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PubMed1995

Flaxseed: a potential treatment for lupus nephritis.

Flaxseed is rich in alpha-linolenic acid (alpha-LA) which has anti-atherogenic properties, and lignans which are platelet activating factor (PAF)-receptor antagonists. These constituents of flaxseed, and its beneficial effects in the MRL/lpr lupus mouse prompted us to perform this dosing study in lupus nephritis patients. Nine patients were enrolled, eight of whom completed the study. After the baseline studies, patients were given 15, 30, and 45 g of flaxseed/day sequentially at four week intervals, followed by a five-week washout period. Compliance, disease activity, blood pressure, plasma lipids, rheology, PAF-induced platelet aggregation, renal function, and serum immunology were assessed. Flaxseed-sachet count and a significant increase of serum alpha-LA indicated good compliance for 15 and 30 g doses. Total and LDL cholesterol, and blood viscosity were significantly reduced with 30 g and to a lesser extent 45 g doses. PAF-induced platelet aggregation was inhibited by all doses. There was a significant decline in serum creatinine with 30 and 45 g, and a concomitant increase in creatinine clearance with increasing flaxseed dose. Proteinuria was reduced with 30 g and to a lesser extent with 45 g of flaxseed. Complement C3 was significantly elevated by all three doses. CD11b expression on neutrophils, a measure of C3bi receptors, was significantly reduced with the 30 g dose. In conclusion, 30 g flaxseed/day was well tolerated and conferred benefit in terms of renal function as well as inflammatory and atherogenic mechanisms important in the pathogenesis of lupus nephritis.

Adult↗
PubMed2004

Protein-only transmission of three yeast prion strains.

Key questions regarding the molecular nature of prions are how different prion strains can be propagated by the same protein and whether they are only protein. Here we demonstrate the protein-only nature of prion strains in a yeast model, the [PSI] genetic element that enhances the read-through of nonsense mutations in the yeast Saccharomyces cerevisiae. Infectious fibrous aggregates containing a Sup35 prion-determining amino-terminal fragment labelled with green fluorescent protein were purified from yeast harbouring distinctive prion strains. Using the infectious aggregates as 'seeds', elongated fibres were generated in vitro from the bacterially expressed labelled prion protein. De novo generation of strain-specific [PSI] infectivity was demonstrated by introducing sheared fibres into uninfected yeast hosts. The cross-sectional morphology of the elongated fibres generated in vitro was indistinguishable from that of the short yeast seeds, as visualized by electron microscopy. Electron diffraction of the long fibres showed the 4.7 A spacing characteristic of the cross-beta structure of amyloids. The fact that the amyloid fibres nucleated in vitro propagate the strain-specific infectivity of the yeast seeds implies that the heritable information of distinct prion strains must be encoded by different, self-propagating cross-beta folding patterns of the same prion protein.

Amyloid↗
PubMed2002

Stabilities and conformations of Alzheimer's beta -amyloid peptide oligomers (Abeta 16-22, Abeta 16-35, and Abeta 10-35): Sequence effects.

Previously, we have studied the minimal oligomer size of an aggregate amyloid seed and the mechanism of seed growth with a multilayer beta-sheet model. Under high temperature simulation conditions, our approach can test the stability of possible amyloid forms. Here, we report our study of oligomers of Alzheimer's amyloid beta-peptide (Abeta) fragments 16-22, 16-35, and 10-35 (abbreviated Abeta(16-22), Abeta(16-35), and Abeta(10-35), respectively). Our simulations indicate that an antiparallel beta-sheet orientation is the most stable for the Abeta(16-22), in agreement with a solid state NMR-based model [Balbach, J. J., Ishii, Y., Antzutkin, O. N., Leapman, R. D., Rizzo, N. W., et al. (2000) Biochemistry 39, 13748-13759]. A model with twenty-four Abeta(16-22) strands indicates a highly twisted fibril. Whereas the short Abeta(16-22) and Abeta(24-36) may exist in fully extended form, the linear parallel beta-sheets for Abeta(16-35) appear impossible, mainly because of the polar region in the middle of the 16-35 sequence. However, a bent double-layered hairpin-like structure (called hook) with the polar region at the turn forms parallel beta-sheets with higher stability. An intra-strand salt-bridge (D23-K28) stabilizes the bent hairpin-like hook structure. The bent double-beta-sheet model for the Abeta(10-35) similarly offers oligomer stability.

Alzheimer Disease↗
PubMed2005

The role of caspase cleavage of tau in Alzheimer disease neuropathology.

Alzheimer disease (AD) is characterized by the accumulation of amyloid plaques and neurofibrillary tangles within selective brain regions. In addition, cell death pathways become active leading to neurodegeneration. Caspase activation, a key step in the programmed cell death pathway known as apoptosis, occurs in AD and leads to the proteolytic cleavage of several neuronal proteins. Previously, it was hypothesized that the development of the classical hallmarks of AD, amyloid plaques and neurofibrillary tangles, occur independently and do not involve the activation of caspases. However, recent studies suggest that plaques, tangles, and caspase activation share a common pathway. Beta-amyloid, the main component of amyloid plaques, activates caspases. Activated caspases can in turn cleave tau, the main component of neurofibrillary tangles. Caspase-cleaved tau (deltatau) may initiate or accelerate the development of tangle pathology. Tau, when cleaved by caspases at Asp421, "seeds" filamentous aggregates in vitro. Caspase-cleaved tau also adopts the MC1 conformation, one of the earliest pathologic events in tangle formation. Importantly, deltatau occurs early in the development of tangle pathology within AD brains and in a transgenic mouse model of AD. This review summarizes recent evidence suggesting that caspase cleavage of tau plays an important role in the development of neurofibrillary tangle pathology. In addition, a model is presented whereby caspase cleavage of tau provides a mechanistic link between the development of amyloid and tangle pathologies.

Alzheimer Disease↗
PubMed1987

Proliferation of human carcinoma cells in calcium-deficient culture medium may depend on autocrine growth factor(s).

The ability to proliferate in media with low calcium concentrations (less than 0.1 mM) at clonal seeding densities is a characteristic of malignant cells. Cells of the carcinoma line C-4I are an exception, and are unable to proliferate in medium with 0.02 mM calcium (LCM) when seeded at low densities. Conditioned medium, derived from a subline of C-4I cells that were adapted to grow in LCM, contained an acid-stable factor of greater than 8 kilodaltons molecular weight that mediated proliferation and colony formation of unadapted C-4I cells in LCM in a concentration-dependent manner. The effect was not related to enhanced cell attachment or spreading, and was maximal when the unadapted C-4I cells were seeded in aggregates of 2-20 cells rather than singly. Thus, calcium independence, a component of the neoplastic phenotype, may be mediated by autocrine tumor-cell-derived factor(s) and may involve long- and short-range cell interactions.

Calcium↗
PubMed2006

Soluble amyloid-beta in the brain: the scarlet pimpernel.

Researchers since the 1990s have predominantly focused on the amyloid hypothesis and the formation of amyloid fibrils as the culprit behind AD when we began working on soluble Abeta (sAbeta). Unexpectedly, this work produced several novel findings. First, we observed that N-terminal truncated peptides are the major components of soluble and insoluble Abeta in AD; secondly, that all sAbeta species belong to the 42 form and the sAbeta x-40 species is virtually absent in AD parenchyma; thirdly, that Abeta42 in the soluble form is non-detectable by immunoblots in plaque-free, normal brains. The later observation that sAbeta 42 species is present in amyloid beta protein precursor (AbetaPP) over-expressing brains of patients with Down syndrome in prenatal and early postnatal development argued that sAbeta is present in brain in abnormal conditions and that its appearance seeds Abeta aggregation and accumulation. Although the sAbeta we described in intact brain tissue appeared to match the soluble Abeta oligomers detected in cell media, which were subsequently shown to be the most toxic form of Abeta, our research has been virtually ignored by the Alzheimer field. It continues nevertheless. Recently we demonstrated that the sAbeta species present in physiologically aging brains are different from those present in brains with sporadic AD as the latter form oligomers more quickly, are more toxic to neurons, and produce more severe membrane damage than the Abeta species associated with normal brain aging. Furthermore, in familial AD, the composition of soluble Abeta appears to dictate distinctive features of the disease phenotype introducing the notion of Abeta strains, a concept well established in prion diseases.

Aging↗
PubMed1994

Mutations associated with a locus for familial Alzheimer's disease result in alternative processing of amyloid beta-protein precursor.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the extracellular deposition of amyloid beta-protein (A beta), a molecule produced by post-translational processing of the beta-amyloid precursor protein (beta APP). Mutations within the gene encoding beta APP have been linked to early onset forms of AD, but the pathogenetic mechanism(s) producing the phenotype are unknown. We analyzed the effects on beta APP processing in vitro of a naturally occurring Ala-->Gly mutation at position 692 of beta APP770 (A692G) (Hendriks, L., Duijin, C., Cras, P., Cruts, M., van Hul, W., van Harskamp, F., Warren, A., McInnis, M., Antonarakis, S., Martin, J.-J., Hofman, A., and van Broeckhoven, C. (1992) Nature Genet. 1, 218-221), as well as the effects of five genetically engineered mutations at or near this site. Substitution of glycine or proline for Ala692, or for Phe690, produced relative increases in secretion of A beta and relative decreases in secretion of the p3 peptide(s) arising after alpha-secretase generation of soluble APP (APPs). The Phe690-->Pro substitution also resulted in the synthesis of truncated APPs molecules. The structurally conservative substitutions Ala692-->Val and Phe690-->Tyr did not exhibit these effects. Certain of the substitutions also resulted in the production of a minor peptides, previously undescribed in vitro, beginning at Ala2, Lys16, and Phe19 of A beta. These data show that beta APP mutations carboxyl-terminal to alpha-secretase and beta-secretase cleavage sites can exert strong control over beta APP processing. Increased secretion of A beta may accelerate amyloidogenesis by providing more precursors for aggregation. It is also possible that truncated A beta peptides resulting from several of these mutations may accelerate amyloidogenesis through self-aggregation and/or seeding the fibrillogenesis of longer, more abundant A beta species.

Alanine↗
PubMed1994

[Organoid forming method--a new in vitro chemosensitivity test].

In 1988, Friedman and coworkers reported a new chemosensitivity test using "organoids" (epithelial cell aggregates) which had a high plating efficiency and short assay period. The authors recognized this test to be useful and studied it experimentally. Human tumor xenografts maintained in the subcutis of BALB/c nu/nu mice were minced into cell aggregates and filtered, then resuspended in enriched NCTC 135 cell culture medium which contained no serum. Petri dishes were coated with a mixture of collagen-I and bovine serum albumin and dried for an hour. The cell aggregates were seeded in these coated dishes and cultured in a condition of low O2 tension (3% O2). Plating efficiency at 24 hours in cultures of three tumors were 20.8 +/- 2.6% on SC-6-JCK (stomach), 37.8 +/- 3.8% on NS-8 (stomach) and 27.2 +/- 1.5% on PAN-1-RITC (pancreas), respectively. The cell number of each organoid increased until 72 hours in culture, although the organoid number of each dish decreased slightly. Flow cytometrical measurement of total DNA content in dishes showed that the amount of human DNA increased more rapidly than that of mouse DNA which was derived from interstitial and infiltrative cells. This culture system appeared to allow a selective growth of epithelial cells. Subsequently, some drug sensitivity was tested using this system. SC-6-JCK tumor is sensitive to mitomycin C (MMC), although resistant to adriamycin (ADM) in a test using nude mice (sc-ip system). Organoids were formed from this tumor and chemosensitivity was tested against MMC and ADM from the viewpoint of change in the organoid number in each dish. After one hour of drug exposure, only a part of the cells in organoids was affected. On the contrary, after an exposure of 24 hours, the ADM-treated group showed the same results as the MMC-treated group. Hence this test was considered to become more appropriate by counting not the organoid number but the total cell number in the dish.

Animals↗
PubMed1999

Colonization pattern of the biocontrol strain Pseudomonas chlororaphis MA 342 on barley seeds visualized by using green fluorescent protein.

Pseudomonas chlororaphis MA 342 is a potent biocontrol agent that can be used against several seed-borne diseases of cereal crops, including net blotch of barley caused by the fungus Drechslera teres. In this study, strain MA 342 was tagged with the gfp gene (encoding the green fluorescent protein) in order to study the fate of cells after seed inoculation. The gfp-tagged strain, MA 342G2, had the same biocontrol efficacy as the wild type when it was applied at high cell concentrations to seeds but was less effective at lower cell concentrations. By comparing cell counts determined by microscopy to the number of CFU, we found that the number of culturable cells was significantly lower than the total number of bacteria on seeds which were inoculated and dried for 20 h. Confocal microscopy and epifluorescence stereomicroscopy were used to determine the pattern of MA 342G2 colonization and cell aggregation on barley seeds. Immediately after inoculation of seeds, bacteria were found mainly under the seed glume, and there was no particular aggregation pattern. However, after the seeds were sown, irregularly distributed areas of bacterial aggregation were found, which reflected epiphytic colonization of glume cells. There was a trend towards bacterial aggregation near the embryo but never within the embryo. Bacterial aggregates were regularly found in the groove of each seed formed by the base of the coleoptile and the scutellum. Based on these results, we suggest that MA 342 colocalizes with the pathogen D. teres, which facilitates the action of the fungistatic compound(s) produced by this strain.

Ascomycota↗
PubMed2005

Heterogeneous nucleation-controlled particulate formation of recombinant human platelet-activating factor acetylhydrolase in pharmaceutical formulation.

Clinical lots of recombinant human platelet-activating factor acetylhydrolase (rhPAF-AH) were prepared in a lyophilized formulation. After reconstitution with sterile water for injection to form an aqueous solution (10 mM sodium citrate, 7.5 w/v% sucrose, and 0.1 w/v% Pluronic-F68, pH 6.5), a few visible, slowly growing particles formed consistently within hours at room temperature. To investigate the mechanism of this phenomenon, immediately after reconstitution, all protein aggregates and exogenous particles were removed by filtration. During 20 days incubation at room temperature, no visible aggregates formed in these filtered samples. In contrast, when nano-sized hydrophilic silica particles were added, they seeded rapid and extensive aggregation of rhPAF-AH. This effect was exacerbated in solutions containing a lower Pluronic-F68 concentration at 0.01%. Aggregation occurred even under conditions where rhPAF-AH adsorption was reversible, and induced no detectable changes to protein secondary and tertiary structures. Decreasing the extent (e.g., adding Pluronic-F68) or affinity (e.g., increasing solution pH) of rhPAF-AH adsorption on nano-sized silica particles was found to be effective at reducing aggregation. Accelerated aggregation was not observed when rhPAF-AH formulation was seeded with aggregated rhPAF-AH. These results show that rhPAF-AH aggregation proceeds through a heterogeneous nucleation-controlled mechanism, where exogenous particles present in solution serve as seeds on which rhPAF-AH adsorb, nucleate, and grow into large aggregates.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1998

Contribution of advanced glycosylation to the amyloidogenicity of islet amyloid polypeptide.

The formation of amyloid within the islets of Langerhans is associated with the development of type II diabetes mellitus and occurs by the aggregation and insolubilization of islet amyloid polypeptide (IAPP). Recent in vitro studies suggest that amyloid formation follows a nucleation-dependent polymerization mechanism, i.e. aggregation is initiated by pre-formed aggregates or nucleation seeds. Modification of the Alzheimer's disease amyloid peptide by advanced glycosylation end products (AGEs), which form spontaneously by the non-enzymatic addition of glucose to protein amino groups, has been shown to enhance peptide aggregation in vitro. To explore the possibility that AGEs contribute to islet amyloid formation, we prepared AGE-modified IAPP (AGE-IAPP) in vitro and studied its properties by biochemical and biophysical techniques. AGE modification induced the formation of high-molecular-mass IAPP aggregates and amyloid formation was demonstrated by Congo red green-gold birefringence and by the presence of a characteristic fibrillar structure by electron microscopy. AGE-IAPP also showed an increase in cytotoxicity toward the astroglioma cell line HTB14. When added to soluble IAPP, AGE-IAPP seeds accelerated IAPP aggregation and abolished the nucleation period required for the polymerization of unseeded IAPP. Circular dichroism spectropolarimetry indicated that AGE-IAPP seeds may act as a template to stabilize the beta-sheet conformation of IAPP, thereby promoting its aggregation. Our studies demonstrate that AGE modification of IAPP results in high-molecular mass, fibrillar amyloid structures that nucleate IAPP amyloid formation and suggest a model for intra-islet amyloid deposition that may occur by the progressive advanced glycosylation of IAPP in vivo.

Amyloid↗
PubMed1970

The aggregation States of phytochrome from etiolated rye and oat seedings.

Initial extracts from etiolated plants contained two aggregates of phytochrome. A major fraction was almost excluded by Sephadex G-200 and was within the fractionation range of Sepharose 4 B. A minor fraction was within the fractionation range of Sephadex G-200. Modifications of a previous isolation procedure which allowed retention of this aggregation state are reported. With respect to gel filtration, the major fraction of phytochrome from oat and rye seedlings was identical. The aggregates of rye and oat phytochrome were also separated by diethylaminoethyl cellulose chromatography.

Journal Article↗
PubMed2006

[A comparative study on seeding methods of human mesenchymal stem cells in bone tissue engineering].

Seeding is the crucial step in bone tissue engineering. In current study, static and dynamic seeding methods for human bone marrow stem cells (hBMSCs) were compared. The methods for assay of DNA content in the constructs after seeding were adopted. The optic microscopy for histological apearance and the scanning electron microscopy (SEM) and fluorescent RT-PCR for osteogenic markers were performed. The maximal initial seeding concentration in static seeding is lower than that in dynamic seeding. Histology and SEM revealed the even distribution and spreading of cells in the dynamically seeded constructs, but showed cell aggregation in the statically seeded counterparts. Fluorescent RT-PCR again revealed stronger osteogenic potential of dynamically seeded constructs. Therefore, this initial study demonstrated that dynamic seeding of human bone marrow stem cells is a promising technique in bone tissue engineering.

Bone Marrow Cells↗
PubMed1987

Differentiation-associated carbohydrate chain on human hematopoietic cells recognized by Clerodendron trichotomum lectin.

In the screening of hematopoietic cell line cell aggregations, the extract of Clerodendron trichotomum seed was found to aggregate K-562 and KG-1 specifically. In the flow cytometric analysis using FITC-conjugated purified CTL, it was confirmed that CTL recognizes the specific carbohydrate(s) which seem to appear only in the early stages of differentiation of myeloid (KG-1) and erythroid (K-562) cell line cells and erythrocytes. The CTL binding to K-562 cells was decreased by TPA treatment which is known to induce retrodifferentiation of K-562. It is also found that this carbohydrate(s) were shaded with NANA on the differentiated cells. In the erythrocyte, CTL receptor was partially shaded by NANA.

Carbohydrate Metabolism↗
PubMed2002

Kinetics and energetics of assembly, nucleation, and growth of aggregates and fibrils for an amyloidogenic protein. Insights into transition states from pressure, temperature, and co-solute studies.

The transition states for prenucleation assembly, nucleation, and growth of aggregates and amyloid fibrils were investigated for a dimeric immunoglobulin light chain variable domain, employing pressure, temperature, and solutes as variables. Pressure-induced aggregation was nucleation-dependent and first-order in protein concentration and could be seeded. The insoluble aggregates were mixtures of amyloid fibrils and amorphous aggregates. Activation volumes, activation surface areas, and activation waters of hydration were larger for aggregate growth than for prenucleation assembly or nucleation, although activation free energies were similar for the three processes. Activation free energies for each of the transition states were dominated by the unfavorable free energy of solvation of newly exposed surfaces. Equilibrium dissociation and unfolding of the dimer showed a much larger volume change than those required to form the transition states for the three processes. Thus, the transition states for these steps are similar to the native state, and their formation requires only small structural perturbations. Finally, the presence of Congo red during amyloid fibril formation shortened lag times and caused pressure insensitivity of nucleation, suggesting that this compound or its analogs may not be effective as inhibitors of amyloidosis.

Amyloid↗
PubMed2005

Regulation by macromolecules of calcium oxalate crystal aggregation in stone formers.

Based on the structure of kidney stones, it is likely that they form as aggregations of preformed crystals, mostly calcium oxalate monohydrate (COM). In this study, we examined the ability of a macromolecular mixture isolated from the urine of normal individuals and stone formers to inhibit aggregation of preformed COM seed crystals in a simple ionic solution using measurements of changes in the particle size distribution (PSD) of preformed COM crystal aggregates. We also examined the effect in this assay of a number of synthetic homopolymers, naturally occurring urine macromolecules, and binary mixtures thereof. The macromolecular mixtures from urine of normals and most stone formers reduced the degree of aggregation of the seed crystals, whereas 22% of stone former urine macromolecules either did not disaggregate or actually promoted further aggregation. Stone formers within one family shared this property, but a non-stone forming sibling did not. Polyanions, either synthetic or naturally occurring, induced disaggregation to an extent similar to that exhibited by normal urine macromolecules, while polycations had no effect on the PSD. However, mixing a polyanion, either poly-aspartate or osteopontin, with the polycation poly-arginine, changed their behavior from disaggregation to aggregation promotion. The disaggregating behavior of normal urinary macromolecules provides a defense against aggregation, but a minority of stone forming individuals lacks this defense, which may contribute to stone formation.

Adult↗
PubMed2003

Theoretical modeling of prion disease incubation.

We apply a theoretical aggregation model to laboratory and epidemiological prion disease incubation time data. In our model, slow growth of misfolded protein aggregates from small initial seeds controls the latent or lag phase; aggregate fissioning and subsequent spreading leads to an exponential growth phase. Our model accounts for the striking reproducibility of incubation times for high dose inoculation of lab animals. In particular, low dose yields broad incubation time distributions, and increasing dose narrows distributions and yields sharply defined onset times. We also explore how incubation time statistics depend upon aggregate morphology. We apply our model to fit the experimental dose-incubation curves for distinct strains of scrapie, and explain logarithmic variation at high dose and deviations from logarithmic behavior at low dose. We use this to make testable predictions for infectivity time-course experiments.

Animals↗