Lawrence K. Duffy

Isolation of Low-Molecular-Weight Proteins from Amyloid Plaque Fibers in Alzheimer's Disease

During aging of the human brain, and particularly in Alzheimers disease, progressive neuronal loss is accompanied by the formation of highly stable intra‐ and extraneuronal protein fibers. Using fluorescence‐activated particle sorting, a method has been developed for purifying essentially to homogeneity the extracellular amyloid fibers that form the cores of senile plaques. The purified plaque cores each contain 60–130 pg of protein. Their amino acid composition shows abundant glycine, trace proline, and ∼50% hydrophobic residues; it resembles that of enriched fractions of the paired helical filaments (PHF) that accumulate intraneuronally in Alzheimers disease. Senile plaque amyloid fibers share with PHF insolubility in numerous protein denaturants and resistance to proteinases. However, treatment of either fiber preparation with concentrated (88%) formic acid or saturated (6.8 M) guanidine thiocyanate followed by sodium dodecyl sulfate causes disappearance of the fibers and releases proteins migrating at 5–7.000 and 11–15.000 Mr which appear to be dimerically related. Following their separation by size‐exclusion HPLC, the proteins solubilized from plaque amyloid and PHF‐enriched fractions have highly similar compositions and, on dialysis, readily aggregate into higher Mr polymers. Antibodies raised to the major low‐Mr protein selectively label both plaque cores and vascular amyloid deposits in Alzheimer brain but do not stain neurofibrillary tangles, senile plaque neurites, or any other neuronal structure. Thus, extraneuronal amyloid plaque filaments in Alzheimers disease are composed of hydrophobic low‐Mr protein(s) which are also present in vascular amyloid deposits. Current evidence suggests that such protein(s) found in PHF‐enriched fractions may derive from copurifying amyloid filament srather than from PHF.

An ultrastructural analysis of tissue surrounding a microdialysis probe

Microdialysis is a widely used in vivo sampling technique commonly used to monitor extracellular levels of a variety of molecules including neurotransmitters and metabolites. To facilitate interpretation of microdialysis results, this study critically examines changes in synaptic morphology induced by microdialysis. Tissue surrounding microdialysis probes was examined using light and electron microscopy at three distances from the probe tract. Microdialysis probes were implanted into rat striatum, and after 40 h of post-operative recovery were perfused with a modified Ringers solution. Light microscope analysis revealed tissue disruption up to 1.4 mm from the probe site. Axonal damage indicative of non-excitotoxic insult was also seen as far away from the probe as was examined. The presence of dark-degenerating neurons was also noted and estimates of neuronal densities revealed loss up to 400 microm from the probe tract. This study, the first qualitative ultrastructural investigation of neuropil surrounding the probe site, indicated swollen processes up to 1.4 mm from the probe tract. Swollen mitochondria and bloated endoplasmic reticulum suggest intracellular chemical disruption. Tissue damage resulting in synaptic and neuronal disruption may affect neurotransmitter efflux or extracellular concentrations of metabolites.

Protein chemical and immunocytochemical studies of meningovascular β-amyloid protein in Alzheimer's disease and normal aging

As a comparison to previous analyses of purified amyloid plaque cores from Alzheimers disease (AD) brain, we performed protein chemical and immunocytochemical studies on amyloid filaments extracted from meningeal blood vessels of patients with Alzheimers disease. Results were compared with those obtained from identically prepared fractions of aged normals without cerebral amyloid angiopathy or other microscopic findings of AD. The amyloid isolation method of Glenner and Wong was modified, including an extraction with sodium dodecyl sulfate (SDS). Gel electrophoresis of purified amyloid from AD meninges yielded bands centered at 4.2 kDa. Sequencing of the HPLC-purified amyloid protein from AD meninges confirmed the published beta-protein sequence for residues 1-30 and 35-40, with the exception of glutamic acid rather than glutamine at position 11. N-terminal heterogeneity was not prominent. No sequence beyond residue 40 was obtained. Proteins of similar but not identical mol. wt. were present in HPLC-purified fractions of normal meninges; neither the beta-protein sequence nor any other interpretable sequence was detected in such fractions. Two antisera raised against the purified AD meningovascular amyloid protein identified the 4.2 kDa band on Western blots of AD preparations; no protein band in this region was labeled in control preparations. The 4.2 kDa band in AD meningeal preparations was also lableled by an antiserum to synthetic beta-peptide but not by an antiserum to the carboxyl terminus of the beta-protein precursor. Both the AD meningovascular amyloid antisera selectively labeled amyloid in cortical and meningeal vessels and plaque cores; tangles, plaque neurites, and cells of normal CNS and numerous non-neural tissues were unstained. The antisera also labeled the occasional deposits of vascular amyloid and less frequent plaque core amyloid found in some aged individuals without AD. We conclude that (1) the meningovascular amyloid beta-protein of AD, whose sequence has been confirmed and extended to residue 40, was not immunocytochemically detectable in neurofibrillary tangles; (2) beta-protein could not be detected in meningeal preparations from aged controls who lack light microscopically visible meningovascular amyloid; and (3) the vascular and plaque core amyloid present in aged normals is antigenically cross-reactive with AD meningovascular amyloid.

Feeding ecology of phocid seals and some walrus in the Alaskan and Canadian Arctic as determined by stomach contents and stable isotope analysis

Feeding habits of ringed (Phoca hispida), bearded (Erignathus barbatus), spotted (Phoca largha) and ribbon (Phoca fasciata) seals and walrus (Odobenus rosmarus) were studied using stomach contents and stable carbon and nitrogen isotopes. Bearded seals fed benthically, primarily crustaceans and mollusks. Both zooplankton and fish were significant prey for ringed seals, while fish was principal spotted seal prey. Few gastric contents were available from ribbon seals. δ15N was positively correlated with age in ribbon seals and δ13C was positively correlated with age in ringed and ribbon seals. δ15N was highest in spotted seals, in agreement with their fish-dominated diet. δ15N was not different between Alaskan-harvested ringed and bearded seals, while δ15N was lowest in ribbon seals and walrus. Carbon-13 was most enriched in bearded seals and walrus reflecting benthic ecosystem use. Canadian ringed seals were depleted in 13C compared to Alaskan pinnipeds, likely because of Beaufort Sea versus Chukchi and Bering seas influence.

SOCIAL BEHAVIOR AND ECOSYSTEM PROCESSES: RIVER OTTER LATRINES AND NUTRIENT DYNAMICS OF TERRESTRIAL VEGETATION

River otters (Lutra canadensis Schreber) inhabiting coastal environments scent-mark specific locations along the coast, known as latrine sites. In this study, we used stable isotope techniques to investigate the effects of this scent-marking behavior on terrestrial vegetation at the terrestrial–marine interface. Our analysis of stable isotope ratios of fur and feces indicated that river otters fed mainly on intertidal and subtidal fish. Eight different species of plants, growing in latrine sites of river otters, had significantly higher values of δ15N compared with the same plant species growing on nonlatrine sites. Elevated N concentrations occurred only in grasses and mosses growing in latrine sites. Our results indicate that, through their scent-marking behavior, coastal river otters transfer marine-derived nitrogen into the beach-fringe forest and thus fertilize the terrestrial vegetation in the terrestrial–marine interface.

Comparison of mercury and methylmercury in northern pike and Arctic grayling from western Alaska rivers

In western Alaska, mercury (Hg) could be a potential health risk to people whose diet is primarily fish-based. In 2000, total Hg (THg) and methylmercury (MeHg) were examined in northern pike (Esox lucius) and Arctic grayling (Thymallus arcticus) from two watersheds in western Alaska, the Yukon and Kuskokwim rivers. Whitefish (Coregonus sp.) were also examined from the Kuskokwim River. Pike from the Yukon and Kuskokwim rivers had mean concentrations of THg in muscle of 1.506 and 0.628 mg/kg wet wt, respectively. The mean concentrations of THg in grayling muscle from these rivers were 0.264 and 0.078 mg/kg, respectfully. Whitefish had a mean THg concentration in muscle of 0.032 mg/kg. MeHg, in pike and grayling constituted nearly 100% of the THg concentrations; the proportion was less in whitefish. A significant positive correlation between Hg levels and fish length was also found. Generally, there were no changes in Hg concentrations in pike or grayling over the last several years. Only pike from theYukon River had THg concentrations that exceeded the USFDA action level for human consumption of edible fish (1 mg/kg). Human hazard index for pike was > or = 1 for both adults and children, indicating a potential for toxic concern, especially among children. Further studies are needed to determine the environmental and human health impacts associated with these Hg concentrations in western Alaska, especially in the context of potentially increased consumption of resident fishes when anadromous salmon catches are reduced.

COMPARISON OF PIGEON GUILLEMOT, CEPPHUS COLUMBA, BLOOD PARAMETERS FROM OILED AND UNOILED AREAS OF ALASKA EIGHT YEARS AFTER THE EXXON VALDEZ OIL SPILL

In 1997, we compared the haematological and plasma biochemical profiles among populations of pigeon guillemots, Cepphus columba, in areas oiled and not oiled by the 1989 Exxon Valdez oil spill (EVOS) that occurred in Prince William Sound (PWS), Alaska. Pigeon guillemot populations in PWS were injured by EVOS and have not returned to pre-spill levels. If oil contamination is limiting recovery of pigeon guillemots in PWS, then we expected that blood parameters of pigeon guillemots would differ between oiled and unoiled areas and that these differences would be consistent with either toxic responses or lower fitness. We collected blood samples from chicks at approximately 20 and 30 days after hatching. Physiological changes associated with chick growth were noted in several blood parameters. We found that only calcium and mean cell volume were significantly different between the chicks in oiled and unoiled areas. Despite these differences, blood biomarkers provided little evidence of continuing oil injury to pigeon guillemot chicks, eight years after the EVOS. Preliminary data from adults indicated elevated aspartate aminotransferase activity in the adults from the oiled area, which is consistent with hepatocellular injury. Because adults have greater opportunities for exposure to residual oil than nestlings, we recommend studies that fully evaluate the health of adults residing in oiled areas.

Multi-generation health risks of persistent organic pollution in the far north: use of the precautionary approach in the Stockholm Convention

Abstract Precautionary regulation of persistent, toxic substances is controversial because of continued and irresolvable uncertainties in ecotoxicology. This is especially an issue for people who eat wild food, still a common practice in the Arctic. Persistent organic pollutants (POPs) have been shown to interfere with hormone function and genetic regulation. In animal studies, myriad dysfunctions can be induced (manifested later in life) by low-dose POPs exposure during development. The ubiquity of POPs in biological tissue makes all organisms subject to developmental exposure. The Arctic, where subsistence living is common, is a sink region for POPs. To curtail bioaccumulation and biomagnification, the United Nations has created the Stockholm Convention in May 2001, which targets 12 chemicals for virtual elimination. Using the precautionary approach, the treaty also enables the listing of new targets as threats are recognized. The “dirty dozen” are well-documented developmental toxics and other POPs are expected to exhibit similar patterns of accumulation and harm. Arctic peoples insist that waiting for irrefutable evidence is poor planning. Nevertheless, the United States, a major signatory, has proved reluctant to ratify the language that would enable this expedient listing of new targets. Such reluctance allows health threats in the Arctic and around the world to grow. This paper reviews the theoretical background for and current evidence regarding the global issues of endocrine disruption and POPs contamination, especially as they relate to wildlife and people in the far north. It is concluded that there is an urgent need for the US to ratify the full text of the Stockholm Convention, including the provision for the listing of new targets.

Partial Sequence of MAP2 in the Region of a Shared Epitope with Alzheimer Neuronbrillary Tangles

Abstract: A 3.3‐kilobase DNA complementary to human microtubule‐associated protein 2 (MAP2) was sequenced by the dideoxy method. The 3’end terminates at an internal EcoRI site before the polyA tail. Due to the arrangement of the cDNA insert in the λX gt 11 vector, the MAP2 fragment is not fused to β‐galactosidase when expressed. The Chou Fasman algorithm for the initial 58 amino acids from the first in‐frame methionine predicts an α helix. Beyond this point, a series of turns is predicted until amino acid 160. The frequent presence of basic residues in proximity to serines or threonines is consistent with multiple phosphorylation sites. The minimum specificity determinant for Ca2+/calmodulin‐dependent kinase is repeated 13 times. The sequence of a region containing a MAP2 epitope that is shared with the Alzheimer neuronbrillary tangle was determined by DNase treatment of the cDNA and antibody selecting the small resultant clones in a λ gt 11 sublibrary. Likewise, a MAP2 epitope that is not shared with the neurofibrillary tangle also has been located. Both epitopes are in the projection portion of the molecule. A bovine MAP2 cyanogen bromide fragment, which contains the epitope shared with the neurofibrillary tangle, is partially insoluble under aqueous conditions, probably due to the aggregation of oppositely charged residues. Thus, rapid cleavage of MAP2 to small peptides is probably necessary in vivo to prevent the aggregation of larger cleavage fragments.

COMPARATIVE BASELINE LEVELS OF MERCURY, HSP 70 AND HSP 60 IN SUBSISTENCE FISH FROM THE YUKON-KUSKOKWIM DELTA REGION OF ALASKA

In subsistence fish; northern pike (Esox lucius), burbot (Lota lota), whitefish (Coregonus nelsoni), grayling (Thymallus arcticus) and sheefish (Stenodus lencichthys), we determined the Hsp 60 and Hsp 70 levels in 31 samples from adult fish gills. A dot-blot analysis using antibodies to either Hsp 70 or Hsp 60 showed the average Hsp 70 concentration was 9.1 microg/mg protein, while the average Hsp 60 concentration was 147.4 microg/mg protein. Mercury levels in muscle tissue in these fish averaged 0.382 ppm. Using a subset of samples (n = 24), we determined that the major component in the muscle of Alaskan subsistence fish was methyl mercury. No correlation was observed between Hsp 60 or Hsp 70 expression in gill tissue and mercury concentrations in muscle tissue. Hsp 60 and Hsp 70 protein levels in the gills were correlated.