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PubMed2005

AlphaB-crystallin, a small heat-shock protein, prevents the amyloid fibril growth of an amyloid beta-peptide and beta2-microglobulin.

AlphaB-crystallin, a small heat-shock protein, exhibits molecular chaperone activity. We have studied the effect of alphaB-crystallin on the fibril growth of the Abeta (amyloid beta)-peptides Abeta-(1-40) and Abeta-(1-42). alphaB-crystallin, but not BSA or hen egg-white lysozyme, prevented the fibril growth of Abeta-(1-40), as revealed by thioflavin T binding, total internal reflection fluorescence microscopy and CD spectroscopy. Comparison of the activity of some mutants and chimaeric alpha-crystallins in preventing Abeta-(1-40) fibril growth with their previously reported chaperone ability in preventing dithiothreitol-induced aggregation of insulin suggests that there might be both common and distinct sites of interaction on alpha-crystallin involved in the prevention of amorphous aggregation of insulin and fibril growth of Abeta-(1-40). alphaB-crystallin also prevents the spontaneous fibril formation (without externally added seeds) of Abeta-(1-42), as well as the fibril growth of Abeta-(1-40) when seeded with the Abeta-(1-42) fibril seed. Sedimentation velocity measurements show that alphaB-crystallin does not form a stable complex with Abeta-(1-40). The mechanism by which it prevents the fibril growth differs from the known mechanism by which it prevents the amorphous aggregation of proteins. alphaB-crystallin binds to the amyloid fibrils of Abeta-(1-40), indicating that the preferential interaction of the chaperone with the fibril nucleus, which inhibits nucleation-dependent polymerization of amyloid fibrils, is the mechanism that is predominantly involved. We found that alphaB-crystallin prevents the fibril growth of beta2-microglobulin under acidic conditions. It also retards the depolymerization of beta2-microglobulin fibrils, indicating that it can interact with the fibrils. Our study sheds light on the role of small heat-shock proteins in protein conformational diseases, particularly in Alzheimer's disease.

Amyloid↗
PubMed2006

Induction and inhibition of preferential enrichment by controlling the mode of the polymorphic transition with seed crystals.

Both induction and inhibition of "preferential enrichment", an unusual symmetry-breaking enantiomeric-resolution phenomenon observed upon simple recrystallization of a certain kind of racemic crystals from organic solvents, have been successfully achieved by controlling the mode of the polymorphic transition during crystallization with appropriate seed crystals. Such control of the polymorphic transition can be interpreted in terms of a novel phenomenon consisting of 1) the adsorption of prenucleation aggregates, 2) the heterogeneous nucleation and crystal growth of a metastable crystalline form, and 3) the subsequent polymorphic transition into the more stable form; these three processes occur on the same surface of a seed crystal. We refer to this phenomenon as an "epitaxial transition", which has been confirmed by means of in situ attenuated total reflection (ATR) FTIR spectroscopy in solution and the solid state, differential scanning calorimetry (DSC) measurements of the deposited crystals, and X-ray crystallographic analysis of the single crystals or the direct-space approach employing the Monte Carlo method with the Rietveld refinement for the structure solution from the powder X-ray diffraction data.

Journal Article↗
PubMed2003

Modified microbatch and seeding in protein crystallization experiments.

The formation of nuclei in a crystallization experiment requires the interaction of protein molecules until a critical size of aggregate is created. In many crystallization screens sufficiently high levels of saturation are never reached to allow this critical nucleation event to occur. There are at least two possibilities to change this situation. The first is to increase the concentration of the protein and precipitating agent during the experiment to levels where spontaneous nucleation will occur. The second is to influence the nucleation event so that crystals can form at lower concentrations. The use of a modified microbatch method has made the first strategy possible and the use of heterogeneous seeding can be used to influence the second.

Crystallization↗
PubMed2003

Cholesterol and Abeta aggregation.

Regarding deposition of amyloid beta-protein (Abeta) in brains with Alzheimer's disease (AD), we previously identified a novel Abeta species that strongly binds to GM1 ganglioside (GM1) in human brains that exhibit early pathological changes of AD. We hypothesized that Abeta undergoes conformational alteration through its binding to GM1 and acts as a seed. We recently found that an increase in the cholesterol concentration in host membranes markedly accelerates Abeta binding to GM1. We then investigated whether the cholesterol concentration in neuronal membranes could be altered under biological conditions that are associated with risk factors for AD development. We attempted to determine the distribution of cholesterol in the synaptic plasma membranes (SPMs) of human apolipoprotein E (apoE)-knock-in mice and found that the cholesterol concentration in the exofacial leaflet of SPMs of the human apoE4-knock-in mice was approximately twice that of human apoE3-knock-in mice. The results of our studies suggest that an increase in the cholesterol concentration in the neuronal membranes accelerates Abeta aggregation through the formation of an endogenous seed.

Aging↗
PubMed2002

Effects of red-yellow soil acidification on seed germination of Chinese pine.

Acid treatments significantly change the physical and chemical properties of red-yellow soil by lowering its pH value and leaching out aluminum(Al) ions that are harmful to the growth of plants. The structure of soil will be damaged, resulting in higher viscosity, higher water retention rate and lower air permeability of the soil. The germination rate of Chinese pine(Pinus tobulacformic Carr.) seeds sowed in soil treated with sulphuric acid(H2SO4) decreased compared to that for untreated soil. The direct cause was the large amount of Al ions leached out because of low pH values(> or = 3.5). The added acid decreased the soil aggregation and increased the number of micro-aggregates(under 250 microns in diameter). Such changes increased the soil's viscosity, which tied the pine needles to the soil after the seeds had germinated and prevented the seedlings from fully developing.

Germination↗
PubMed1998

Surface hydrolysis of poly(glycolic acid) meshes increases the seeding density of vascular smooth muscle cells.

A procedure for surface hydrolysis of poly(glycolic acid) (PGA) meshes was developed to increase cell seeding density and improve attachment of vascular smooth muscle cells. Hydrolysis of PGA in 1N NaOH transformed ester groups on the surface of PGA fibers to carboxylic acid and hydroxyl groups. After hydrolysis, the polymer scaffold retained its original gross appearance and dimensions while the fiber diameter decreased. A plot of fiber diameter versus the hydrolysis time showed a linear relationship, with a rate of decrease in fiber diameter of 0.65 microm/min. The molecular weight and thermal properties of the polymer did not change significantly following surface hydrolysis. In cell seeding experiments, surface-hydrolyzed mesh was seeded with more than twice as many cells as unmodified PGA mesh. Vascular smooth muscle cells attached to the surface-hydrolyzed PGA mesh both as individual cells and as cell aggregates while only cell aggregates were observed on the unmodified mesh. Control experiments indicated that adsorption of serum proteins onto the surface-hydrolyzed PGA fibers was correlated with the increase in cell seeding density. These results demonstrate that optimization of biomaterial-cell interactions provides a strategy for increasing the initial cell seeding density for the engineering of tissues of high cell density.

Animals↗
PubMed2002

Persistence of tertiary structure in 7.9 M guanidinium chloride: the case of endo-beta-1,3-glucanase from Pyrococcus furiosus.

The Pyrococcus furiosus endo-beta-1,3-glucanase belongs to the subfamily of laminarinase, which can be classified as "all beta proteins" as confirmed by deconvolution of far-UV CD and FTIR spectra. The persistence of a significant amount of tertiary structure in 7.9 M GdmCl, as indicated by near-UV CD spectroscopy, accompanied by a red-shift of the maximum fluorescence emission wavelength is a peculiar property of this hyperthermophilic endoglucanase. The possibility to observe tertiary structure elements under extremely denaturing conditions is notable and is limited to only a few examples. The unusual resistance toward guanidinium chloride denaturation is paralleled by a notable stability at extremely low pH and at high temperature. The analysis of the protein spectral properties indicates that the secondary structure elements are preserved down to pH 1.0 and up to 90 degrees C at pH 7.4 and pH 3.0. The study of the conditions that determine the persistence of residual structure at high denaturant concentration and the examination of these structures are particularly interesting because these state(s) may be preliminary or coincident with the coalescence of protein aggregates or to the formation of amyloid-like fibrils, and they may serve as seeds of protein folding.

Circular Dichroism↗
PubMed2005

RhoA and cytoskeletal disruption mediate reduced osteoblastogenesis and enhanced adipogenesis of human mesenchymal stem cells in modeled microgravity.

UNLABELLED: Spaceflight, aging, and disuse lead to reduced BMD. This study shows that overexpression of constitutively active RhoA restores actin cytoskeletal arrangement, enhances the osteoblastic phenotype, and suppresses the adipocytic phenotype of human mesenchymal stem cells cultured in modeled microgravity. INTRODUCTION: Reduced BMD during spaceflight is partly caused by reduced bone formation. However, mechanisms responsible for this bone loss remain unclear. We have previously shown reduced osteoblastogenesis and enhanced adipogenesis of human mesenchymal stem cells (hMSCs) cultured in modeled microgravity (MMG). The small GTPase, RhoA, regulates actin stress fiber formation and has been implicated in the lineage commitment of hMSCs. We examined the effects of MMG on actin cytoskeletal organization and RhoA activity and the ability of constitutively active RhoA to reverse these effects. MATERIALS AND METHODS: hMSCs were seeded onto plastic microcarrier beads at a density of 10(6) and allowed to form aggregates in DMEM containing 10% FBS for 7 days. Aggregates were incubated in DMEM containing 2% FBS for 6 h with or without an adenoviral vector containing constitutively active RhoA at a multiplicity of infection (moi) of 500 and allowed to recover in 10% FBS for 24 h. Cells were transferred to the rotary cell culture system to model microgravity or to be maintained at normal gravity for 7 days in DMEM, 10% FBS, 10 nM dexamethasone, 10 mM beta-glycerol phosphate, and 50 muM ascorbic acid 2-phosphate. RESULTS: F-actin stress fibers are disrupted in hMSCs within 3 h of initiation of MMG and are completely absent by 7 days, whereas monomeric G-actin is increased. Because of the association of G-actin with lipid droplets in fat cells, the observed 310% increase in intracellular lipid accumulation in hMSCs cultured in MMG was not unexpected. Consistent with these changes in cellular morphology, 7 days of MMG significantly reduces RhoA activity and subsequent phosphorylation of cofilin by 88+/-2% and 77+/-9%, respectively. Importantly, introduction of an adenoviral construct expressing constitutively active RhoA reverses the elimination of stress fibers, significantly increases osteoblastic gene expression of type I collagen, alkaline phosphatase, and runt-related transcription factor 2, and suppresses adipocytic gene expression of leptin and glucose transporter 4 in hMSCs cultured in MMG. CONCLUSION: Suppression of RhoA activity during MMG represents a novel mechanism for reduced osteoblastogenesis and enhanced adipogenesis of hMSCs.

Adipogenesis↗
PubMed2001

Generation of aggregated beta-amyloid in the rat hippocampus impairs synaptic transmission and plasticity and causes memory deficits.

We injected a combination of the beta-amyloids (Abetas) Abeta40 and Abeta43 to "seed" formation of amyloid deposits in the dorsal dentate gyrus of rats in vivo, on the basis of a theory of Jarrett and Landsbury (1993). Rats were tested on several different learning tasks, and synaptic transmission and plasticity were assessed in vivo. Between 7 and 16 weeks after injection, we found aggregated amyloid material, reactive astrocytosis, microgliosis, and cell loss around the sites of injection. Rats were impaired specifically in working memory type tasks in accordance with the type of memory deficit observed in the early stages of Alzheimer's disease. Synaptic transmission and long-term potentiation, a candidate cellular mechanism for memory, were severely impaired in vivo. Injections of the same dose of fragments individually did not induce these effects. These findings suggest that aggregated amyloid material induces cognitive deficits similar to those observed in the early phases of Alzheimer's disease via an alteration in neuronal transmission and plasticity.

Alzheimer Disease↗
PubMed2006

Mesenchymal stem cells cultured on a collagen scaffold: In vitro osteogenic differentiation.

OBJECTIVE: Management of periodontal defects has always been a challenge in clinical periodontics. Recently mesenchymal stem cells (MSC) have been proposed for tissue regeneration in periodontal disease and repair of large bone defects. Bone regeneration has to be supported by a scaffold which has to be biocompatible, biodegradable, and able to support cell growth and differentiation. The aim of this study was to evaluate osteogenic differentiation of MSC seeded on a collagen scaffold. DESIGN: MSC were obtained from adult rat bone marrow, expanded and cultured in plastic dishes or seeded in a collagen scaffold (Gingistat). MSC were induced towards osteogenic differentiation using osteogenic supplements. Cell differentiation and calcium deposits were evaluated by immunoblotting, immunohistochemistry, histochemical techniques, enzymatic activity assay, and SEM-EDX analysis. Biomaterial in vitro degradation was evaluated by measuring mass reduction after incubation in culture medium. RESULTS: Rat MSC osteogenic differentiation was demonstrated by osteopontin and osteocalcin expression and an increase in alkaline phosphatase activity. MSC were distributed homogeneously in the collagen scaffold. Nodular aggregates and alizarin red stained calcium deposits were observed in MSC induced towards osteogenic differentiation cultured in dishes or seeded in the collagen scaffold. SEM-EDX analysis demonstrated that calcium co-localized with phosphorous. The biomaterial in vitro degraded in 4-5 weeks. CONCLUSIONS: MSC from bone marrow differentiate towards osteogenic lineage, representing a suitable cell source for bone formation in periodontal regeneration. Gingistat collagen scaffold supports MSC distribution and differentiation, but its short degradation time may be a limitation for a future application in bone tissue regeneration.

Alkaline Phosphatase↗
PubMed1971

Biochemical differentiation in reaggregating brain cell culture.

Dissociated cells from embryonic mouse brain reassociate in rotation culture to form aggregates. During cell culture the specific activities of choline acetyl-transferase (EC 2.3.1.6), acetylcholinesterase (EC 3.1.1.7), and glutamate decarboxylase (EC 4.1.1.15) in the aggregates increase up to twenty-fold, a phenomenon that approximates some of the biochemical events in the development of the mouse brain.

Acetylcholinesterase↗
PubMed2003

Co-incorporation of A beta 40 and A beta 42 to form mixed pre-fibrillar aggregates.

Senile plaques, the invariable hallmark and likely proximal cause of Alzheimer's disease (AD), are structured depositions of the 40- and 42-residue forms of the A beta peptide. Conversely, diffuse plaques, which are not associated with neurodegeneration, consist mainly of unstructured A beta 42. We have investigated the interaction between A beta 40 and A beta 42 through an assay, which involves labeling both variants with an environment-sensitive fluorophore. We have monitored association of A beta without fibrillar seeds, which allows investigation of molecular species preceding fibrils. Immediately upon mixture, A beta 40 and A beta 42 associate into mixed aggregates, in which the peptides are unstructured and relatively accessible to water. When left to incubate for an extended period, larger, more tightly packed aggregates, which show secondary structure, replace the small, unstructured aggregates formed earlier. Our results show that in vitro the two A beta variants coassemble early in the fibrillogenesis pathway. The ease of formation for mixed and homogeneous aggregates is similar. A change in the local A beta variant ratio can therefore have a significant impact on A beta aggregation; indeed such a change has been reported in some types of familial AD.

Alzheimer Disease↗
PubMed2005

Recombinant adeno-associated virus (rAAV) expressing TFPI-2 inhibits invasion, angiogenesis and tumor growth in a human glioblastoma cell line.

Recombinant adeno-associated viruses (rAAV) have become the vector of choice for many gene therapy protocols. rAAVs have a number of attractive features including long-term transgene expression and the ability to transduce both dividing and non-dividing cells. We have shown previously the anti-cancer role of tissue factor pathway inhibitor-2 (TFPI-2), a matrix-associated serine protease inhibitor, in human glioblastomas. As a result of our present study, in which 0.8-kb fragment of human TFPI-2 was cloned into the adeno-associated viral vectors (rAAA-TFPI-2), rAAV-TFPI-2 infection of SNB19 cells significantly increased TFPI-2 as determined by Western blotting. As assessed by spheroid and Matrigel assays, infection of SNB19 cells with rAAV-TFPI-2 significantly reduced migration and invasion in a dose-dependent manner. Tumor spheroids infected with rAAV-TFPI-2 and co-cultured with fetal rat brain aggregates did not invade rat brain aggregates, whereas 90-95% of the mock and AAV-CMV infected cells invaded rat brain aggregates. In vitro angiogenesis studies (tumor cells co-cultured with endothelial cells or endothelial cells seeded on matrigel) showed reduction of capillary-like structure formation in rAAV-TFPI-2-treated cells as compared to parental and mock-transfected cells. In in vivo angiogenesis results demonstrated the formation of microvessels in SNB19 parental cells and this formation was inhibited when the SNB19 cells were infected with rAAV-TFPI-2. Further, we observed a large reduction of tumor growth in SNB19 cells treated with rAAV-TFPI-2 virus injected intracerebrally when compared to controls. Our study demonstrates that rAAV-TFPI-2-mediated gene therapy offers a novel tool for the treatment of brain tumors.

Animals↗
PubMed2004

An intersheet packing interaction in A beta fibrils mapped by disulfide cross-linking.

Most models for the central cross-beta folding unit in amyloid fibrils of the Alzheimer's plaque protein Abeta align the peptides in register in H-bonded, parallel beta-sheet structure. Some models require the Abeta peptide to undergo a chain reversal when folding into the amyloid core, while other models feature very long extended chains, or zigzag chains, traversing the protofilament. In this paper we introduce the use of disulfide bond cross-linking to probe the fold within the core and the packing interactions between beta-sheets. In one approach, amyloid fibrils grown under reducing conditions from each of three double cysteine mutants (17/34, 17/35, and 17/36) of the Abeta(1-40) sequence were subjected to oxidizing conditions. Of these three mutants, only the Leu17Cys/Leu34Cys peptide could be cross-linked efficiently while resident in fibrils. In another approach, double Cys mutants were cross-linked as monomers before aggregation, and the resulting fibrils were assessed for stability, antibody binding, dye binding, and cross-seeding efficiency. Here too, fibrils from the 17/34 double Cys mutant most closely resemble wild-type Abeta(1-40) fibrils. These data support models of the Abeta fibril in which the Leu17 and Leu34 side chains of the same peptide pack against each other at the beta-sheet interface within the amyloid core. Related cross-linking strategies may reveal longer range spatial relationships. The ability of the cross-linked 17/35 double Cys mutant Abeta to also make amyloid fibrils illustrates a remarkable plasticity of the amyloid structure and suggests a structural mechanism for the generation of conformational variants of amyloid.

Amino Acid Sequence↗
PubMed2006

New insights into the self-assembly of insulin amyloid fibrils: an H-D exchange FT-IR study.

The solvent protection of the amide backbone in bovine insulin fibrils was studied by FT-IR spectroscopy. In the mature fibrils, approximately 85 +/- 2% of amide protons are protected. Of those "trapped" protons, a further 25 +/- 2 or 35 +/- 2% is H-D exchanged after incubation for 1 h at 1 GPa and 25 degrees C or 0.1 MPa and 100 degrees C, respectively. In contrast to the native or unfolded protein, fibrils do not H-D exchange upon incubation at 65 degrees C. A complete deuteration of H(2)O-grown fibrils occurs when the beta-sheet structure is reassembled in a 75 wt % DMSO/D(2)O solution. Our findings suggest a densely packed environment around the amide protons involved in the intermolecular beta-sheet motive. In disagreement with the concept of "amyloid fibers as water-filled nanotubes" [Perutz, M. F., et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 5591-5595], elution of D(2)O-grown fibrils with H(2)O is complete, which is reflected by the vanishing of D(2)O bending vibrations at 1214 cm(-)(1). This implies the absence of "trapped water" within insulin fibrils. The rigid conformations of the native and fibrillar insulin contrast with transient intermediate states docking at the fibrils' ends. Room-temperature seeding is accompanied by an accelerated H-D exchange in insulin molecules in the act of docking and integrating with the seeds, proving that the profound structural disruption is the sine qua non of forming an aggregation-competent conformation.

Amyloid↗
PubMed2002

Cholesterol-dependent aggregation of amyloid beta-protein.

One of the fundamental pathological processes of Alzheimer's disease (AD) is the aggregation of the amyloid beta-protein (Abeta). In the case of familial AD, the expression of genes responsible for this disease is likely to enhance aggregation of Abeta through its enhanced generation. However, there is no evidence to indicate thus far that in the case of sporadic AD, a major form of the disease, the generation of Abeta is altered. Thus, one could assume that the aggregation of Abeta in AD is induced by unknown posttranslational modification or by an altered clearance mechanism, or both. We previously identified a novel Abeta species in the human brain that exhibited early pathological changes of AD. This Aalpha is characterized by its tight binding to GM1 ganglioside (GM1). Based on its unique molecular characteristics, including its extremely high aggregation potential and altered immunoreactivity, we hypothesized that Abeta undergoes conformational alteration and acts as a seed for Abeta fibrillogenesis. In regard to the molecular mechanism underlying the formation of GM1-Abeta, we recently found that binding of Abeta to GM1 was facilitated in cholesterol-rich environments and, furthermore, it was dependent on the cholesterol-induced clustering of GM1 in the host membranes. Recently, increasing evidence indicates that cholesterol is a risk factor for AD development. The results of our current studies may provide a new insight into the molecular mechanism underlying the cholesterol-dependent development of AD.

Alzheimer Disease↗
PubMed1978

Cell junction and ultrastructural development of reaggregated mouse brain cultures.

The morphological development of reaggregated embryonic mouse brain cells was followed by scanning and transmission electron microscopy. Initially, the reaggregated cells are loosely packed and lack specialized cell junction; however, after 2-4 days puncta adhaerens junctions are numerous. The first immature synapses are seen at 8-9 days of culture. These synaptic complexes mature in appearance and increase approximately 3-fold in number during the next 3 weeks of culture. Two distinct, electron dense, intercellular specialization, apparently unique to neural cell cultures, are observed in these older cultures. Brain cell reaggregates observed at 2, 3, and 5 months of culture show signs of aging, i.e., a gradual decrease in the number of synapses, myelin degeneration and increased lipofuscin granules.

Aging↗