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.     

Alzheimer beta-amyloid peptides: normal and abnormal localization

Alzheimer's disease (AD) neuropathology is characterized by accumulation of "senile" plaques (SPs) and neurofibrillary tangles (NFTs) in vulnerable brain regions. SPs are principally composed of aggregates of up to 42/43 amino acid beta-amyloid (A beta) peptides. The discovery of familial AD (FAD) mutations in the genes for the amyloid precursor protein (APP) and presenilins (PSs), all of which increase A beta42 production, support the view that A beta is centrally involved in the pathogenesis of AD. A beta42 aggregates readily, and is thought to seed the formation of fibrils, which then act as templates for plaque formation. A beta is generated by the sequential intracellular cleavage of APP by beta-secretase to generate the N-terminal end of A beta, and intramembranous cleavage by gamma-secretase to generate the C-terminal end. Cell biological studies have demonstrated that A beta is generated in the ER, Golgi, and endosomal/lysosomal system. A central question involving the role of A beta in AD concerns how A beta causes disease and whether it is extracellular A beta deposition and/or intracellular A beta accumulation that initiates the disease process. The most prevalent view is that SPs are composed of extracellular deposits of secreted A beta and that A beta causes toxicity to surrounding neurons as extracellular SP. The recent emphasis on the intracellular biology of APP and A beta has led some investigators to consider the possibility that intraneuronal A beta may directly cause toxicity. In this review we will outline current knowledge of the localization of both intracellular and extracellular A beta.     

Histopathological studies of senile plaques and cerebral amyloidosis in cynomolgus monkeys

Senile plaques (SPs) and cerebral amyloid angiopathy (CAA), pathological hallmarks of Alzheimer's disease, have not been thoroughly investigated histopathologically in nonhuman primates. To determine the onset age and histopathological characteristics of SPs and CAA, we examined the brains of 64 cynomolgus monkeys (Macaca fascicularis) from 2 to 35 years old. Mature (classical and primitive) plaques appeared in 16 out of 25 monkeys that were >20 years old. Moreover, mature plaques were observed more frequently than diffuse plaques and were located in the temporal cortex of the superior or inferior gyri and amygdala. Diffuse plaques in contrast to mature plaques did not show definite tendencies in onset age and distribution. CAA appeared in more than 22-year-old monkeys in 10 out of 16 animals and was frequently observed in capillaries and often found adjoining mature plaques. During immunohistochemical examination, an antiserum for amyloid β protein (Aβ) 1–40 could detect all SPs, whereas a monoclonal antibody for Aβ 8–17 could not detect any diffuse plaques and only one third of the primitive plaques. As for CAA, the polyclonal antiserum was more sensitive than the monoclonal antibody. The present study describes the histopathological features of SPs and CAA in old cynomolgus monkeys.     

非遗传性老年痴呆的动物模型及行为学,病理学改变

通过毁损Ｗｉｓｔａｒ大鼠迈内特基底核（ｎｂＭ）建立非遗传性老年痴呆（ＡＤ）动物模型。用Ｍｏｒｒｉｓ水迷宫、六胺银染色、ａｃｅｔｙｌｃｈｏｌｉｎｅｓｔｅｒａｓｅ（ＡＣｈＥ）细胞化学、β淀粉蛋白（βＡＰ）和τ蛋白免疫细胞化学及超微结构观察等方法进行行为学及病理学变化研究。结果证明：毁损ｎｂＭ鼠学习、记忆能力下降,乙酰胆碱酯酶ＡＣｈＥ锐减,βＡＰ和τ蛋白样免疫神经元增加,细胞水胞、溶解,溶酶体、微管增加     

Analysis of GC-rich repetitive nucleotide sequences in great apes

The genomes of four primate species, belonging to the families Pongidae (chimpanzee, gorilla, and orangutan) and Hylobatidae (gibbons), have been analyzed for the presence and organization of two human GC-rich heterochromatic repetitive sequences: Satellite (Sat) and LongSau (LSau) repeats. By Southern blot hybridization and PCR, both families of repeats were detected in all the analyzed species, thus indicating their origin in an ape ancestor. In the chimpanzee and gorilla, as in man, Sat sequences showed a 68-bp Sau3A periodicity and were preferentially organized in large clusters, whereas in the orangutan, they were organized in DNA fragments of 550 bp, which did not seem to be characterized by a tandem organization. On the contrary, in each of the analyzed species, the bulk of LSau sequences showed a longer Sau3A periodicity than that observed in man (450–550 bp). Furthermore, only in the chimpanzee genome some of LSau repeats seemed to be interspersed within blocks of Sat sequences. This sequence organization, which also characterizes the human genome, is probably absent in the gorilla. In fact, the analysis of a gorilla genomic library suggested that LSau repeats are not preferentially in linkage with Sat sequences. Moreover, LSau sequences were found in a genomic sector characterized by the simultaneous presence of L1 and (CA) repeats, as well as of anonymous sequences and known genes. In spite of the different sequence organization, the nucleotide differences between complete human and gorilla LSau repeats were very few, whereas one gorilla LSau repeat, interrupted by a probably-truncated L1 transposon, showed a higher degree of divergence. Besides the gorilla, this unusual sequence organization was detected in man, and, to a lesser extent, in the chimpanzee. Correspondence to: R. Meneveri     

Acetylcholine receptors and tau phosphorylation

Alzheimer's disease (AD) is characterized by the presence, in the brain of the patients, of two aberrant structures: intracellular neurofibrillary tangles (NFTs), containing an abnormal hyperphosphorylated form of tau protein, and extracellular senile plaques (SPs), mainly composed by fibrillar amyloid beta peptide. Another feature of AD is the neurodegeneration and dysfunction of basal forebrain cholinergic system. A possible connection among those AD characteristics could occur. Thus, the purpose of this short review is to summarize the involvement of nicotinic (nAChR) and muscarinic (mAChR) receptors on tau phosphorylation, in a direct way, or through the previous interaction of some of these receptors with amyloid beta. Several studies have demonstrated that nAChR activation results in a significantly increase of tau phosphorylation, whereas mAChR activation, may prevent tau phosphorylation.     

Age-related changes of Alzheimer's disease-associated proteins in cynomolgus monkey brains

We characterized senile plaques (SPs) immunohistochemically in cynomolgus monkey brains and also examined age-related biochemical changes of Alzheimer's disease (AD)-associated proteins in these brains from monkeys of various ages. In the neocortex of aged monkeys (>20 years old), we found SPs but no neurofibrillary tangles (NFTs). Antibodies against beta-amyloid precursor protein (APP) or apolipoprotein E (ApoE) stained SPs; however, the pattern of immunostaining was different for the two antigens. APP was present only in swollen neurites, but ApoE was present throughout all parts of SPs. Western blot analysis revealed that the pattern of APP expression changed with age. Although full-length APP695 protein was mainly expressed in brains from young monkeys (4-years-old), the expression of full-length APP751 protein was increased in brains from older monkeys (>20 years old). Biochemical analyses also showed that levels of various AD-associated proteins increased significantly with age in nerve ending fractions. Both SP-associated (APP) and NFT-associated proteins (tau, activated glycogen synthase kinase 3beta, cyclin dependent kinase 5, p35, and p25) accumulated in the nerve ending fraction with increasing age; however, we found no NFTs or paired helical filaments of tau in aged cynomolgus monkey brains. This age-related accumulation of these proteins in the nerve ending fraction was similar to that observed in our laboratory previously for presenilin-1 (PS-1). The accumulation of these SP-associated proteins in this fraction may be a causal event in the spontaneous formation of SPs; thus, SPs may be formed initially in nerve endings. Taken together, these results suggest that intensive investigation of age-related changes in the nerve ending and in axonal transport will contribute to a better understanding of the pathogenesis of neurodegenerative disorders such as AD.     

Reversion of Q beta RNA phage mutants by homologous RNA recombination.

Q beta phage RNAs with inactivating insertion (8-base) or deletion (17-base) mutations within their replicase genes were prepared from modified Q beta cDNAs and transfected into Escherichia coli spheroplasts containing Q beta replicase provided in trans by a resident plasmid. Replicase-defective (Rep-) Q beta phage produced by these spheroplasts were detected as normal-sized plaques on lawns of cells containing plasmid-derived Q beta replicase, but were unable to form plaques on cells lacking this plasmid. When individual Rep- phage were isolated and grown to high titer in cells containing plasmid-derived Q beta replicase, revertant (Rep+) Q beta phage were obtained at a frequency of ca. 10(-8). To investigate the mechanism of this reversion, a point mutation was placed into the plasmid-derived Q beta replicase gene by site-directed mutagenesis. Q beta mutants amplified on cells containing the resultant plasmid also yielded Rep+ revertants. Genomic RNA was isolated from several of the latter phage revertants and sequenced. Results showed that the original mutation (insertion or deletion) was no longer present in the phage revertants but that the marker mutation placed into the plasmid was now present in the genomic RNAs, indicating that recombination was one mechanism involved in the reversion of the Q beta mutants. Further experiments demonstrated that the 3' noncoding region of the plasmid-derived replicase gene was necessary for the reversion-recombination of the deletion mutant, whereas this region was not required for reversion or recombination of the insertion mutant. Results are discussed in terms of a template-switching model of RNA recombination involving Q beta replicase, the mutant phage genome, and plasmid-derived replicase mRNA.     

Elevated A beta and apolipoprotein E in A betaPP transgenic mice and its relationship to amyloid accumulation in Alzheimer's disease

BACKGROUND: Amyloid-beta (A beta) accumulates in plaques and as cerebral amyloid angiopathy (CAA) in the brains of both Alzheimer's disease (AD) patients and transgenic A betaPPswe/tg2576 (tg2576) mice. Increasingly, evidence in humans and mice shows this process to be modulated by apolipoprotein E (apoE). MATERIALS AND METHODS: To explore this relationship, we measured apoE and A beta levels in brains of tg2576 mice and controls at intervals between 2 and 20 months. In addition, A beta concentrations in plasma and muscle of these animals were also quantified. RESULTS: Quite strikingly, we found that the amount of tg2576 mice brain apoE was elevated by an average of 45%, relative to the control mice from 2 months on. The level of brain apoE soared after 14 months to almost 60% greater than the level found in control mice. A beta concentrations in brains before 9 months were less than 2 ng/mg of protein, but by 14 months concentrations rose to 8.7 ng/mg, and by 20 months to 47 ng/mg. In plasma, we noted that the levels of A beta in tg2576 mice declined from above 30 ng/ml prior to 12 months to 14 ng/ml by 14 months. Histology showed that A beta plaques and CAA began to be discernible in the tg2576 mice at about 9 and 20 months of age, respectively. CONCLUSIONS: ApoE was immunocytochemically detected in neuritic plaques that were positive for thioflavine-S. We suggest that the elevation of brain apoE in tg2576 mice participates in an age-related dysregulation of A beta clearance and signals the start of A beta sequestration during the time of cognitive dysfunction.     

Altered localization and cytoplasmic domain-binding properties of tyrosine-phosphorylated beta 1 integrin

We describe a novel approach to study tyrosine-phosphorylated (PY) integrins in cells transformed by virally encoded tyrosine kinases. We have synthesized a peptide (PY beta 1 peptide) that represents a portion of the cytoplasmic domain of the beta 1 integrin subunit and is phosphorylated on the tyrosine residue known to be the target of oncogenic tyrosine kinases. Antibodies prepared against the PY beta 1 peptide, after removal of cross-reacting antibodies by absorption and affinity purification, recognized the PY beta 1 peptide and the tyrosine-phosphorylated form of the intact beta 1 subunit, but did not bind the nonphosphorylated beta 1 peptide, the nonphosphorylated beta 1 subunit or other unrelated tyrosine-phosphorylated proteins. The anti- PY beta 1 antibodies labeled the podosomes of Rous sarcoma virus- transformed fibroblasts, but did not detectably stain nontransformed fibroblasts. The localization of the tyrosine phosphorylated beta 1 subunits appeared distinct from that of the beta 1 subunit. Adhesion plaques were stained by the anti-beta 1 subunit antibodies in Rous sarcoma virus-transformed fibroblasts plated on fibronectin, whereas neither podosomes nor adhesion plaques were labeled on vitronectin or on uncoated plates. Anti-phosphotyrosine antibodies labeled podosomes, adhesion plaques and cell-cell boundaries regardless of the substratum. One of the SH2 domains of the p85 subunit of phosphatidylinositol-3- kinase bound to the PY beta 1 peptide, but not to the non- phosphorylated beta 1 cytoplasmic peptide. Other SH2 domains did not bind to the PY beta 1 peptide. These results show that the phosphorylated form of the beta 1 integrin subunit is detected in a different subcellular localization than the nonphosphorylated form and suggest that the phosphorylation on tyrosine of the beta 1 subunit cytoplasmic domain may affect cellular signaling pathways.     

Immunocytochemical distribution of extracellular matrix receptors in human osteoclasts: a beta 3 integrin is colocalized with vinculin and talin in the podosomes of osteoclastoma giant cells

Human osteoclasts (OCLs) obtained from cell suspensions of surgically excised giant cell bone tumors (osteoclastomas) were attached to glass coverslips and analyzed by immunofluorescence with antibodies to integrins and cytoskeletal proteins. It was found that in OCLs (i) podosomes, identified by their F-actin core and by interference reflection microscopy, were predominantly found in a peripheral belt as described in avian OCLs; (ii) each F-actin core was surrounded by a ring of vinculin and talin; (iii) beta 1 integrin was diffuse in the ventral membrane; (iv) beta 3 integrin was distributed in intensely fluorescent rings surrounding F-actin cores; (v) beta 2 integrin was absent; (vi) beta 4 integrin was absent. The macrophages detected in the same coverslips displayed podosomes containing beta 2 but not beta 3, fibroblasts showed adhesion plaques positive for beta 1 and beta 3 but not for beta 2, and platelets were intensely positive for beta 3. These results indicate that OCLs produce an integrin complex that is absent in the monocyte-macrophage lineage.     

Increased 5-Lipoxygenase Immunoreactivity in the Hippocampus of Patients With Alzheimer's Disease

The proinflammatory enzyme 5-lipoxygenase (5-LOX) is upregulated in Alzheimer''s disease (AD), but its localization and association with the hallmark lesions of the disease, β-amyloid (Aβ) plaques and neurofibrillary tangles (NFTs), is unknown. This study examined the distribution and cellular localization of 5-LOX in the medial temporal lobe from AD and control subjects. The spatial relationship between 5-LOX immunoreactive structures and AD lesions was also examined. We report that, in AD subjects, 5-LOX immunoreactivity is elevated relative to controls, and its localization is dependent on the antibody-targeted portion of the 5-LOX amino acid sequence. Carboxy terminus–directed antibodies detected 5-LOX in glial cells and neurons, but less frequently in neurons with dystrophic (NFT) morphology. In contrast, immunoreactivity observed using 5-LOX amino terminus–directed antibodies was virtually absent in neurons and abundant in NFTs, neuritic plaques, and glia. Double-labeling studies showed a close association of 5-LOX–immunoreactive processes and glial cells with Aβ immunoreactive plaques and vasculature and also detected 5-LOX in tau immunoreactive and amyloid containing NFTs. Different immunolabeling patterns with antibodies against carboxy vs amino terminus of 5-LOX may be caused by post-translational modifications of 5-LOX protein in Aβ plaques and NFTs. The relationship between elevated intracellular 5-LOX and hallmark AD pathological lesions provides further evidence that neuroinflammatory pathways contribute to the pathogenesis of AD. (J Histochem Cytochem 56:1065–1073, 2008)     

Overexpression of the neuritotrophic cytokine S100beta precedes the appearance of neuritic beta-amyloid plaques in APPV717F mice

Homozygous APPV717F transgenic mice overexpress a human beta-amyloid precursor protein (betaAPP) minigene encoding a familial Alzheimer's disease mutation. These mice develop Alzheimer-type neuritic beta-amyloid plaques surrounded by astrocytes. S100beta is an astrocyte-derived cytokine that promotes neurite growth and promotes excessive expression of betaAPP. S100beta overexpression in Alzheimer's disease correlates with the proliferation of betaAPP-immunoreactive neurites in beta-amyloid plaques. We found age-related increases in tissue levels of both betaAPP and S100beta mRNA in transgenic mice. Neuronal betaAPP overexpression was found in cell somas in young mice, whereas older mice showed betaAPP overexpression in dystrophic neurites in plaques. These age-related changes were accompanied by progressive increases in S100beta expression, as determined by S100beta load (percent immunoreactive area). These increases were evident as early as 1 and 2 months of age, months before the appearance of beta-amyloid deposits in these mice. Such precocious astrocyte activation and S100beta overexpression are similar to our earlier findings in Down's syndrome. Accelerated age-related overexpression of S100beta may interact with age-associated overexpression of mutant betaAPP in transgenic mice to promote development of Alzheimer-like neuropathological changes.     

A survey of the apes in the Dzanga-Ndoki National Park, Central African Republic: a comparison between the census and survey methods of estimating the gorilla (Gorilla gorilla gorilla) and chimpanzee (Pan troglodytes) nest group density

A survey of apes was carried out between October 1996 and May 1997 in the Dzanga sector of the Dzanga‐Ndoki National Park, Central African Republic (CAR), to estimate gorilla (Gorilla gorilla gorilla) and chimpanzee (Pan troglodytes) densities. The density estimates were based on nest counts. The strip transect census and the line transect survey method (Standing Crop Nest Count) were used to estimate the gorilla nest group density. The strip transect has been most commonly used to date. It assumes that all nest groups within the width of the strip are detected, but as this assumption is easily violated in the dense tropical rain forest, the line transect survey was also used. In this method, only the nest groups on the transect line itself should be detected. This method proved to be an adequate and easy technique for estimating animal densities in dense vegetation. The gorilla density of 1.6 individuals km^?2 (line transect survey method) found for the Dzanga sector is one of the highest densities ever reported in the literature for the Western lowland gorilla. The density estimate for chimpanzees was 0.16 individuals km^?2 (census method). The results of this study confirm the importance of the Dzanga‐Ndoki National Park for primate conservation.     

Codetection of Respiratory Syncytial Virus in Habituated Wild Western Lowland Gorillas and Humans During a Respiratory Disease Outbreak

Pneumoviruses have been identified as causative agents in several respiratory disease outbreaks in habituated wild great apes. Based on phylogenetic evidence, transmission from humans is likely. However, the pathogens have never been detected in the local human population prior to or at the same time as an outbreak. Here, we report the first simultaneous detection of a human respiratory syncytial virus (HRSV) infection in western lowland gorillas (Gorilla gorilla gorilla) and in the local human population at a field program in the Central African Republic. A total of 15 gorilla and 15 human fecal samples and 80 human throat swabs were tested for HRSV, human metapneumovirus, and other respiratory viruses. We were able to obtain identical sequences for HRSV A from four gorillas and four humans. In contrast, we did not detect HRSV or any other classic human respiratory virus in gorilla fecal samples in two other outbreaks in the same field program. Enterovirus sequences were detected but the implication of these viruses in the etiology of these outbreaks remains speculative. Our findings of HRSV in wild but human-habituated gorillas underline, once again, the risk of interspecies transmission from humans to endangered great apes.     

Bioinformatic analysis of the SPs and NFTs proteomes unravel putative biomarker candidates for Alzheimer's disease

Aging is the main risk factor for the appearance of age-related neurodegenerative diseases, including Alzheimer's disease (AD). AD is the most common form of dementia, characterized by the presence of senile plaques (SPs) and neurofibrillary tangles (NFTs), the main histopathological hallmarks in AD brains. The core of these deposits are predominantly amyloid fibrils in SPs and hyperphosphorylated Tau protein in NFTs, but other molecular components can be found associated with these pathological lesions. Herein, an extensive literature review was carried out to obtain the SPs and NFTs proteomes, followed by a bioinformatic analysis and further putative biomarker validation. For SPs, 857 proteins were recovered, and, for NFTs, 627 proteins of which 375 occur in both groups and represent the common proteome. Gene Ontology (GO) enrichment analysis permitted the identification of biological processes and the molecular functions most associated with these lesions. Analysis of the SPs and NFTs common proteins unraveled pathways and molecular targets linking both histopathological events. Further, validation of a putative phosphotarget arising from the in silico analysis was performed in serum-derived extracellular vesicles from AD patients. This bioinformatic approach contributed to the identification of putative molecular targets, valuable for AD diagnostic or therapeutic intervention.     

Profiling of amyloid beta peptide variants using SELDI Protein Chip arrays

The profile of amyloid beta (A beta) peptide variants secreted into the media of human cultured cells that express the amyloid precursor protein was examined by Surface Enhanced Laser Desorption/Ionization (SELDI) ProteinChip technology from Ciphergen Biosystems using biologically active ProteinChip Arrays. An anti-A beta polyclonal antibody (anti-NTA4) was used to capture and purify multiple immunoreactive A beta fragments from a single microliter of media onto the ProteinChip Array. Fragments retained on the surface of the ProteinChip Array were detected directly by mass in the ProteinChip System to provide detailed information on the identity of different A beta variants secreted from the cultured cells. We discuss existing and potential applications of this immunoassay for the detection and relative quantitation of A beta species from both cultured cell systems and clinical samples.     

C-Terminal Fragments of Amyloid-β Peptide Cause Cholinergic Axonal Degeneration by a Toxic Effect Rather Than by Physical Injury in the Nondemented Human Brain

Previous experimental studies have indicated that amyloid-b peptide (A) may cause axonal degeneration in the brain of individuals with Alzheimer's disease (AD) by physical injury, mass lesion, or membrane perturbation. In this study, acetylcholinesterase histochemical, and A and tau immunohistochemical double-staining were performed in nondemented elderly human hippocampal and entorhinal brain samples, to demonstrate the presence of dystrophic neurites caused by the C-terminal or N-terminal fragments of A. The early interactions between the A-stained senile plaques (SPs) and the enzyme-positive axons were investigated. The double-stained samples revealed that A deposition occurs first, followed by the development of cholinergic axonal damage. Most of the dystrophic axonal processes are incorporated in the peripheral area of the SPs and are positive for phosphorylated tau [pS²⁰²] and tau-5. The result suggests that C-terminal fragments are more harmful than N-terminal fragments of A and may induce the development of dystrophic neurites by a toxic effect rather than by physical injury.     

Growth of beta-amyloid(1-40) protofibrils by monomer elongation and lateral association. Characterization of distinct products by light scattering and atomic force microscopy

Amyloid plaques in brain tissue are a hallmark of Alzheimer's disease. Primary components of these plaques are 40- and 42-residue peptides, denoted A beta(1-40) and A beta(1-42), that are derived by proteolysis of cellular amyloid precursor protein. Synthetic A beta(1-40) and A beta(1-42) form amyloid fibrils in vitro that share many features with the amyloid in plaques. Soluble intermediates in A beta fibrillogenesis, termed protofibrils, have been identified previously, and here we describe the in vitro formation and isolation of A beta(1-40) protofibrils by size exclusion chromatography. In some experiments, the A beta(1-40) was radiomethylated to better quantify various A beta species. Mechanistic studies clarified two separate modes of protofibril growth, elongation by monomer deposition and protofibril-protofibril association, that could be resolved by varying the NaCl concentration. Small isolated protofibrils in dilute Tris-HCl buffers were directed along the elongation pathway by addition of A beta(1-40) monomer or along the association pathway by addition of NaCl. Multi-angle light scattering analysis revealed that protofibrils with initial molecular masses M(w) of (7-30) x 10(3) kDa grew to M(w) values of up to 250 x 10(3) kDa by these two growth processes. However, the mass per unit length of the associated protofibrils was about 2-3 times that of the elongated protofibrils. Rate constants for further elongation by monomer deposition with the elongated, associated, and initial protofibril pools were identical when equal number concentrations of original protofibrils were compared, indicating that the original number of protofibril ends had not been altered by the elongation or association processes. Atomic force microscopy revealed heterogeneous initial protofibrils that became more rodlike following the elongation reaction. Our data indicate that protofibril elongation in the absence of NaCl results from monomer deposition only at the ends of protofibrils and proceeds without an increase in protofibril diameter. In contrast, protofibril association occurs in the absence of monomer when NaCl is introduced, but this association involves lateral interactions that result in a relatively disordered fibril structure.     

Immunoreactive dynorphin-A in ovine anterior pituitary and effects of estradiol-17 beta administration

Anterior lobe (AL) tissue of the ovine pituitary gland contained a form of immunoreactive dynorphin-A (ir-DYN-A) larger than that found in pituitary neurointermediate lobe. Administration of estradiol-17 beta or estradiol-17 beta plus progesterone to ovariectomized sheep decreased AL tissue concentrations of ir-DYN-A but did not affect any LH parameter. Enzymatically dispersed AL cells also contained ir-DYN-A, but specific release during in vitro incubation was too low to be detected even when cells were exposed to gonadotropin-releasing hormone.