Human P301L-Mutant Tau Expression in Mouse Entorhinal-Hippocampal Network Causes Tau Aggregation and Presynaptic Pathology but No Cognitive Deficits

Accumulation of hyperphosphorylated tau in the entorhinal cortex (EC) is one of the earliest pathological hallmarks in patients with Alzheimer’s disease (AD). It can occur before significant Aβ deposition and appears to “spread” into anatomically connected brain regions. To determine whether this early-stage pathology is sufficient to cause disease progression and cognitive decline in experimental models, we overexpressed mutant human tau (hTauP301L) predominantly in layer II/III neurons of the mouse EC. Cognitive functions remained normal in mice at 4, 8, 12 and 16 months of age, despite early and extensive tau accumulation in the EC. Perforant path (PP) axon terminals within the dentate gyrus (DG) contained abnormal conformations of tau even in young EC-hTau mice, and phosphorylated tau increased with age in both the EC and PP. In old mice, ultrastructural alterations in presynaptic terminals were observed at PP-to-granule cell synapses. Phosphorylated tau was more abundant in presynaptic than postsynaptic elements. Human and pathological tau was also detected within hippocampal neurons of this mouse model. Thus, hTauP301L accumulation predominantly in the EC and related presynaptic pathology in hippocampal circuits was not sufficient to cause robust cognitive deficits within the age range analyzed here.     

Genetic characterization of the mei-41 locus in Drosophila melanogaster

Summary The mutagen-sensitive mutant mus(1)104 ^D1 of Drosophila melanogaster maps to a position on the X chromosome very close to the meiotic mutant mei-41 ^D5 . Both mutants have been characterized as mutagen-sensitive and defective in post-replication repair. In the present report we show by complementation studies that mus(1)104 and mus(1)103 are allelic with mei-41. In addition, two reported alleles of mus(1)104 lie between the mei-41 alleles A10 and D5. The size of the mei-41 locus is estimated to be about 0.1 centimorgans (cM). Because several alleles of mei-41 have been shown to reduce recombination and increase meiotic chromosome loss and nondisjunction, mus(1)104 ^D1 females were examined for defects in meiosis. Although there was no evidence for reduced recombination on the second chromosome in homozygous mus(1)104 ^D1 females, heterozygous mus(1)104 ^D1 /mei-41 ^>D5 and mus(1)104 ^D1 /deficiency females showed reduced levels of recombination. However, there was no evidence of an increase in nondijunction in these females.We dedicate this article to the memory of Larry Sandler, who passed away suddenly on February 7, 1987     

Galactosialidosis: molecular heterogeneity in biosynthesis and processing of protective protein for β-galactosidase

Summary Biosynthesis and processing of the protective protein for -galactosidase in normal and galactosialidosis fibroblasts were investigated using specific antiserum preparations. A 45-kd precursor was processed to a mature 30-kd protein in normal fibroblasts. The mature protective protein was not detected in any of the twelve galactosialidosis fibroblast strains examined in this study. The precursor was not detected in two cases and in the others was of heterogeneous molecular weight, i.e., normal, abnormally low, or abnormally high. These molecular abnormalities were not correlated with clinical manifestations of the patients.     

Light and electron microscopic immunocytochemistry on the localization of 3β-hydroxysteroid dehydrogenase/isomerase in the bovine adrenal cortical cells

Summary The localization of 3-hydroxysteroid dehydrogenase/isomerase (3-HSD) was studied in bovine adrenal glands by light as well as electron microscopic immunocytochemistry, using anti-bovine adrenal 3-HSD antibody. With light microscopy the cytoplasm of the glomerulosa cells was weakly immunostained, while that of the fasciculata-reticularis cells was intensely immunostained though both the capsular connective tissue cells and the medullary cells were entirely negative for this reaction. Electron microscopic immunocytochemistry revealed that the positive reaction products for 3-HSD were present on the membrane of smooth endoplasmic reticulum of the cortical cells, especially that of the fasciculata and reticularis cells. Other cell organelles such as mitochondria and Golgi apparatus were entirely negative. The present results indicate that 3-HSD is present in the membrane of smooth endoplasmic reticulum of bovine adrenal cortical cells.Supported by grants from the Ministry of Education Science and Culture, Japan     

Rotation and protein-protein interactions of cytochrome P-450 in the inner membrane of adrenocortical mitochondria

Rotational diffusion of the total cytochrome P-450 (P-450scc plus P-45011 beta) in bovine adrenocortical mitochondria was examined by observing the decay of absorption anisotropy, r(t), after photolysis of the hemo.CO complex by a vertically polarized laser flash. Analysis of r(t) was based on a "rotation-about-membrane normal" model. The measurements were used to investigate intermolecular interactions of cytochrome P-450 with other membrane proteins. The absorption anisotropy decayed within 1 ms to a time-independent value. Rotational diffusion of cytochrome P-450 was dependent on the presence and absence of deoxycorticosterone (DOC), a substrate for cytochrome P-45011 beta. The observed value for the normalized time-independent anisotropy r(infinity)/r(0) and the average rotational relaxation time phi are r(infinity)/r(0) = 0.88 and phi = 233 microseconds when DOC is absent, and r(infinity)/r(0) = 0.65 and phi = 350 microseconds when DOC is present. Judging from the phi value, rotating P-450 is not a monomeric molecule, but would be a small microaggregate with an average diameter of about 120 A. A significantly high value of r(infinity)/r(0) implies co-existence immobile populations of cytochrome P-450. Based on the assumption that the heme angle tilts 55 degrees from the membrane plane (Gut et al. (1983) J. Biol. Chem. 258, 8588-8594), 65% (when DOC is present) or 88% (when DOC is absent) of cytochrome P-450 in mitochondria is immobilized within the experimental time range of 2 ms due to the presence of immobile protein microaggregates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coordination structures and reactivities of compound II in iron and manganese horseradish peroxidases. A resonance Raman study

Resonance Raman investigations on compound II of native, diacetyldeuteroheme-, and manganese-substituted horseradish peroxidase (isozyme C) revealed that the metal-oxygen linkage in the compound differed from one another in its bond strength and/or structure. Fe(IV) = O stretching frequency for compound II of native enzyme was pH sensitive, giving the Raman line at 772 and 789 cm-1 at pH 7 and 10, respectively. The results confirmed the presence of a hydrogen bond between the oxo-ligand and a nearby amino acid residue (Sitter, A. J., Reczek, C. M., and Terner, J. (1985) J. Biol. Chem. 260, 7515-7522). The Fe(IV) = O stretch for compound II of diacetylheme-enzyme was located at 781 cm-1 at pH 7 which was 9 cm-1 higher than that of native enzyme compound II. At pH 10, however, the Fe(IV) = O stretch was found at 790 cm-1, essentially the same frequency as that of native enzyme compound II. The pK value for the pH transition, 8.5, was also the same as that of native compound II. Unlike in native enzyme, D2O-H2O exchange did not cause a shift of the Fe(IV) = O frequency of diacetylheme-enzyme. Thus, the metal-oxygen bond at pH 7 was stronger in diacetylheme-enzyme due to a weaker hydrogen bonding to the oxo-ligand, while the Fe(IV) = O bond strength became essentially the same between both enzymes at alkaline pH upon disruption of the hydrogen bond. A much lower reactivity of the diacetylheme-enzyme compound II was accounted to be due to the weaker hydrogen bond. Compound II of manganese-substituted enzyme exhibited Mn(IV)-oxygen stretch about 630 cm-1, which was pH insensitive but down-shifted by 18 cm-1 upon the D2O-H2O exchange. The finding indicates that its structure is in Mn(IV)-OH, where the proton is exchangeable with a water proton. These results establish that the structure of native enzyme compound II is Fe(IV) = O but not Fe(IV)-OH.     

Properties of porcine platelet myosin. II. Shape change of the molecule

Porcine platelet myosin molecules were examined by electron microscopy for changes in their shape. At high ionic strength, the molecules were morphologically indistinguishable from skeletal muscle myosin, except for a slight difference in the bent regions of their tails. At physiological ionic strength, however, the following important difference was observed between the two myosins. Unlike skeletal muscle myosin, the filaments of nonphosphorylated platelet myosin could be disassembled by stoichiometric ATP into a monomeric form with sharply bent or folded tail, and reassembled after ATP hydrolysis. Similar disassembly changes could be induced by various nucleotide triphosphates (CTP, GTP, ITP, and UTP) and to a lesser extent by ADP, AMP, and AMPPNP. These results suggest that ATP binds to the hydrolytic sites in platelet myosin molecule and induces the molecular shape change.     

Infrared spectra of carbon monoxide complexes of indoleamine 2,3-dioxygenase and L-tryptophan 2,3-dioxygenases. Effects of substrates on the CO-stretching frequencies

Carbonmonoxy indoleamine 2,3-dioxygenase from rabbit small intestine exhibited two CO stretch bands at 1953 and 1933 cm-1 with half-band widths (delta v 1/2) of both approximately 15 cm-1. Upon addition of an excess amount of L-tryptophan, the substrate, the spectrum changed into that with an intense single band at 1902 cm-1 with the delta v 1/2 of 15 cm-1. Carbonmonoxy L-tryptophan 2,3-dioxygenase of Pseudomonas acidovorans in the absence of L-tryptophan showed a fused CO stretch band which consists of two components at 1965 and 1958 cm-1 (delta v 1/2 for the fused band; 25 cm-1), which was converted into a sharp single band at 1968 cm-1 (delta v 1/2; 10 cm-1) upon addition of excess L-tryptophan. On the other hand, CO complex of rat liver L-tryptophan 2,3-dioxygenase in the absence of L-tryptophan gave a spectrum with a poorly defined peak around 1961 cm-1. By the addition of L-tryptophan, the spectrum changed into that with two distinct bands at 1972 and 1920 cm-1 (delta v 1/2; 6 and 13 cm-1, respectively). These spectra were insensitive to pH in a range where the enzymes were not denatured (neutral to near pH 9). The infrared spectra of the carbonmonoxy enzymes were also affected by the addition of certain effectors such as skatole and alpha-methyl-DL-tryptophan, which facilitate the binding of L-tryptophan to the catalytic site of intestinal and Pseudomonas enzymes, respectively. However, the changes were of different types from those by the saturating amount of L-tryptophan. Possible mechanisms for these phenomena are discussed in relation to the structure of the heme-CO complex in these heme-containing dioxygenases.     

On the relationship between two indices (“Bulk density” and “dry-matter content”) of dry-matter accumulation in plant organs

“Bulk density” and “dry-matter content” are useful indices of dry-matter accumulation in plant organs. A theoretical equation describing the relationship between these two indices was put forward. To examine the reliability of this equation, the seasonal changes of these two indices were investigated in the leaves and stems of different ages ofAucuba japonica. In each organ both indices varied seasonally almost parallel to each other, but the seasonal changes of dry-matter content were less obvious than those of bulk density. The observed bulk density was always larger than that calculated from the observed dry-matter content by the theoretical equation. A drying experiment showed that this discrepancy was caused by the decrement of the volume of the plant material by water loss during the period from the weight measurement to the volume measurement. When the water loss was negligible, the equation described well the relationships between the two indices of the experimental materials. It was also shown that this equation was useful for the estimation of the amount of air space in plant materials.     

Stereospecific incorporation of hydrogens from NADPH in elongation of very long fatty acyl-CoA by swine cerebral microsomes

The elongation of arachidoyl-CoA by swine cerebral microsomes resulted in the production of behenic acid (22:0) and lignoceric acid (24:0) concomitantly. When 4S-[4-²H₁]NADPH was used for the elongation of arachidoyl-CoA, the incorporation of two deuterium atoms into 22:0 was observed by the technique of mass fragmentography. Furthermore, the incorporation of four deuterium atoms into 24:0 was also detected. On the other hand, when 4R-[4-²H₁]NADPH was used, no deuterium was incorporated into the elongated products.