Amyloid-β Protofibrils: Size,Morphology and Synaptotoxicity of an Engineered Mimic

Structural and biochemical studies of the aggregation of the amyloid-β peptide (Aβ) are important to understand the mechanisms of Alzheimer''s disease, but research is complicated by aggregate inhomogeneity and instability. We previously engineered a hairpin form of Aβ called Aβcc, which forms stable protofibrils that do not convert into amyloid fibrils. Here we provide a detailed characterization of Aβ₄₂ cc protofibrils. Like wild type Aβ they appear as smooth rod-like particles with a diameter of 3.1 (±0.2) nm and typical lengths in the range 60 to 220 nm when observed by atomic force microscopy. Non-perturbing analytical ultracentrifugation and nanoparticle tracking analyses are consistent with such rod-like protofibrils. Aβ₄₂ cc protofibrils bind the ANS dye indicating that they, like other toxic protein aggregates, expose hydrophobic surface. Assays with the OC/A11 pair of oligomer specific antibodies put Aβ₄₂ cc protofibrils into the same class of species as fibrillar oligomers of wild type Aβ. Aβ₄₂ cc protofibrils may be used to extract binding proteins in biological fluids and apolipoprotein E is readily detected as a binder in human serum. Finally, Aβ₄₂ cc protofibrils act to attenuate spontaneous synaptic activity in mouse hippocampal neurons. The experiments indicate considerable structural and chemical similarities between protofibrils formed by Aβ₄₂ cc and aggregates of wild type Aβ₄₂. We suggest that Aβ₄₂ cc protofibrils may be used in research and applications that require stable preparations of protofibrillar Aβ.     

Cellular localization and age-dependent changes in mRNA for cyclic adenosine 3',5'-monophosphate-dependent protein kinases in rat testis

Gonadotropin activation of cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinases plays an important role in the regulation of testicular function. This study was undertaken to establish the expression of various subunits of cAMP-dependent protein kinases in different testicular cell types as well as during sexual maturation. RNA was extracted from cultured Sertoli cells, cultured peritubular cells, germ cells (pachytene spermatocytes, round spermatids), tumor Leydig cells, as well as whole testis from rats of various ages. Messenger RNA levels were studied by Northern analysis using available cDNA probes. The regulatory subunit (R) designated RII51 was found to be predominantly expressed in cAMP-stimulated Sertoli cells and tumor Leydig cells. Much lower levels were found in cultured peritubular cells and germ cells. A 2.9- and 3.2-kb mRNA for the RI subunit were found at about similar levels in all cell types, whereas the smaller 1.7-kb mRNA was expressed in high levels in germ cells. Also, the catalytic subunit (C) of cAMP-dependent protein kinase, designated C alpha, was expressed in all cell types; the highest mRNA levels for this subunit were found in germ cells and in tumor Leydig cells. The 1.7-kb mRNA for androgen-binding protein (ABP) was abundant in cAMP-stimulated Sertoli cells and was not present in other cell types of the testis. Furthermore, the cellular localization of the cAMP-dependent protein kinase subunits was also supported by developmental studies. The mRNA level of the RII51 3.2-kb species was relatively constant until Day 30, after which there was a tendency to decrease. A 1.6-kb message first appeared at greater ages. The mRNA for the smaller 1.7-kb species of RI, as well as the C alpha, showed a significant increase during development, supporting an enrichment of these mRNAs in germ cells. Messenger RNA levels for ABP were not detected in testis from 5- to 10-day-old rats but increased up to Day 30. After this age, mRNA for ABP revealed an age-dependent decrease, which parallels the relative increase of germ cells in the testis. In summary, these results demonstrate a clear pattern of cellular localization of the various mRNA species for subunits of the cAMP-dependent protein kinase in the rat testis.     

Complex ganglioside expression and tetanus toxin binding by PC12 pheochromocytoma cells

Ganglioside expression and tetanus toxin binding were studied in the rat pheochromocytoma cell line PC12. Seven ganglioside species were readily detected in extracts of PC12 cells; two were identified as tri- and tetrasialogangliosides, which are common brain constituents but unusual components of neuronal cell lines. Carbohydrate composition, acid and enzyme hydrolyses, and mass spectral analysis revealed that the major species is GT 1b, a predominant mammalian brain ganglioside previously reported to support high affinity tetanus toxin binding (Rogers, T. B., and Snyder, S. H. (1981) J. Biol. Chem. 256, 2402-2407). Direct binding of 125I-tetanus toxin to PC12 gangliosides on TLC plates revealed selective binding to the tri- and tetrasialogangliosides. Radioiodinated toxin also bound with high affinity to intact PC12 cells or their isolated membranes. The binding affinity (Kd = 1.25 nM), density of receptors (Bmax = 238 pmol/mg of membrane protein), and dependence on pH, ionic strength, and temperature were similar to those previously reported for toxin binding to rat brain synaptic membranes. Differentiation of PC12 cells caused an increase in expression of the tri- and tetrasialogangliosides and a closely matched increase in tetanus toxin binding to cell membranes. These data provide evidence that complex gangliosides may act as tetanus toxin receptors, and demonstrate the utility of the PC12 cell line for studies of tetanus toxicity and complex ganglioside expression.     

Phototoxicity of protoporphyrin as related to its subcellular localization in mice livers after short-term feeding with griseofulvin.

The substrate specificities of three cellulases and a beta-glucosidase purified from Thermoascus aurantiacus were examined. All three cellulases partially degraded native cellulose. Cellulase I, but not cellulase II and cellulase III, readily hydrolyzed the mixed beta-1,3; beta-1,6-polysaccharides such as carboxymethyl-pachyman, yeast glucan and laminarin. Both cellulase I and the beta-glucosidase degraded xylan, and it is proposed that the xylanase activity is an inherent feature of these two enzymes. Lichenin (beta-1,4; beta-1,3) was degraded by all three cellulases. Cellulase II cannot degrade carboxymethyl-cellulose, and with filter paper as substrate the end product was cellobiose, which indicates that cellulase II is an exo-beta-1,4-glucan cellobiosylhydrolase. Degradation of cellulose (filter paper) can be catalysed independently by each of the three cellulases; there was no synergistic effect between any of the cellulases, and cellobiose was the principal product of degradation. The mode of action of one cellulase (cellulase III) was examined by using reduced cellulodextrins. The central linkages of the cellulodextrins were the preferred points of cleavage, which, with the rapid decrease in viscosity of carboxymethyl-cellulose, confirmed that cellulase III was an endocellulase. The rate of hydrolysis increased with chain length of the reduced cellulodextrins, and these kinetic data indicated that the specificity region of cellulase III was five or six glucose units in length.     

Immunohistochemical localization of porcine diazepam-binding inhibitor (DBI) to rat endocrine pancreas

Summary The occurrence of diazepam-binding inhibitor (DBI), isolated and characterized from porcine upper intestine, was examined in the pancreas of Sprague-Dawley albino rats using indirect immunofluorescence. The polypeptide was found in the endocrine Langerhans islets and, utilizing double-labelling controls, it was shown to be present within the peripherally located glucagon-containing cells. Regulation of islet hormone production may therefore be under DBI control.