Improving the electrostatic design of the Jefferson Lab 300 kV DC photogun.

The 300 kV DC high voltage photogun at Jefferson Lab was redesigned to deliver electron beams with a much higher bunch charge and improved beam properties. The original design provided only a modest longitudinal electric field (Ez) at the photocathode, which limited the achievable extracted bunch charge. To reach the bunch charge goal of approximately few nC with 75 ps full-width at half-maximum Gaussian laser pulse width, the existing DC high voltage photogun electrodes and anode-cathode gap were modified to increase Ez at the photocathode. In addition, the anode aperture was spatially shifted with respect to the beamline longitudinal axis to minimize the beam deflection introduced by the non-symmetric nature of the inverted insulator photogun design. We present the electrostatic design of the original photogun and the modified photogun and beam dynamics simulations that predict vastly improved performance. We also quantify the impact of the photocathode recess on beam quality, where recess describes the actual location of the photocathode inside the photogun cathode electrode relative to the intended location. A photocathode unintentionally recessed/misplaced by sub-millimeter distance can significantly impact the downstream beam size.

Pulsed-laser nonlinear Thomson scattering for general scattering geometries.

In a recent paper it has been shown that single electron Thomson backscatter calculations can be performed including the effects of pulsed high intensity lasers. In this paper we present a more detailed treatment of the problem and present results for more general scattering geometries. In particular, we present new results for 90 degrees Thomson scattering. Such geometries have been increasingly studied as x-ray sources of short-pulse radiation. Also, we present a clearer physical basis for these different cases.

Spectral distributions of thomson-scattered photons from high-intensity pulsed lasers.

General formulas for the far-field spectral distribution of photons Thomson scattered by a single electron have been obtained. Effects due to the pulsed nature of the laser beam are explicitly allowed, simultaneously with intensity high enough that harmonic generation is possible. For realistic pulsed photon beams, the spectrum of backscattered radiation is considerably broadened because of changes in the longitudinal velocity of the electrons during the pulse. Such ponderomotive broadening is especially pronounced at higher harmonics, eventually leading to a continuous emission spectrum.

Energy-recovery linac project at Cornell University.

There is considerable interest in using superconducting electron linacs with energy recovery as synchrotron radiation sources. Such energy recovery linacs (ERLs) would open new regimes of X-ray science because they are capable of producing ultra-brilliant X-ray beams [>5 x 10(22) photons s(-1) (0.1% bandwidth)(-1) mm(-2) mrad(-2) at 10 keV], maintaining a very small source size ( approximately 3 micro m r.m.s.) suitable for micro X-ray beams, and making very intense fast ( approximately 100 fs) X-ray pulses. Each of these characteristics would permit the execution of experiments that are not feasible with existing synchrotron sources. Many technical issues must be satisfactorily resolved before the potential of a full-scale ERL can be realised, including the generation of high average current (10 to 100 mA), high-brightness electron beams (0.015 to 0.15 nm rad emittances, respectively); acceleration of these beams to energies of 5-7 GeV without unacceptable emittance degradation; stable and efficient operation of superconducting linear accelerators at very high gradients etc. Cornell University, in collaboration with Jefferson Laboratory, has proposed to resolve these issues by the construction of a 100 MeV, 100 mA prototype ERL. The intention is to then utilize the information that is learned from the prototype to propose the construction of a full-scale ERL light source.

Vaccination with soluble Abeta oligomers generates toxicity-neutralizing antibodies.

In recent studies of transgenic models of Alzheimer's disease (AD), it has been reported that antibodies to aged beta amyloid peptide 1-42 (Abeta(1-42)) solutions (mixtures of Abeta monomers, oligomers and amyloid fibrils) cause conspicuous reduction of amyloid plaques and neurological improvement. In some cases, however, neurological improvement has been independent of obvious plaque reduction, and it has been suggested that immunization might neutralize soluble, non-fibrillar forms of Abeta. It is now known that Abeta toxicity resides not only in fibrils, but also in soluble protofibrils and oligomers. The current study has investigated the immune response to low doses of Abeta(1-42) oligomers and the characteristics of the antibodies they induce. Rabbits that were injected with Abeta(1-42) solutions containing only monomers and oligomers produced antibodies that preferentially bound to assembled forms of Abeta in immunoblots and in physiological solutions. The antibodies have proven useful for assays that can detect inhibitors of oligomer formation, for immunofluorescence localization of cell-attached oligomers to receptor-like puncta, and for immunoblots that show the presence of SDS-stable oligomers in Alzheimer's brain tissue. The antibodies, moreover, were found to neutralize the toxicity of soluble oligomers in cell culture. Results support the hypothesis that immunizations of transgenic mice derive therapeutic benefit from the immuno-neutralization of soluble Abeta-derived toxins. Analogous immuno-neutralization of oligomers in humans may be a key in AD vaccines.

Targeting small Abeta oligomers: the solution to an Alzheimer's disease conundrum?

Amyloid beta (Abeta) is a small self-aggregating peptide produced at low levels by normal brain metabolism. In Alzheimer's disease (AD), self-aggregation of Abeta becomes rampant, manifested most strikingly as the amyloid fibrils of senile plaques. Because fibrils can kill neurons in culture, it has been argued that fibrils initiate the neurodegenerative cascades of AD. An emerging and different view, however, is that fibrils are not the only toxic form of Abeta, and perhaps not the neurotoxin that is most relevant to AD: small oligomers and protofibrils also have potent neurological activity. Immuno-neutralization of soluble Abeta-derived toxins might be the key to optimizing AD vaccines that are now on the horizon.

Purification and characterization of the yeast glycosylphosphatidylinositol-anchored, monobasic-specific aspartyl protease yapsin 2 (Mkc7p).

The Saccharomyces cerevisiae YPS2 (formerly MKC7) gene product is a glycosylphosphatidylinositol-linked aspartyl protease that functions as a yeast secretase. Here, the glycosylphosphatidylinositol-linked form of yapsin 2 (Mkc7p) was purified to homogeneity from the membrane fraction of an overexpressing yeast strain. Purified yapsin 2 migrated diffusely in SDS-polyacrylamide gel electrophoresis (molecular mass approximately 200 kDa), suggesting extensive, heterogeneous glycosylation. Studies using internally quenched fluorogenic peptide substrates revealed cleavage by the enzyme carboxyl to Lys or Arg. No cleavage was seen when both Lys and Arg were absent. No significant enhancement was seen with multiple basic residues. However, cleavage always occurred carboxyl to the most COOH-terminal basic residue. V(max)/K(m) was insensitive to P(2) and P(3) residues except that Pro at P(2) blocked cleavage entirely. These results suggest that yapsin 2 is a monobasic amino acid-specific protease that requires a basic residue at P(1) and excludes basic residues from P(1)'. The pH dependence of V(max)/K(m) for a substrate containing a pro-alpha factor cleavage site was bell-shaped, with a maximum near pH 4.0. However, V(max)/K(m) for a substrate mimicking the alpha-secretase site in human beta amyloid precursor protein was optimal near pH 6.0, consistent with cleavage of beta amyloid precursor protein by yapsin 2 when expressed in yeast.

Involvement of cell surface glycosyl-phosphatidylinositol-linked aspartyl proteases in alpha-secretase-type cleavage and ectodomain solubilization of human Alzheimer beta-amyloid precursor protein in yeast.

Human beta-amyloid precursor protein (APP) introduced into yeast undergoes alpha-secretase-type cleavage, suggesting that yeast have alpha-secretase-like protease(s). Here we report that two structurally and functionally related glycosyl-phosphatidylinositol-linked yeast aspartyl proteases, Mkc7p and Yap3p (collectively termed yapsin), are responsible for alpha-secretase-type cleavage of APP expressed in yeast, resulting in release of soluble APP into the extracellular space. Disruption of MKC7 and YAP3 in a vacuolar protease-deficient strain abolished this APP cleavage/release, and APP cleavage/release could be restored by introduction of MKC7 or YAP3 on a single copy plasmid. Purified Mkc7p cleaved an internally quenched fluorogenic APP peptide substrate at the alpha-secretase cleavage site. Measurement of proteolytic activity either in yeast homogenates or on the yeast cell surface revealed that most Mkc7p and Yap3p activities were localized at the cell surface. These results establish a molecular basis for alpha-secretase-type cleavage in yeast and support the generally held concept that alpha-secretase cleavage of APP occurs at the cell surface.

Amyloid-beta peptide activates cultured astrocytes: morphological alterations, cytokine induction and nitric oxide release.

A common feature of many neurodegenerative disorders is an abundance of activated glial cells (astrocytes and microglia). In Alzheimer's disease (AD), activated astrocytes are in close apposition to and surrounding the amyloid plaques. The mechanisms by which the astrocytes become activated in AD and the consequences of reactive astrocytosis to disease progression are not known. We examined the possibility that the amyloid-beta (Abeta) peptide, a major constituent of the amyloid plaque, could act as a stimulus leading to activation. We found that treatment of rat cortical astrocyte cultures with aggregated Abeta 1-42 peptide induces activation, as assessed by reactive morphological changes and upregulation of selective glial mRNA and proteins, such as the inflammatory cytokine interleukin-1beta. Abeta also stimulates inducible nitric oxide synthase (iNOS) mRNA levels and nitric oxide (NO) release. Abeta 1-42, a major form of amyloid associated with neurotoxicity, activated astrocytes in a time- and dose-dependent manner, whereas a scrambled Abeta 1-42 sequence or Abeta 17-42 had little or no effect. We also determined that the Abeta activity can be found in a supernatant fraction containing soluble Abeta oligomers. Our data suggest that Abeta plays a role in the reactive astrocytosis of AD and that the inflammatory response induced upon glial activation is a critical component of the neurodegenerative process.

Diffusible, nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins.

Abeta1-42 is a self-associating peptide whose neurotoxic derivatives are thought to play a role in Alzheimer's pathogenesis. Neurotoxicity of amyloid beta protein (Abeta) has been attributed to its fibrillar forms, but experiments presented here characterize neurotoxins that assemble when fibril formation is inhibited. These neurotoxins comprise small diffusible Abeta oligomers (referred to as ADDLs, for Abeta-derived diffusible ligands), which were found to kill mature neurons in organotypic central nervous system cultures at nanomolar concentrations. At cell surfaces, ADDLs bound to trypsin-sensitive sites and surface-derived tryptic peptides blocked binding and afforded neuroprotection. Germ-line knockout of Fyn, a protein tyrosine kinase linked to apoptosis and elevated in Alzheimer's disease, also was neuroprotective. Remarkably, neurological dysfunction evoked by ADDLs occurred well in advance of cellular degeneration. Without lag, and despite retention of evoked action potentials, ADDLs inhibited hippocampal long-term potentiation, indicating an immediate impact on signal transduction. We hypothesize that impaired synaptic plasticity and associated memory dysfunction during early stage Alzheimer's disease and severe cellular degeneration and dementia during end stage could be caused by the biphasic impact of Abeta-derived diffusible ligands acting upon particular neural signal transduction pathways.

Internally consistent libraries of fluorogenic substrates demonstrate that Kex2 protease specificity is generated by multiple mechanisms.

Kex2 protease from the yeast Saccharomyces cerevisiae is the prototype for a family of eukaryotic proprotein processing proteases. To clarify understanding of the interactions responsible for substrate recognition in this family of enzymes, we have carried out a systematic examination of Kex2 substrate specificity using internally consistent sets of substrates having substitutions at only one or two positions. We examined Kex2 sequence recognition for residues at P3, P2, and P1 using two types of fluorogenic peptide substrates, peptidyl-methylcoumarinamides and internally quenched substrates in which cleavage occurs at an actual peptide bond. Kinetic analysis of the two sets of substrates gave comparable data on specificity at these three positions. For the best substrate sequences, high catalytic constants (kCM/KM) of (2-5) x 10(7) M-1 s-1 were seen for cleavage of both peptidyl-methylcoumarinamides and peptide bonds. While no evidence for positive interactions with the P3 residue emerged, Kex2 was found to discriminate against at least one residue Asp. at this position. Specificity at P2 was shown to rely primarily on recognition of a positive charge, although steric constraints on the P2 side chain were also apparent. Kex2 was demonstrated to be exquisitely selective for Arg at P1. Substitutions with similar charge (Lys, ornithine) or similar hydrogen-bonding capability (citrulline) do not confer efficient catalysis. Comparison of otherwise identical substrates having either Arg or citrulline at P1 showed that the positive charge of the Arg guanidinium group stabilizes the transition state by approximately 6.8 kcal/mol.

Rapid impact of beta-amyloid on paxillin in a neural cell line.

Beta-amyloid1-42 (Abeta) is a naturally occuring peptide whose accumulation in the brain is putatively coupled to Alzheimer's disease pathogenesis. Deleterious effects of Abeta on neurons have been linked to the inappropriate activation of signaling pathways within the cell (reviewed in Yankner, 1996), including tyrosine phosphorylation of focal adhesion kinase (FAK) (Zhang et al., 1994, 1996a,b). Here we have investigated the effects of Abeta on paxillin in a neural cell line. Paxillin, a substrate for FAK, is thought to act as a signal "integrator," functioning to link other proteins into multi-molecular signaling complexes (reviewed in Turner, 1994). Treatment of the rat central nervous system B103 cell line with aggregates of Abeta was found to induce the tyrosine phosphorylation of paxillin within 30 min, nearly 24 hr prior to significant cell death. Particularly striking was a subsequent "mobilization" of paxillin to the cytoskeleton in Abeta-treated cells. The amount of paxillin associated with the cytoskeleton in Abeta-treated cells was increased 10-fold over controls. The Abeta-induced paxillin accumulation could be visualized immunocytochemically, with an increase in number and size of paxillin-labeled focal contacts upon treatment with Abeta. This effect was specific, in that vinculin, another focal contact protein, was unaffected by Abeta. Disruption of f-actin, which inhibits both Abeta-induced neurotoxicity (Furukawa and Mattson, 1995) and focal contact signaling in B103 cells (Zhang et al., 1996b) was found to block the cytoskeletal paxillin accumulation. The rapid tyrosine phosphorylation and cytoskeletal mobilization of paxillin links Abeta to the activation of focal contact signaling events that may influence neuronal cytoskeletal architecture, gene expression, synaptic plasticity and cell viability.

Morphology and toxicity of Abeta-(1-42) dimer derived from neuritic and vascular amyloid deposits of Alzheimer's disease.

In the course of analyzing the chemical composition of Alzheimer's disease neuritic and vascular amyloid, we have purified stable dimeric and trimeric components of Abeta peptides. These peptides (molecular mass 9.0 and 13.5 kDa) were separated by size exclusion chromatography in the presence of 80% formic acid or 5 guanidine thiocyanate, pH 7.4. The average ratio of monomers, dimers, and trimers was 55:30:15, respectively. Similar structures were produced over time upon incubation of synthetic Abeta-(1-42) at pH 7.4. The stability of these oligomeric forms was also demonstrated by Western blot and mass spectrometry. Atomic force microscopy and electron microscopy rotary shadowing revealed that the monomers polymerized into 8-10-nm filaments, whereas the dimers generated prolate ellipsoids measuring 3-4 nm in diameter. The pathogenic effects of the dimeric Abeta-(1-40/42) were tested in cultures of rat hippocampal neuron glia cells. Only in the presence of microglia did the dimer elicit neuronal killing. It is possible that these potentially pathogenic Abeta-(1-40/42) dimers and trimers from Alzheimer's disease amyloid represent the soluble oligomers of Abeta recently described in Alzheimer's disease brains (Kuo, Y.-M., Emmerling, M. R., Vigo-Pelfrey, C., Kasunic, T. C., Kirkpatrick, J. B., Murdoch, G. H., Ball, M. J., and Roher, A. E. (1996) J. Biol. Chem., 271, 4077-4081).

A beta peptide enhances focal adhesion kinase/Fyn association in a rat CNS nerve cell line.

Neurotoxicity of the amyloid beta protein (A beta) is known to correlate with a selective change in protein tyrosine phosphorylation (Tyr(P)) of focal adhesion kinase (FAK) (Zhang et al., J. Biol. Chem., 269 (1994) 25247-25250). The current work has found that exposure of neuronal cells to A beta upregulates the stable association of FAK with Fyn, a neuronally-enriched protein tyrosine kinase of the Src-family. In cells incubated with aged A beta 1-42, the amount of immunoprecipitable FAK-Fyn complex increased approximately 280%. Equivalent results were obtained whether anti-FAK or anti-Fyn was used to precipitate the complex. Cells incubated with non-toxic A beta 17-42, which makes aggregates and attaches to cells but does not upregulate FAK Tyr(P), exhibited no increase in FAK-Fyn complex. Aberrant Fyn activity due to the A beta evoked association with FAK could play a role in neuronal degeneration and also cause anomalies in synaptic plasticity. These possibilities are of particular significance because of the reported increase in Fyn immunoreactivity in Alzheimer's-afflicted neurons (Shirazi and Wood, NeuroReport, 4 (1993) 435-437).

Protein kinase C and F-actin are essential for stimulation of neuronal FAK tyrosine phosphorylation by G-proteins and amyloid beta protein.

Focal adhesion kinase (FAK) is a protein tyrosine kinase implicated in signal transduction pathways for integrins, neuropeptides, and lysophosphatidic acid. FAK, first discovered in non-neuronal cells, recently has been reported to occur in neurons, where its tyrosine phosphorylation is upregulated by fibronectin and by the Alzheimer's Abeta peptide. The current work has elucidated molecular events leading to tyrosine phosphorylation of FAK in the rat B103 CNS nerve cell line. Activation of receptor-coupled G-proteins by Mas-7 was found to evoke rapid upregulation of FAK tyrosine phosphorylation (Tyr(P)). Upregulation by Mas-7 was blocked by GF109203X, a potent inhibitor of protein kinase C (PKC). Phorbol ester also upregulated FAK-YP, verifying a role for PKC in the transduction cascade. Upregulation of FAK-YP by activation of G-proteins and PKC was dependent upon intact F-actin, as cytochalasin D abolished stimulation by Mas-7 and by phorbol ester. The relatively slow increase in FAK-YP evoked by chronic exposure to Abeta also was abolished by GF109203X and by cytochalasin D. The results show that tyrosine phosphorylation of FAK in neurons is regulated positively by PKC, functioning down-stream from G-proteins through an F-actin-dependent mechanism. The Alzheimer's Abeta peptide is capable of activating elements of this same signal transduction pathway, via membrane events that remain to be determined.

The nanometer-scale structure of amyloid-beta visualized by atomic force microscopy.

Amyloid-beta (A beta) is the major protein component of neuritic plaques found in Alzheimer's disease. Evidence suggests that the physical aggregation state of A beta directly influences neurotoxicity and specific cellular biochemical events. Atomic force microscopy (AFM) is used to investigate the three-dimensional structure of aggregated A beta and characterize aggregate/fibril size, structure, and distribution. Aggregates are characterized by fibril length and packing densities. The packing densities correspond to the differential thickness of fiber aggregates along a zeta axis (fiber height above the x-y imaging surface). Densely packed aggregates ( > or = 100 nm thick) were observed. At the edges of these densely packed regions and in dispersed regions, three types of A beta fibrils were observed. These were classified by fibril thickness into three size ranges: 2-3 nm thick, 4-6 nm thick, and 8-12 nm thick. Some of the two thicker classes of fibrils exhibited pronounced axial periodicity. Substructural features observed included fibril branching or annealing and a height periodicity which varied with fibril thickness. When identical samples were visualized with AFM and electron microscopy (EM) the thicker fibrils (4-6 nm and 8-12 nm thick) had similar morphology. In comparison, the densely packed regions of approximately > or = 100 nm thickness observed by AFM were difficult to resolve by EM. The small, 2- to 3-nm-thick, fibrils were not observed by EM even though they were routinely imaged by AFM. These studies demonstrate that AFM imaging of A beta fibrils can, for the first time, resolve nanometer-scale, zeta-axis, surface-height (thickness) fibril features. Concurrent x-y surface scans of fibrils reveal the surface submicrometer structure and organization of aggregated A beta. Thus, when AFM imaging of A beta is combined with, and correlated to, careful studies of cellular A beta toxicity it may be possible to relate certain A beta structural features to cellular neurotoxicity.

Synthetic chemical diversity: solid phase synthesis of libraries of C2 symmetric inhibitors of HIV protease containing diamino diol and diamino alcohol cores.

Solid phase synthesis of non-oligomeric organic compounds has been pursued for high-efficiency generation of large numbers of structurally diverse compounds for drug screening. Known as chemical diversity libraries or combinatorial libraries (when the synthesis is carried out in a combinatorial fashion), these compounds can be used for de novo discovery of drug leads or for expedient structure--activity relationship (SAR) studies. To expand the scope of solid phase synthesis beyond the capability of the traditional method of solid phase synthesis for peptides, a strategy was developed for bi-directional solid phase synthesis starting with diamino alcohol or diamino diol core structures. The strategy relies on using bifunctional linkers to modify the core structures, simultaneously protecting the hydroxyl group or the diol moiety of the core and providing a carboxyl group for attachment of the modified cores to a solid support. The two NH2 groups of the modified cores attached to the solid support were then deprotected and reacted with a wide variety of amine-reactive reagents (carboxylic acids, sulfonyl chlorides, isocyanates, chloroformates, etc.) to extend the molecule in both directions. This strategy was successfully applied to automated parallel synthesis of a library of C2 symmetric inhibitors of HIV protease containing the known symmetry-based diamino diol and diamino alcohol core structures, thus enabling expedient access of large numbers of analogs in this series. A library of over 300 discrete compounds was synthesized using this methodology in order to identify potent (IC50 < 100 nM) HIV protease inhibitors with reduced size. This paper describes the technical aspects of this technology.

Cathepsin D from Alzheimer's-diseased and normal brains.

An acid protease activity from human brain was found to cleave a fluorogenic peptide substrate encompassing the amino terminus of Alzheimer's amyloid-beta peptide (A beta). The protease was isolated and determined to be cathepsin D based on chromatographic, immunological, and enzymatic data. Analysis of the cleavage sites indicated that cathepsin D hydrolyzed the methionine--aspartate bond generating the in vivo amino terminus of A beta. These data suggested that cathepsin D could be involved in amyloidogenic processing of the amyloid precursor protein. Consequently, cathepsin D from both Alzheimer's-diseased and control brains was compared to determine whether there were any differences which could account for an increase in A beta production in Alzheimer's disease. No differences were detected in isoform composition or tissue content of cathepsin D as measured by 2-D IEF-SDS-PAGE. Enzymological characterization of brain cathepsin D demonstrated that it could undergo a previously undescribed pH-dependent reversible activation. However, that activation appeared identical for both AD and normal brain enzymes. These data demonstrate that concentration, isoform distribution, and several enzymological characteristics of cathepsin D are not distinguishable between AD and normal brain. The pH dependence of cathepsin D activity suggests, however, that its intracellular localization may be important in considering the potential role of cathepsin D in Alzheimer's disease.

Evidence against a role for the Kunitz domain in amyloidogenic and secretory processing of the amyloid precursor protein.

The effect of the Kunitz proteinase inhibitor (KPI) on potential beta-amyloid precursor protein (beta PP)-processing activities from control and Alzheimer's disease (AD) brains was examined using fluorogenic substrates designed to mimic the secretory and amyloidogenic cleavages in beta PP. In addition, the level of secretion of KPI-containing beta PP751 and KPI-lacking beta PP695 from transfected cells was examined to assess the effect of the KPI on beta PP secretion. beta PP751 and beta PP695, obtained from conditioned media of transfected cells, had no effect on proteinase activities against the secretory and amyloidogenic substrates in extracts from control and AD brains. At similar concentrations beta PP751, but not beta PP695, completely inhibited the activity of trypsin against these substrates. Serine proteinase inhibitors had only modest effects on activities from brain, whereas cysteine modification completely inhibited them, indicating that these proteinase activities were not of the serine type. Thus, the results do not support a role for the KPI in the secretion of beta PP or in the amyloidogenic cleavage of beta PP. The amounts of beta PP695 and beta PP751 collected from the media of transfected cells after 48 h of growth were similar, indicating an equal rate of secretion. This result suggests that the KPI domain in beta PP751 did not inhibit the secretory cleavage in transfected cells.