AMPK Activation Restores Salivary Function Following Radiation Treatment.

Head and neck cancers represent a significant portion of cancer diagnoses, with head and neck cancer incidence increasing in some parts of the world. Typical treatment of early-stage head and neck cancers includes either surgery or radiotherapy; however, advanced cases often require surgery followed by radiation and chemotherapy. Salivary gland damage following radiotherapy leads to severe and chronic hypofunction with decreased salivary output, xerostomia, impaired ability to chew and swallow, increased risk of developing oral mucositis, and malnutrition. There is currently no standard of care for radiation-induced salivary gland dysfunction, and treatment is often limited to palliative treatment that provides only temporary relief. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is an enzyme that activates catabolic processes and has been shown to influence the cell cycle, proliferation, and autophagy. In the present study, we found that radiation (IR) treatment decreases tissue levels of phosphorylated AMPK following radiation and decreases intracellular NAD+ and AMP while increasing intracellular adenosine triphosphate. Furthermore, expression of sirtuin 1 (SIRT1) and nicotinamide phosphoribosyl transferase (NAMPT) was lower 5 d following IR. Treatment with AMPK activators, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and metformin, attenuated compensatory proliferation (days 6, 7, and 30) following IR and reversed chronic (day 30) salivary gland dysfunction post-IR. In addition, treatment with metformin or AICAR increased markers of apical/basolateral polarity (phosphorylated aPKCζT560-positive area) and differentiation (amylase-positive area) within irradiated parotid glands to levels similar to untreated controls. Taken together, these data suggest that AMPK may be a novel therapeutic target for treatment of radiation-induced salivary damage.

A monomer-dimer equilibrium of a cellular prion protein (PrPC) not observed with recombinant PrP.

Both the purified normal (protease-sensitive) isoform of the prion protein (PrP(C)) (Pergami, P., Jaffe, H., and Safar, J. (1996) Anal. Biochem. 236, 63-73) and recombinant prion protein (PrP) have been found to be in monomeric form (Mehlhorn, I., Groth, D., Stockel, J., Moffat, B., Reilly, D., Yansura, D., Willet, W. S., Baldwin, M., Fletterick, R., Cohen, F. E., Vandlen, R., Henner, D., and Prusiner, S. B. (1996) Biochemistry 35, 5528-5537; and this paper), and therefore PrP(C)-PrP(C) interactions were previously unknown. In this report we confirm recombinant PrP to be a monomer by analytical ultracentrifugation. However, by three lines of evidence (enzyme-linked immunosorbent assay (ELISA), cross-linking experiments, and size exclusion chromatography) we could also demonstrate that, under native conditions, at least part of the native bovine PrP(C) exists as a monomer-dimer equilibrium. A bovine PrP(C)-specific immuno-sandwich ELISA was developed and calibrated with recombinant PrP (Meyer, R. K., Oesch, B., Fatzer, R., Zurbriggen, A., and Vandevelde, M. (1999) J. Virol. 73, 9386-9392). By this ELISA we identified a distinct PrP(C) fraction and partially purified this protein. When serial dilutions of brain homogenate or partially purified PrP(C) were measured, using the peptide antibody C15S, a nonlinear dose-response curve was obtained. This nonlinearity was shown not to be due to an artifact of the procedure but to a monomer-dimer equilibrium of PrP(C) with preferential binding of the antibody to the dimer. From the curvature we could deduce the association constant (3.9 x 10(8) M(-1) at 37 degrees C). Accordingly, DeltaG degrees of the reaction was calculated (-48.6 kJ M(-1)), and DeltaH degrees (9.5 kJ M(-1)) as well as DeltaS degrees (0.2 kJ K(-1) M(-1)) were extrapolated from the van't Hoff plot. When serial dilutions of monomeric recombinant PrP were tested, only a straight line was obtained, supporting our hypothesis. Additional evidence of dimer formation was revealed by Western blotting of partially purified PrP(C) cross-linked by the homobifunctional cross-linker BS(3). Finally, size exclusion chromatography of partially purified PrP(C) fractions revealed an additional shoulder not observed with recombinant PrP. The difference in respect of dimer formation between native PrP(C) and recombinant PrP could be explained by the lack of glycosylation of the latter.

Conceptual and motor learning in music performance.

Are the mental plans for action abstract or specified in terms of the movements with which they are produced? We report motor independence for expert music performance but not for novice performance in a transfer-of-learning task. Skilled adult pianists practiced musical pieces and transferred to new pieces with the same or different motor (hand and finger) requirements and conceptual (melodic) relations. Greatest transfer in sequence duration was observed when the same conceptual relations were retained from training to transfer, regardless of motor movements. In a second experiment, novice child pianists performed the same task. More experienced child pianists showed transfer on both the motor and the conceptual dimensions; the least experienced child pianists demonstrated transfer only to sequences with identical motor and conceptual dimensions. These findings suggest that mental plans for action become independent of the required movements only at advanced skill levels.

Detection of bovine spongiform encephalopathy-specific PrP(Sc) by treatment with heat and guanidine thiocyanate.

The conversion of a ubiquitous cellular protein (PrP(C)), an isoform of the prion protein (PrP), to the pathology-associated isoform PrP(Sc) is one of the hallmarks of transmissible spongiform encephalopathies such as bovine spongiform encephalopathy (BSE). Accumulation of PrP(Sc) has been used to diagnose BSE. Here we describe a quantitative enzyme-linked immunosorbent assay (ELISA) that involves antibodies against epitopes within the protease-resistant core of the PrP molecule to measure the amount of PrP in brain tissues from animals with BSE and normal controls. In native tissue preparations, little difference was found between the two groups. However, following treatment of the tissue with heat and guanidine thiocyanate (Gh treatment), the ELISA discriminated BSE-specific PrP(Sc) from PrP(C) in bovine brain homogenates. PrP(Sc) was identified by Western blot, centrifugation, and protease digestion experiments. It was thought that folding or complexing of PrP(Sc) is most probably reversed by the Gh treatment, making hidden antigenic sites accessible. The digestion experiments also showed that protease-resistant PrP in BSE is more difficult to detect than that in hamster scrapie. While the concentration of PrP(C) in cattle is similar to that in hamsters, PrP(Sc) sparse in comparison. The detection of PrP(Sc) by a simple physicochemical treatment without the need for protease digestion, as described in this study, could be applied to develop a diagnostic assay to screen large numbers of samples.

High-resolution fluorescent particle-tracking flow visualization within an intraventricular axial flow left ventricular assist device.

Flow visualization is typically applied in blood pump development to both confirm the design expectations and identify regions that may be predisposed to blood element deposition and trauma. Rotary pumps, in particular, place high demands on the technique chosen to visualize the flow given the limited visual accessibility of the flow path and the high impeller speeds. Fluorescent image-tracking velocimetry currently is used at the University of Pittsburgh Medical Center to visualize flow accurately inside of these pumps both qualitatively and quantitatively. Flow patterns under steady conditions within an intraventricular axial flow, left ventricular assist pump (prototype No. 7, SUN Medical Technology Research Corporation, Nagano, Japan) were investigated using this technique. The flow fields at the impeller-stator interface and at the pump outlet were given specific attention. This allowed the assessment of the fluid dynamics throughout the hydrodynamic design limits of the pump.

Neuronal degeneration in brain stem nuclei in bovine spongiform encephalopathy.

In 57% of 151 BSE positive brains of Swiss cattle, degenerating neurons were found in BSE predilection sites, either single scattered (34%) or involving large parts of brain stem nuclei (23%). The lesion consisted of central chromatolysis, shrinkage, karyolysis or nuclear pyknosis. In 43% of the BSE positive brains no neuronal changes besides vacuolation were present. Neuronal degeneration other than vacuolation alone is not pathognomonic for BSE but, according to our findings, seems to be of some importance together with the typical vacuolation of neuropil and neurons. There is no correlation between extent of these degenerative changes and accumulation of PrPSc protein, determined by immunohistochemistry. The significance remains obscure, yet as it had not been seen in cattle brains before the outbreak of BSE in Switzerland in 1990 it certainly is linked to the disease. Possible pathogenetic mechanisms are discussed.

In situ hybridization and immunohistochemistry for prion protein (PrP) in bovine spongiform encephalopathy (BSE).

In about 5% of the cows showing clinical signs of bovine spongiform encephalopathy (BSE) the histopathological examination is not conclusive. In order to rule out BSE in these cases, additional methods are necessary. For that reason, non-radioactive in situ hybridization (ISH) was performed using a riboprobe against the messenger RNA coding for the prion protein (PrP). In addition, a polyclonal antibody for immunohistochemistry (IHC) was generated against a synthetic peptide derived from bovine PrP. ISH and IHC were used to analyse brain sections of cattle suffering from BSE and various neurological diseases including four cows with clinically suspect but histologically unconfirmed BSE. ISH revealed no differences in localization, distribution and neuronal levels of PrP mRNA between BSE positive and negative cattle. However, there was a BSE-specific staining pattern in IHC allowing to exclude BSE the four suspected cases. Additionally, IHC for PrP is an elegant alternative to search for scrapie associated fibrils by electron microscopy.

Scrapie prion rod formation in vitro requires both detergent extraction and limited proteolysis.

Scrapie prion infectivity can be enriched from hamster brain homogenates by using limited proteolysis and detergent extraction. Purified fractions contain both scrapie infectivity and the protein PrP 27-30, which is aggregated in the form of prion rods. During purification, PrP 27-30 is produced from a larger membrane protein, PrPSc, by limited proteolysis with proteinase K. Brain homogenates from scrapie-infected hamsters do not contain prion rods prior to exposure to detergents and proteases. To determine whether both detergent extraction and limited proteolysis are required for the formation of prion rods, microsomal membranes were prepared from infected brains in the presence of protease inhibitors. The isolated membranes were then detergent extracted as well as protease digested to evaluate the effects of these treatments on the formation of prion rods. Neither detergent (2% Sarkosyl) extraction nor limited proteinase K digestion of scrapie microsomes produced recognizable prion amyloid rods. Only after combining detergent extraction with limited proteolysis were numerous prion rods observed. Rod formation was influenced by the protease concentration, the specificity of the protease, and the duration of digestion. Rod formation also depended upon the detergent; some combinations of protease and detergent did not produce prion amyloid rods. Similar results were obtained with purified PrPSc fractions prepared by repeated detergent extractions in the presence of protease inhibitors. These fractions contained amorphous structures but not rods; however, prion rods were produced upon conversion of PrPSc to PrP 27-30 by limited proteolysis. We conclude that the formation of prion amyloid rods in vitro requires both detergent extraction and limited proteolysis. In vivo, amyloid filaments found in the brains of animals with scrapie resemble prion rods in their width and their labeling with prion protein (PrP) antisera; however, filaments are typically longer than rods. Whether limited proteolysis and some process equivalent to detergent extraction are required for amyloid filament formation in vivo remains to be established.

Bundling of actin filaments by alpha-actinin depends on its molecular length.

Cross-linking of actin filaments (F-actin) into bundles and networks was investigated with three different isoforms of the dumbbell-shaped alpha-actinin homodimer under identical reaction conditions. These were isolated from chicken gizzard smooth muscle, Acanthamoeba, and Dictyostelium, respectively. Examination in the electron microscope revealed that each isoform was able to cross-link F-actin into networks. In addition, F-actin bundles were obtained with chicken gizzard and Acanthamoeba alpha-actinin, but not Dictyostelium alpha-actinin under conditions where actin by itself polymerized into disperse filaments. This F-actin bundle formation critically depended on the proper molar ratio of alpha-actinin to actin, and hence F-actin bundles immediately disappeared when free alpha-actinin was withdrawn from the surrounding medium. The apparent dissociation constants (Kds) at half-saturation of the actin binding sites were 0.4 microM at 22 degrees C and 1.2 microM at 37 degrees C for chicken gizzard, and 2.7 microM at 22 degrees C for both Acanthamoeba and Dictyostelium alpha-actinin. Chicken gizzard and Dictyostelium alpha-actinin predominantly cross-linked actin filaments in an antiparallel fashion, whereas Acanthamoeba alpha-actinin cross-linked actin filaments preferentially in a parallel fashion. The average molecular length of free alpha-actinin was 37 nm for glycerol-sprayed/rotary metal-shadowed and 35 nm for negatively stained chicken gizzard; 46 and 44 nm, respectively, for Acanthamoeba; and 34 and 31 nm, respectively, for Dictyostelium alpha-actinin. In negatively stained preparations we also evaluated the average molecular length of alpha-actinin when bound to actin filaments: 36 nm for chicken gizzard and 35 nm for Acanthamoeba alpha-actinin, a molecular length roughly coinciding with the crossover repeat of the two-stranded F-actin helix (i.e., 36 nm), but only 28 nm for Dictyostelium alpha-actinin. Furthermore, the minimal spacing between cross-linking alpha-actinin molecules along actin filaments was close to 36 nm for both smooth muscle and Acanthamoeba alpha-actinin, but only 31 nm for Dictyostelium alpha-actinin. This observation suggests that the molecular length of the alpha-actinin homodimer may determine its spacing along the actin filament, and hence F-actin bundle formation may require "tight" (i.e., one molecule after the other) and "untwisted" (i.e., the long axis of the molecule being parallel to the actin filament axis) packing of alpha-actinin molecules along the actin filaments.

Vinculin-lipid monolayer interactions: a model for focal contact formation.

Vinculin-lipid interactions were investigated in a modified Langmuir trough. Provided proper conditions, vinculin had the potential to penetrate into phospholipid monolayers and to form rigid, cohesive protein films even at phospholipid monolayer pressures similar to those assumed to exist in living cell membranes. The equilibrium constant for this reaction was estimated to be on the order of 2.5 X 10(-9) to 2.2 X 10(-7) mol/liter (for pressures between 25 and 35 mN/m). Penetration velocity depended on lipid composition: it was high with acidic phospholipids, intermediate with mixtures of acidic and neutral phospholipids, and low with neutral phospholipids. Electron microscopy of freeze-dried/metal-shadowed vinculin films, recovered from the phospholipid monolayer surface, revealed relatively tightly packed globular particles, 13 to 18 nm in diameter, on average significantly larger than the particles seen in glycerol-sprayed and rotary metal-shadowed preparations of soluble vinculin. The lipid monolayer penetration ability of vinculin appears to depend on its conformation. Acid treatment or low salt buffers induced reversible changes in vinculin conformation such that it abolished its lipid penetration potential. These conformational changes could be documented by both circular dichroism and fluorescence spectroscopy. These results indicate that in the focal contact area vinculin may act like a "glue" and link, in a reversible way, stress fibers of cultured cells via their anchor proteins to the extracellular matrix.

Scrapie and cellular prion proteins share polypeptide epitopes.

Purified preparations of scrapie prions contain one major protein, PrP 27-30, and aggregates of rod-shaped structures. On the basis of the NH2-terminal amino acid sequence of PrP 27-30, a synthetic peptide (PrP-P1) was constructed. Monospecific rabbit antisera to PrP-P1 were found by immunoblotting to react with PrP 27-30 and its precursor (PrP 33-35Sc), as well as with a related protease-sensitive cellular homologue (PrP 33-35C). An enzyme-linked immunosorbent assay showed that rabbit antiserum to PrP 27-30 was more reactive with PrP 27-30 than with PrP-P1; conversely, antiserum to PrP-P1 was more reactive with the peptide than with the prion proteins. In addition, antibodies to PrP-P1 decorate purified prion rods and stain amyloid plaques in scrapie-infected hamster brain. The peptide epitopes shared by PrP 27-30, PrP 33-35Sc, and PrP 33-35C clearly establish a relationship among these three proteins.

Separation and properties of cellular and scrapie prion proteins.

Purified preparations of scrapie prions contain a sialoglycoprotein of Mr 27,000-30,000, designated PrP 27-30, which is derived from the scrapie prion protein [Mr, 33,000-35,000 (PrP 33-35Sc)] by limited proteolysis. Under these same conditions of proteolysis, a cellular protein of the same size (PrP 33-35C) is completely degraded. Subcellular fractionation of hamster brain showed that both PrP 33-35Sc and PrP 33-35C were found only in membrane fractions. NaCl, EDTA, and osmotic shock failed to release the prion proteins from microsomal membranes. Electron microscopy of these microsomal fractions showed membrane vesicles but not prion amyloid rods. Detergent treatment of scrapie-infected membranes solubilized PrP 33-35C, while PrP 33-35Sc aggregated into amyloid rods; the concentration of PrP 33-35C was similar to that recovered from analogous fractions prepared from uninfected control brains. The apparent amphipathic character of the PrP 33-35Sc may explain the association of scrapie infectivity with both membranes and amyloid filaments.

Diacylglycerol in large alpha-actinin/actin complexes and in the cytoskeleton of activated platelets.

The interaction of the cytoskeleton with plasma membranes may be mediated by vinculin, alpha-actinin and other proteins; alpha-actinin can interact specifically with model membranes only if they contain diacylglycerol and palmitic acid. On stimulation of platelets by thrombin, which leads to a reorganization of the cytoskeleton, diacylglycerol is produced rapidly, simultaneously with the disappearance of phosphatidylinositol. One important function of the diacylglycerol produced in platelets may be the activation of the Ca2+-and phospholipid-dependent protein kinase C. We show here that, in the presence of diacylglycerol and palmitic acid, a supramolecular complex between alpha-actinin and actin is formed in vitro. In the electron microscope, this complex displays substructures similar to those of microfilament bundles in vivo. Furthermore, such alpha-actinin/lipid complexes can also be formed in situ during the stimulation of blood platelet aggregation. Thus, alpha-actinin may be one of the proteins directly involved in structures connecting the cytoskeleton to cell membranes.

Scrapie PrP 27-30 is a sialoglycoprotein.

The major scrapie prion protein, designated PrP 27-30, exhibited both charge and size heterogeneity after purification from infected hamster brains. Eight or more discrete charge isomers of PrP 27-30 with isoelectric points ranging from approximately pH 4.6 to 7.9 were found by using non-equilibrium pH gradient electrophoresis in the first dimension followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. The charge isomers were detected by silver staining as well as by radioiodination. The procedures used to disaggregate PrP 27-30 before electrophoresis in the first dimension do not appear to be responsible for the charge heterogeneity. However, heating PrP 27-30 to 100 degrees C for 15 min in 0.1 N NaOH or 0.1 N HCl resulted in modification of the protein and alteration of its electrophoretic pattern. A PrP 27-30 fragment (molecular weight, 17,100 to 21,900) obtained by cyanogen bromide cleavage also exhibited charge and size heterogeneity. Periodic acid-Schiff staining of PrP 27-30 electrophoresed into sodium dodecyl sulfate-polyacrylamide gels demonstrated that carbohydrate residues are attached to the protein. Digestion of PrP 27-30 with neuraminidase and endo-beta-N-acetylglucosaminidase H resulted in significant changes in the isoelectric pH of PrP 27-30 isomers, whereas digestion with alkaline phosphatase had no effect. Our results demonstrate that PrP 27-30 is a sialoglycoprotein; this is consistent with several properties of this protein and of the scrapie prion.

Scrapie and Creutzfeldt-Jakob disease prions.

Scrapie and Creutzfeldt-Jakob disease are caused by prions which can be distinguished from both viruses and viroids. Aggregates of prions are ultrastructurally and histochemically identical to amyloid. Extracellular collections of prions form amyloid plaques within scrapie-infected brain. Prion amyloid plaques seem analogous to viral inclusion bodies in that they are composed of causative pathogens and are not merely a consequence of the disease.

alpha-Actinin interacts specifically with model membranes containing glycerides and fatty acids.

A method was developed to identify specific protein-lipid interactions of complex lipid mixtures and to assess their effect upon the arrangement of such complexes in monolayers at an air-water interface. Its application to striated muscle alpha-actinin revealed that just two lipids selectively interact with alpha-actinin. One molecule of glyceride and one molecule of fatty acid were found to be associated in a constant stoichiometry with one molecule of the alpha-actinin dimer. In the presence of both glycerides and fatty acids unexpectedly rigid monolayer areas formed. This lipid specificity could be confirmed by brief protease digestion of alpha-actinin liposome mixtures followed by peptide analysis; the peptide patterns of alpha-actinin depended on the presence or absence of only these two lipids. Possible implications of these findings are discussed in the context of Z-line formation.

Actin filament bundles in vaccinia virus infected fibroblasts.

When monolayer cultures of mouse 3T3 cells were infected with vaccinia virus, a rapid decrease of microfilament bundles was observed. The major disappearance of the cellular microfilaments occurred within the first hour after vaccinia virus infection, which is the period of uncoating of the virus core and early viral protein synthesis. The microfilament bundles of infected cells were significantly preserved in the presence of puromycin which inhibits viral protein synthesis.