     

Prion uptake in the gut: identification of the first uptake and replication sites

After oral exposure, prions are thought to enter Peyer's patches via M cells and accumulate first upon follicular dendritic cells (FDCs) before spreading to the nervous system. How prions are actually initially acquired from the gut lumen is not known. Using high-resolution immunofluorescence and cryo-immunogold electron microscopy, we report the trafficking of the prion protein (PrP) toward Peyer's patches of wild-type and PrP-deficient mice. PrP was transiently detectable at 1 day post feeding (dpf) within large multivesicular LAMP1-positive endosomes of enterocytes in the follicle-associated epithelium (FAE) and at much lower levels within M cells. Subsequently, PrP was detected on vesicles in the late endosomal compartments of macrophages in the subepithelial dome. At 7-21 dpf, increased PrP labelling was observed on the plasma membranes of FDCs in germinal centres of Peyer's patches from wild-type mice only, identifying FDCs as the first sites of PrP conversion and replication. Detection of PrP on extracellular vesicles displaying FAE enterocyte-derived A33 protein implied transport towards FDCs in association with FAE-derived vesicles. By 21 dpf, PrP was observed on the plasma membranes of neurons within neighbouring myenteric plexi. Together, these data identify a novel potential M cell-independent mechanism for prion transport, mediated by FAE enterocytes, which acts to initiate conversion and replication upon FDCs and subsequent infection of enteric nerves.