Immunohistochemical characteristics of the constituents of senile plaques and amyloid angiopathy in aged cynomolgus monkeys

Abstract: In this study, we immunohistochemically examined the several constituents of senile plaques (SPs) and cerebral amyloid angiopathy (CAA) in aged cynomolgus monkeys. Apolipoprotein E (apoE) deposited in all mature plaques and CAA, and in half of the diffuse plaques. Alpha-1-antichymotripsin (αACT) deposited in half of the mature plaques and in one third of the CAA. Amyloid precursor protein (APP), ubiquitin (Ub), and microtubule-associated protein-2 (MAP-2) accumulated in the swollen neurites of mature plaques. Glial fibrillary acidic protein (GFAP) was detected in the astrocytes and their processes surrounding the mature plaques. Tau was detected in neither the SPs nor CAA. Therefore, mature plaques involved extracellular Aβ, apoE, and αACT, and also astrocytes and swollen neurites. However, diffuse plaques involved only extracellular Aβ and apoE. Since these features, except for tau, were consistent with those in humans, this animal model will be useful for studying the pathogenesis of cerebral amyloid deposition.     

Growth kinetics of a bacteriophage in continuous culture

Lytic coliphage Qbeta was grown in continuously cultured host bacteria using a cascade of stirred flow reactors. The apparatus was constructed so that the steady stream of exponentially growing bacterial cells passing through the stirred flow reactors served to prevent coevolution brought about by host-parasite interactions. Wall growth was the primary cause for deviation from ideal continuous culture conditions and is largely dependent on the surface structure of the host bacteria. Using an Escherichia coli strain deficient in adhesive type I pili expression, the desynchronization of single burst events could easily be followed over the course of four infection latency periods. Computer simulations based on a two-stage model for the Qbeta infection cycle were in perfect agreement with the experimental data. Applications of the optimized system to strategies of molecular evolution are discussed. (c) 1996 John Wiley & Sons, Inc.     

The Ca2+ influx induced by β-amyloid peptide 25–35 in cultured hippocampal neurons results from network excitation

Although a neurotoxic role has been postulated for the β-amyloid protein (βAP), which accumulates in brain tissues in Alzheimer's disease, a precise mechanism underlying this toxicity has not been identified. The peptide fragment consisting of amino acid residues 25 through 35 (βAP25-35), in particular, has been reported to be toxic in cultured neurons. We report that βAP25-35, applied to rat hippocampal neurons in culture, caused reversible and repeatable increases in the intracellular Ca²⁺ concentration ([Ca²⁺]_i), as measured by fura 2 fluorimetry. Furthermore, βAP25-35 induced bursts of excitatory potentials and action potential firing in individual neurons studied with whole cell current clamp recordings. The βAP25-35–induced [Ca²⁺]_i elevations and electrical activity were enhanced by removal of extracellular Mg²⁺, and they could be blocked by tetrodotoxin, by non-N-methyl-D -aspartate (NMDA) and NMDA glutamate receptor antagonists, and by the L-type Ca²⁺ channel antagonist nimodipine. Similar responses of bursts of action potentials and [Ca²⁺]_i increases were evoked by βAP1-40. Responses to βAP25-35 were not prevented by pretreatment with pertussis toxin. Excitatory responses and [Ca²⁺]_i elevations were not observed in cerebellar neuron cultures in which inhibitory synapses predominate. Although the effects of βAP25-35 depended on the activation of glutamatergic synapses, there was no enhancement of kainate- or NMDA-induced currents by βAP25-35 in voltage-clamp studies. We conclude that βAP25-35 enhances excitatory activity in glutamatergic synaptic networks, causing excitatory potentials and Ca²⁺ influx. This property may explain the toxicity of βAP25–35. © 1995 John Wiley & Sons, Inc.     

Gene transfer and expression in mouse preimplantation embryos by recombinant adenovirus vector

Replication-defective recombinant adenovirus, Adex4SRLacZL, was used as a vector for transferring exogenous genes in mouse zona pellucida-free eggs at the pronuclear stage. The vector contained the E. coli LacZ reporter gene under the control of the SRα promoter (SV40 early promoter-fused HTLV-I LTR), and the expression of the reporter gene was examined during preimplantation development in culture. Histochemical staining of the embryos for β-galactosidase activity showed that the exogenous LacZ gene as expressed in 98% of the embryos at the morula-blastocyst stages. As in the microinjection method, the exogenous genes could be pursued from the 2-cell stage. Neither apparent morphological changes nor cytotoxic effects were observed. Both the percentages of embryos expressing reporter genes and the rate of development to the blastocyst stage were higher in the adenovirus vector-treated embryos than in the microinjected ones. These results suggest that the adenovirus vector system is a useful tool in investigating the genetic control of early mammalian development. © 1995 wiley-Liss, Inc.     

The use of N-urethane-protected N-carboxyanhydrides (UNCAs) in amino acid and peptide synthesis

N-Urethane-protected N-carboxyanhydrides (UNCAs) are very reactives. They have been successfully used in peptide synthesis, in both solution and solid phase. We have demonstrated that UNCAs are interesting starting materials for the synthesis of various amino acid derivatives. Chemoselective reduction of UNCAs with sodium borohydride led the corresponding N-protected β amino alcohols. Reaction of UNCAs with Meldrum's acid, followed by cyclisation, yielded enantiomerially pure tetramic acid derivatives. Diastereoselective reduction of tetramic acid derivatives produced (4S,5S)-N-alkoxycarbonyl-4-hydroxy-5-alkylpyrrolidin-2-ones derived from amino acids, which after hydrolysis yielded statine and statine analogues. Tetramic acid derivatives could also be obtained by reaction of UNCAs with benzyl ethyl followed by hydrogenolytic deprotection and decarboxylation. UNCAs also reacted with phosphoranes to produce the ketophosphorane in excellent yields. Subsequent oxidation with oxone or with [bis(acetoxy)-iodol]-benzene produced vicinal tricarbonyl derivatives. These reactions usually proceeded smoothly and with high yields.     

Lactam bridge stabilization of α-helical peptides: Ring size,orientation and positional effects

A series of 14 residue amphipathic α-helical peptides, in which the sidechains of glutamic acid and lysine have been covalently joined, was synthesized in order to determine the effect of spacing, position and orientation of these lactam bridges. It was found that although an (i, i+3) spacing would position the lactam bridge on the same face of the helix, these lactams with 18-member rings were actually helix-destabilizing regardless of position or location. On the other hand, (i, i+4) lactams with 21-member rings were helix-stabilizing but this was dependent on orientation. Glutamic acid-lysine lactams increased the helical content of the peptide when compared with their linear homologue in benign conditions (50 mM KH₂PO₄, 100 mM KCl, pH 7). Two Glu-Lys (i, i+4) lactams located at the N- and C-termini gave rise to a peptide with greater than 99% helical content in benign conditions. Peptides with Lys-Glu oriented lactams were random structures in benign conditions but in the presence of 50% TFE could be induced into a helical conformation. The stability of the single-stranded α-helices, as measured by thermal denaturations in 25% TFE indicated that Glu-Lys oriented lactam bridges stabilized the helical conformation relative to the linear unbridged peptide. One Glu-Lys lactam in the middle of the peptide was more effective at stabilizing helical structure than two Glu-Lys lactams positioned one at each end of the molecule. The lactams with the Lys-Glu orientation were destabilizing relative to the unbridged peptide. This study demonstrates that correct orientation and position of a lactam bridge is critical in order to design peptides with high helical content in aqueous media.     

Comparative biochemical studies of carotenoids in sea urchins—III. Relationship between developmental mode and carotenoids in the Australian echinoids Heliocidaris erythrogramma and H. tuberculata and a comparison with Japanese species

The sea urchin Heliocidaris tuberculata is typical of most echinoids in having a small egg and a feeding larva, while H. erythrogramma has a large egg and modified development through a non-feeding larvae. The carotenoids in the gonads of these two species were investigated from the comparative biochemical points of view. The carotenoid content of the buoyant eggs of H. erythrogramma was approximately 60 times that of the negatively-buoyant eggs of H. tuberculata. With respect to cytoplasmic volume, however, the carotenoid concentration in the eggs of H. tuberculata was approximately twice that in the eggs of H. erythrogramma. In both species β-echinenone was the principal carotenoid found and their carotenoid patterns were similar. It is very interesting from a functional point of view that carotenoid levels per cytoplasmic volume are conserved across most of the species we have examined irrespective of phylogeny and egg size. In light of this result we suggest that carotenoids may play an important role in developing stage in all echinoids including indirect and direct developers.     

Age-related differences in TGF-α and proto-oncogenes expression in rat liver after a low dose of carbon tetrachloride

The resiliency of rats during early postnatal development to CCl₄ or to an interactive hepatotoxicity of chlordecone (CD) + CCl₄ has been shown to be due to an efficient stimulation of tissue repair. The objective of the current study was to investigate if this is due to efficient expression of transforming growth factor-α (TGF-α) and proto-oncogenes. Postnatally developing (20 day old) and adult (60 day old) male Sprague–Dawley rats were challenged with a single low dose of CCl₄ (100 μL/kg, ip) or corn oil. Liver samples were collected during a time course (0–96 h) after the administration of CCl₄ and used to examine TGF-α and early (c-fos) and late (H-ras and K-ras) proto-oncogenes mRNA expressions. Significant increases in TGF-α, H-ras, and K-ras gene expressions were evident as early as 12 hours after CCl₄ and peaked between 24 and 48 hours in an age-dependent manner as detected by slot-blot analysis. Results of the study revealed three- and twofold increases in TGF-α gene expression in 20 and 60 day old rats, respectively, after CCl₄. There were 3.5- and 2.5-fold increases in H-ras and 4.4- and 3.4-fold increases in K-ras in 20 and 60 day old rats, respectively. In contrast, a 10-fold increase in c-fos mRNA expression was evident in 20 day old rats 1 hour after CCl₄ treatment, returning to the baseline value by 3 hours, whereas in 60 day old rats, this increase was less than twofold. The overall findings of this study indicate that TGF-α and the early and late proto-oncogene mRNA expressions were enhanced in an age- and time-dependent manner in response to a low dose of CCl₄. These results further strengthen the view that the remarkable resiliency of rats to hepatotoxicants during early postnatal development is due to substantial increases in stimulation of hepatocellular regeneration and tissue repair mechanisms, leading to regression of liver injury and recovery. © 1996 John Wiley & Sons, Inc.