Richard Rubenstein

Small, highly structured RNAs participate in the conversion of human recombinant PrPSen to PrPRes in vitro

We have identified a small, highly structured (shs)RNA that binds human recombinant prion protein (hrPrP) with high affinity and specificity under physiological conditions (e.g. 10% bovine calf serum (BCS), neutral pH, nanomolar concentrations of RNA and hrPrP). We also demonstrate the ability of this shsRNA to form highly stable nucleoprotein complexes with hrPrP and cellular PrP (PrP(C)) from various cell extracts and mammalian brain homogenates. The apparent mass of the nucleoprotein complex is dependent on the molar ratio of hrPrP to RNA during complex formation. The hrPrP in these complexes acquires resistance to degradation by Proteinase K (PK). Other shsRNAs, however, under identical conditions, neither form stable complexes with hrPrP nor do they induce resistance to PK digestion. We also demonstrate that the RNAs in these nucleoprotein complexes become resistant to ribonuclease A hydrolysis. These interactions between shsRNAs and hrPrP suggest possible roles of RNAs in the modulation of PrP structure and perhaps disease development. ShsRNAs that bind to hrPrP with high affinity and induce resistance to PK digestion can be used to develop molecular biology assays for the screening of compounds associated with PrP structure transformation or for drugs that inhibit this process.

Cells of monocyte/microglial lineage are involved in both microvessel amyloidosis and fibrillar plaque formation in APPsw tg mice.

Ultrastructural three-dimensional reconstruction indicates that deposition of amyloid in the wall of capillaries and in perivascular plaques in APP(SW) transgenic mice (Tg2576) represents two steps of one pathological process associated with inflammation of the vascular wall and perivascular space with cells of monocyte/microglia lineage and fibrillar amyloid-beta deposition. Plaque growth is associated with an increase in the number of microglial cells from two in the smallest plaque to 113 in the largest plaque; however, the growth in the number of microglial cells does not result in amyloid deposit degradation. On the contrary, an increase in the number and volume of microglial cells correlates with the growth of amyloid star from 62 to 34,460 microm(3), and an increase of the plaque volume from 1555 to 284,497 microm(3) (r=0.9). Growth in the number of microglial cells in the absence of morphological evidence of fibrillar amyloid internalization and phagocytosis indicates that microglial cells do not remove amyloid in Tg2576 mice. The study suggests that (a) the mechanism of capillary amyloidosis and plaque formation is similar, (b) the cells of monocyte/macrophage lineage play a critical role in fibrillar amyloid deposition in both types of lesions, and (c) treatment of one of these two forms of brain amyloidosis may affect both types of pathological changes.

Immune surveillance and antigen conformation determines humoral immune response to the prion protein immunogen

Transmissible spongiform encephalopathies (TSE) are progressive degenerative disorders of the central nervous system. PrP(Sc) is a TSE-specific marker derived from the host-encoded glycoprotein, PrPc. The generation of antibodies to PrP plays an important role in the diagnosis of these diseases. In this study the role of the PrP immunogen and the species being immunized was examined in relation to specific epitopes. Various mammals (mice, hamsters, rabbits and PrP null mice) were immunized with formic acid-treated PrP(Sc) isolated from mice, hamsters and sheep. Both the species being immunized and the source of immunogen played an important role in the antibody response. Response to a limited number of linear epitopes was seen among the various immunized animals. One region in the C-terminal portion of PrP appeared highly immunogenic in all species. Comparison of immunoreactivity and the pepscan-defined linear epitope sites suggests both linear and conformational directed responses in many of the animals. Information on the forces directing immune responses to PrP will lead to a better understanding of host-PrP interactions. It will also assist in the development of new strategies for generating additional tools for immunodiagnosis.

Simple and specific detection of abnormal prion protein by a magnetic bead-based immunoassay coupled with laser-induced fluorescence spectrofluorometry.

Transmissible spongiform encephalopathies (TSEs), also termed prion diseases, are fatal neurodegenerative conditions that affect both humans and animals. The transmissibility and fatal nature of TSEs necessitate their rapid and accurate diagnosis. Laser-induced fluorescence (LIF) spectrofluorometry is useful for obtaining measurements on fluorescence-labeled targets with a high degree of sensitivity. In the present study, we applied this technology to the immunological detection of abnormal prion protein, PrPSc, which is a universal diagnostic marker for TSEs. The assay format consists of a magnetic bead-based sandwich immunoassay utilizing a biotin-conjugated capture antibody and a fluorophore-labeled detector antibody. By using one pair of anti-PrP monoclonal antibodies (MAbs), PrPSc in brain homogenates from various experimental and natural TSEs can be easily detected with high specificity. Furthermore, the assay proved to be applicable for the detection of PrPSc in the lymph nodes from deer with TSE. The sensitivity of the assay was shown to be comparable to standard immunoblotting, but has several advantages over conventional tests, in terms of flexibility, simplicity, specificity, and run time. These results provide an important basis for the development of an early diagnostic test with potential for multi-sample analysis.

The nature of the scrapie agent. Biological characteristics of scrapie in different scrapie strain-host combinations.

They are chronic mainly in the sense that they are slow. On the other hand, these diseases follow a course which is just as regular as the course of the acute infections, only the time factor is different. In the first place the so-called incubation period, although extremely long, apparently does not vary with very wide limits. The appearance and the progression of the clinical signs follow a set pattern.

Characteristics of scrapie isolates derived from hay mites.

Previous epidemiological evidence suggested that in some instances a vector and/or reservoir is involved in the occurrence and spread of transmissible spongiform encephalopathies (TSEs). In a preliminary study, hay mite preparations from five Icelandic farms with a history of scrapie were injected into mice, and some of these mice became sick after long incubation periods. To confirm that the disease was scrapie, subsequent passages in mice were performed. In addition, the characteristics of the disease process in these passages were assessed and the results compared to those findings with standard scrapie strains. As expected for scrapie, subsequent passages in the same host led to shortened incubation periods compared to those in primary isolate mice, and all mice had spongiform changes in brain. Results were similar for three of four isolates with regard to clinical manifestations, the incubation periods in mice of the three scrapie incubation-period genotypes (s7s7, s7p7, p7p7), and the PrPSc Western blot (WB) pattern. The characteristics of the fourth isolate were markedly different from the other three isolates with regard to these parameters. Comparison of the characteristics of standard mouse-adapted scrapie strains and the four isolates revealed differences; these differences were particularly pronounced for the fourth isolate.

Determining the density matrix of a molecular beam using a longitudinal matter wave interferometer

Two separated oscillatory fields, if tuned to different frequencies, can generate or interrogate longitudinal momentum coherences in a beam of two-state particles. We demonstrate that use of differentially detuned separated oscillatory fields is an efficient method to determine the longitudinal density matrix of a particle beam.

Optics and Interferometry with Atoms and Molecules

Publisher Summary nThis chapter discusses recent accomplishments in the atom and molecular optics and interferometry at MIT. The chapter begins with a discussion of the details of an experimental apparatus and gives an overview of recent accomplishments in atom and molecular optics. It then describes the atom and molecule interferometer, which is unique in that the two interfering components of the atom wave are spatially separated and can be physically isolated by a metal foil. The interferometer is especially well suited for the study of atomic and molecular properties as it enables one to apply different interactions to each of the two components of the wave function, which in turn permits spectroscopic precision in the study of interactions that shift the energy or phase of a single state of the atom. The chapter also describes an experiment in which this capability is used to determine the ground state polarizability of sodium to 0.3%—an order of magnitude improvement—by measuring the energy shift due to a uniform electric field applied to one component of the wave function. The chapter also provides an overview of the relativistic effects in electromagnetic interactions, and differential force interferometry.

Replication of Scrapie Strains in Vitro and Their Influence on Neuronal Functions

Scrapie is a progressive neurodegenerative disease occurring naturally in sheep and goats which serves as a prototype for the unconventional slow infectious diseases, a group consisting of at least six other animal and human diseases caused by similar agents. Scrapie has been transmitted experimentally t o mice and hamsters, which serve as useful animal model systems to study the disease. Diseasespecific structures termed scrapie-associated fibrils (SAF) and protease-resistant protein (PrP). which are the major components of these structures, are uniquely associated with these diseases. However. the nature of the agent as well a s its association with S A F and PrP remains a mystery. Depending on the scrapie strain-host strain used. differences exist in biological, biochemical, and histopathological parameters. , The existence of scrapie strains was established from long term studies of serially passaged scrapie from natural and experimental sources in inbred mouse strains. In mice, more than 20 scrapie strains have been identified. More recently, the establishment of distinct scrapie strains in hamsters has also been described. Furthermore, the pathogenesis associated with the various scrapie strains is due to neural spread and neuroinvasion of infectivity.5- The establishment of a tissue culture system which supports the replication of scrapie strains would enhance our understanding of the nature of the agent, aid in examining and characterizing scrapie strain variations, and help in deciphering the neuropathogenesis associated with this and related diseases. The cell line, termed PC12, is a cloned tumor cell line derived from a rat pheochromocytoma. In the continued presence of nerve growth factor (NGF) these cells cease dividing and acquire the morphological, biochemical. and electrophysiological properties of differentiated neurons. -I5 We have previously reported that differentiated PC12 cells infected at a multiplicity of 1 with diluted mouse-adapted 139A scrapie brain homogenate supported scrapie strain replication.. Using the incubation period bioassay, scrapie infectivity did not change from 0 to 20 days post infection (p.i.). The levels of infectivity increased significantly by 27 and 34 days p.i. Endpoint titration studies of scrapie infectivity in PC12 cells have indicated that by 56 days p.i. up to 4 LDx units per

Coherence of large gratings and electron‐beam fabrication techniques for atom‐wave interferometry

We describe the fabrication of slotted, free‐standing structures used as amplitude gratings in a separated‐beam interferometer. Improvements in electron‐beam writing techniques have allowed us to compensate for electron‐beam system drift, making practical the exposure of 800×800 μm gratings with period as small as 0.14 μm. Alignment marks are used for periodic drift compensation. Finite element analysis of fracture formation in silicon nitride films gives us a tool for the prediction of structural failure in arbitrarily shaped free‐standing structures.