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At least 199 records · Page 11Linked to original sources
PubMed2000

Aggregation of an amyloidogenic fragment of human islet amyloid polypeptide.

Native human islet amyloid polypeptide (hIAPP) has been identified as the major component of amyloid plaques found in the pancreatic islets of Langerhans of persons affected by type 2 diabetes mellitus. Early studies of hIAPP determined that a segment of the molecule, amino acids 20-29, is responsible for its aggregation into amyloid fibrils. The present study demonstrates that the aggregation of hIAPP 20-29-Trp is a nucleation-dependent process, displaying a distinct lag time before the onset of rapid aggregation. Moreover, the lag time can be eliminated by seeding the sample of unaggregated peptide with preformed fibrils. In contrast to the expectation from the conventional model of nucleation-dependent aggregation, however, the lag time of hIAPP aggregation does not depend on peptide concentration. To explain this observation, a modified version of the standard model of nucleation-dependent aggregation is presented in which the monomeric peptide concentration is buffered by an off-aggregation-pathway formation of peptide micelles.

Amyloid↗
PubMed2005

Formation of melanocyte spheroids on the chitosan-coated surface.

The search for biocompatible materials that can maintain function of melanocytes as the cellular patch is a feasible alternative for use in the autologous melanocyte transplantation for vitiligo. In this study, we demonstrated that the surface of chitosan-coated polystyrene wells supported the growth and phenotype expression of melanocytes. Depending on the seeding density and culture time, melanocytes were monolayered or spheroidal in morphology. At seeding densities above 10 x 10(3) cells/cm2, human melanocytes started to aggregate on the surface of chitosan after 2 days in culture. These aggregates grew into compact melanocyte spheroids on day 3 and more melanocyte spheroids were observed when a higher seeding density was used. Cells remained viable in the spheroids and grew into dendritic melanocytes when they were reinoculated on polystyrene wells. Conversely, the time for the formation of melanocyte spheroids needed a longer period at lower seeding density. For example, melanocytes at as low as 1.25 x 10(3) cells/cm2 did not aggregate until the 20th day of culture. In order to interpret the phenomenon further, we proposed the formation of melanocyte spheroids on the chitosan is mediated by a balance between two competing forces: the interactions of cell-chitosan and cell-cell.

Cell Adhesion↗
PubMed2000

eNOS-overexpressing endothelial cells inhibit platelet aggregation and smooth muscle cell proliferation in vitro.

Endothelial cell seeding of synthetic small diameter vascular grafts (SSDVG) has been shown to diminish thrombosis and intimal hyperplasia, resulting in improved graft patency. However, endothelial cell retention on seeded grafts when exposed to physiological shearing conditions remains poor. We report that the genetic engineering of endothelial cells to overexpress endothelial nitric oxide synthase (eNOS), may create improved anti-thrombotic and anti-hyperplastic endothelial cell phenotypes for SSDVG seeding. eNOS-overexpressing endothelial cells may potentially overcome the biochemical loss due to shear induced reduction in endothelial cell coverage on SSDVG. Bovine aortic endothelial cells (BAEC) were transfected with the human eNOS gene, and co-incubated with either human whole blood or bovine aortic smooth muscle cells (BASMC) in vitro. eNOS-transfected BAEC significantly overexpressed eNOS compared to control beta-Gal-transfected and untransfected BAEC up to 120 h post transfection. In co-incubation and co-culture assays, human platelet aggregation decreased by 46% and BASMC proliferation decreased by 67.2% when compared to incubation with untransfected BAEC.

Animals↗
PubMed2004

Hsp25, a member of the Hsp30 family, promotes inclusion formation in response to stress.

Protein aggregates are oligomeric complexes of misfolded proteins, and serve as the seeds of inclusion bodies termed aggresomes in the cells. Heat shock proteins (Hsps) prevent misfolding and aggregate formation. Here, we found that only avian Hsp25 dominantly accumulated in the aggresomes induced by proteasome inhibition. Molecular cloning of chicken Hsp25 (cHsp25) revealed that it belongs to the Hsp30 family, which is a subfamily of the alpha-crystallin/small Hsp gene family. Unexpectedly, overexpression of cHsp25 into HeLa cells promoted inclusion formation whereas overexpression of mouse Hsp27 and its chicken homologue did not. These results suggest that cHsp25 acts differently from other small Hsps on protein aggregates.

Amino Acid Sequence↗
PubMed1986

The influence of serum and serum proteins on calcium oxalate crystal growth and aggregation.

The effects of serum, albumin, alpha-globulin, and a mixture of alpha- and beta-globulin on the growth and aggregation of calcium oxalate crystals were measured in a standard seeded crystallisation system. At concentrations below 0.01% blood, which corresponded to microscopic haematuria, all were potent inhibitors of crystal aggregation whilst only having a minor effect on crystal growth. It was found that albumin, alpha-globulin and beta-globulin can account for the total inhibitory effect of serum on crystal growth and crystal aggregation.

Alpha-Globulins↗
PubMed1997

Effect of seed crystals of uric acid and monosodium urate on the crystallization of calcium oxalate in undiluted human urine in vitro.

1. The aim of this study was to determine whether seed crystals of uric acid or monosodium urate promote the epitaxial deposition of calcium oxalate in undiluted human urine. The effects of seed crystals of uric acid, monosodium urate or calcium oxalate on calcium oxalate crystallization induced in pooled 24-h urine samples collected from six healthy men were determined by [14C]oxalate deposition and Coulter counter particle analysis. The precipitated crystals were examined by scanning electron microscopy. 2. Seed crystals of uric acid, monosodium urate and calcium oxalate increased the precipitated particle volume in comparison with the control containing no seeds by 13.6%, 56.8% and 206.5% respectively, whereas the deposition of [14C]oxalate in these samples relative to the control was 1.4% (P < 0.05), 5.2% (P < 0.01) and 54% (P < 0.001) respectively. The crystalline particles deposited in the presence of monosodium urate seeds were smaller than those in the control samples. Scanning electron microscopy showed that large aggregates of calcium oxalate were formed in the presence of calcium oxalate seeds, which themselves were not visible. In contrast, monosodium urate and, to a lesser extent, uric acid seeds were scattered free on the membrane surfaces and attached like barnacles upon the surface of the calcium oxalate crystals. 3. It was concluded that seed crystals of monosodium urate and uric acid do not promote calcium oxalate deposition to a physiologically significant degree in urine. However, binding of monosodium urate and uric acid crystals and their subsequent enclosure within actively growing calcium oxalate crystals might occur in vivo, thereby explaining the occurrence of mixed urate/oxalate stones.

Calcium Oxalate↗
PubMed1993

[The inhibitory effects of Takusha on the formation, growth and aggregation of calcium oxalate crystals in vitro].

The inhibitory effects of Takusha on the formation, growth and aggregation of calcium oxalate crystals were estimated by the microplate method and the use of Coulter counter TAII in the diluted urine system and undiluted urine system. In the diluted urine system, the inhibitory effect on aggregation and growth was calculated from the changes in the number and volume of crystals by the seed crystal method. Takusha had a strong inhibitory effect on the aggregation and growth when the concentration was above 10 micrograms/ml. In measuring the metastable limit by the microplate method, Takusha had a mild inhibitory effect on the formation of crystals above the concentration of 1 mg/ml. In the undiluted urine system, after determining the metastable limit, the formation and growth of calcium oxalate crystals which precipitated in response to a load of sodium oxalate were measured. Takusha had a strong inhibitory effect on the formation and growth at the concentration of 0.1-1 mg/ml. Takusha had no glycosaminoglicans in the dimethylmethylene blue assay and had an inhibitory effect on aggregation and growth when its molecular weight was over 10,000. Therefore, Takusha might be useful for preventing stones in recurrent stone formers.

Calcium Oxalate↗
PubMed2003

Nuclear and chloroplast genetic structure indicate fine-scale spatial dynamics in a neotropical tree population.

Dicorynia guianensis is a canopy tree, endemic to the tropical rain forest of French Guiana. We compared generational and spatial genetic structure for maternally and biparentally inherited markers in two cohorts (adult and seedling) in order to infer processes shaping the distribution of genetic diversity. The study was conducted on a 40 ha study plot located at Paracou near Kourou, where 172 adults trees and 375 saplings were sampled. Aggregation of trees was therefore suggested at different distances, ranging from 100 to 400 m. There was a strong link between demographic and genetic spatial structures at small distances (less than 100 m) that is likely to be the consequence of restricted seed dispersal. Genetic differentiation was more pronounced between spatial aggregates than between cohorts. Despite the spatial differentiation, the species was able to maintain high levels of diversity for maternal genomes, suggesting rapid turnover of aggregates. Spatial autocorrelation was larger for chloroplast than nuclear markers indicating a strong asymmetry between pollen and seed flow. Fixation indices indicated a lower heterozygote deficiency for the adults, maybe because of gradual elimination of selfed trees. Genetic relatedness at lower distances was higher in adult trees than in saplings, as a result of generation overlapping in the adult cohort. Overall, our results confirm earlier biological knowledge about the dispersion mechanisms of the species, and lead to an enhanced role of spatial processes in the dynamics of genetic diversity of D. guianensis.

Cell Nucleus↗
PubMed1991

[Inventions for preservation of hormonal function in long-term culture of human functioning pituitary adenoma].

This study was designed to establish in vitro model systems in human hormone-producing pituitary adenomas that are analogous to the in vivo cellular environment. Mechanically dispersed cells composed of single cells and aggregates from 6 pituitary adenomas (3 GH producing adenomas and 3 prolactinomas) were cultured on microporous membrane cell culture inserts (Millicell-CM) coated with Basement Membrane Matrigel for up to 6 months. Growth hormone or prolactin in the medium was measured during the culture, and morphological feature in vitro was also compared with that of the original tumor at intervals. Not only single cells but also large aggregated cells which usually float in the medium when seeded on conventional plastic, were flattened and firmly attached to coated microporous membrane under the control of medium volume in culture. In both type adenomas, especially prolactinomas, surviving aggregated adenoma cells revealed preserved hormone activity and no dedifferentiation of cell characteristics after 6 months in culture. Particularly during the first 2 months in culture, close similarity existed between in vivo and in vitro conditions with regard to cell morphology and hormone release. These results indicate that this new culture method may further aid the investigation of in vitro cellular structure and function in human pituitary adenomas under conditions which closely mimic the in vivo cellular environment.

Adenoma↗
PubMed1989

Neurons in primary cultures from five defined rat brain regions--cellular composition and morphological appearance.

Primary cultures from 15-17 days old fetal rat cerebral cortex, striatum, hippocampus, substantia nigra and brain stem were grown for ten days. Cell aggregates were formed one to two days after seeding. The cell bodies migrated peripherally from the clusters during development and networks of processes were formed. The cultures from the different brain regions contained predominantly neurons, stained by an antiserum against the neuron-specific enolase (NSE). There were differences in morphological appearance of the aggregates and also of the single neuronal cells cultivated from the various brain regions. On the bottom of the culture dishes a monolayer was formed of predominantly undifferentiated (mesenchymal-like) cells. Some cells of the monolayer stained for the astrocyte markers glial fibrillary acidic protein (GFAp) or S-100. The majority of the cells were, however, unstained to these markers. Very few endothelial cells and macrophages were observed.

Animals↗
PubMed2002

Grape seed and grape skin extracts elicit a greater antiplatelet effect when used in combination than when used individually in dogs and humans.

Grape products, rich in polyphenolics, inhibit platelet aggregation (PA), a risk factor for coronary artery disease. We postulated that combining extracts of grape seed (GSD) and grape skin (GSK), primary sources of grape polyphenolics, individually shown to inhibit PA, might enhance their individual antiplatelet effects. This hypothesis was examined in vitro (human platelets) and ex vivo (dog platelets) by studying the effects of the extracts on collagen-induced whole blood PA. In vitro, threshold concentration of only GSD, individually incubated with blood, significantly inhibited PA; PA was inhibited by 12.7 +/- 3.5% (P < or = 0.01). No significant changes in Pa were observed with threshold concentrations of GSK, used individually. In two dose combinations, GSD and GSK inhibited PA 40.5 +/- 10.1% (P < or = 0.005) and 96.5 +/- 3.1% (P < or = 0.001). In the ex vivo study, seven dogs were fed threshold doses of GSD or GSK individually, in combination or in combination with a proprietary enzyme blend (EB; thought to enhance bioavailability) for 8 d. PA was measured before and after each treatment. PA measurements were also repeated 24 h after the final dose of GSD + GSK + EB. Feeding the extracts individually did not affect PA, whereas feeding them in combination inhibited PA by 31.9 +/- 7.1% (P < or = 0.05). Feeding EB in addition to GSD + GSK inhibited PA by 56.2 +/- 8.1% (P < or = 0.005); 24 h later, PA was still inhibited by 31.5 +/- 10.5% (P < or = 0.05), suggesting a residual antiplatelet effect from the administration of the final dose. The results suggest that the components of GSD and GSK, when present in combination as in red wine, grape juice or in a commercial preparation containing both extracts, exhibit a greater antiplatelet effect than when present individually.

Animals↗
PubMedDate not supplied

Alpha-toxin production by Clostridium septicum at different culture conditions.

Clostridium septicum is responsible for a number of diseases, the most serious of which are a frequently fatal non-traumatic gas gangrene in man and braxy malignant oedema and blackquarter in farm animals. Immunity to these diseases is mediated mainly by antitoxin raised against C. septicum alpha toxin, which has hemolytic, lethal and necrotizing activities. Clostridium septicum produces lethal antigen only in low titre and in a variable way. In addition, the immunogenicity of native toxic filtrates is weak, which results in poor antibody response in animals. The aim of this work was to determine the best culture conditions for obtention of the protective antigen. To do this, the presence of the alpha toxin was identified in culture filtrates by means of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under different culture conditions. The strain used was C. septicum 3606. The cultures were performed in anaerobic conditions at 37 degrees C. Two systems were employed: (a) batch (0.5% initial glucose concentration); and (b) batch with partial feeding of the carbon source (0.1% glucose concentration) at controlled pH. The hemolytic activity of supernatants was determined using human erythrocytes, and the final biomass concentration was estimated by dry weight determination. The proteins present in supernatants were concentrated by ultrafiltration and identified in 10% SDS-polyacrylamide gels. With partial feeding of the carbon source the final biomass (2.22 g/L) was three times higher than the amount reached in batch system (0.70 g/L), however, no difference was found in the hemolytic activity (16 HU50/mL) showing no correlations between cell growth and hemolytic activity of the supernatants. Electrophoresis of filtrate cultures obtained with partial feeding of the carbon source allowed us to identify several bands, two of them corresponded to non-active alpha-protoxin (46 kDa) and the active toxin (42 kDa) and the aggregate of the active toxin at the seeding line. On the other hand, the ultrafiltrate from batch system showed less number of bands and the 46 kDa band was much weaker, suggesting a lower toxin production in this system. It becomes important to determine culture conditions and correlate with extracellular protein presence to optimize C. septicum antigen protector production.

Journal Article↗
PubMed2006

The physical basis of how prion conformations determine strain phenotypes.

A principle that has emerged from studies of protein aggregation is that proteins typically can misfold into a range of different aggregated forms. Moreover, the phenotypic and pathological consequences of protein aggregation depend critically on the specific misfolded form. A striking example of this is the prion strain phenomenon, in which prion particles composed of the same protein cause distinct heritable states. Accumulating evidence from yeast prions such as [PSI+] and mammalian prions argues that differences in the prion conformation underlie prion strain variants. Nonetheless, it remains poorly understood why changes in the conformation of misfolded proteins alter their physiological effects. Here we present and experimentally validate an analytical model describing how [PSI+] strain phenotypes arise from the dynamic interaction among the effects of prion dilution, competition for a limited pool of soluble protein, and conformation-dependent differences in prion growth and division rates. Analysis of three distinct prion conformations of yeast Sup35 (the [PSI+] protein determinant) and their in vivo phenotypes reveals that the Sup35 amyloid causing the strongest phenotype surprisingly shows the slowest growth. This slow growth, however, is more than compensated for by an increased brittleness that promotes prion division. The propensity of aggregates to undergo breakage, thereby generating new seeds, probably represents a key determinant of their physiological impact for both infectious (prion) and non-infectious amyloids.

Amyloid↗
PubMed2005

Morphological transition between diffusion-limited and ballistic aggregation growth patterns.

In this work, the transition between diffusion-limited (DLA) and ballistic aggregation (BA) models was reconsidered using a model in which biased random walks simulate the particle trajectories. The bias is controlled by a parameter lambda, which assumes the value lambda=0 (1) for the ballistic (diffusion-limited) aggregation model. Patterns growing from a single seed were considered. In order to simulate large clusters, an efficient algorithm was developed. For lambda (not equal to) 0 , the patterns are fractal on small length scales, but homogeneous on large ones. We evaluated the mean density of particles (-)rho in the region defined by a circle of radius r centered at the initial seed. As a function of r, (-)rho reaches the asymptotic value rho(0)(lambda) following a power law (-)rho = rho(0) +Ar(-gamma) with a universal exponent gamma=0.46 (2) , independent of lambda . The asymptotic value has the behavior rho(0) approximately |1-lambda|(beta) , where beta=0.26 (1) . The characteristic crossover length that determines the transition from DLA- to BA-like scaling regimes is given by xi approximately |1-lambda|(-nu) , where nu=0.61 (1) , while the cluster mass at the crossover follows a power law M(xi) approximately |1-lambda(-alpha) , where alpha=0.97 (2) . We deduce the scaling relations beta=nugamma and beta=2nu-alpha between these exponents.

Journal Article↗
PubMed2006

N-terminal domain of yeast Hsp104 chaperone is dispensable for thermotolerance and prion propagation but necessary for curing prions by Hsp104 overexpression.

Hsp104 is a hexameric protein chaperone that resolubilizes stress-damaged proteins from aggregates. Hsp104 promotes [PSI(+)] prion propagation by breaking prion aggregates, which propagate as amyloid fibers, into more numerous prion "seeds." Inactivating Hsp104 cures cells of [PSI(+)] and other amyloid-like yeast prions. Overexpressing Hsp104 also eliminates [PSI(+)], presumably by completely resolubilizing prion aggregates. Inexplicably, however, excess Hsp104 does not cure the other prions. Here we identify missense mutations in Hsp104's amino-terminal domain (NTD), which is conserved among Hsp100 proteins but whose function is unknown, that improve [PSI(+)] propagation. Hsp104Delta147, engineered to lack the NTD, supported [PSI(+)] and functioned normally in thermotolerance and protein disaggregation. Hsp104Delta147 failed to cure [PSI(+)] when overexpressed, however, implying that excess Hsp104 does not eliminate [PSI(+)] by direct dissolution of prion aggregates. Curing of [PSI(+)] by overexpressing catalytically inactive Hsp104 (Hsp104KT), which interferes with endogenous Hsp104, did not require the NTD. We further found that Hsp104 mutants defective in threading peptides through the hexamer pore had reduced ability to support [PSI(+)] in proportion to protein resolubilization defects, suggesting that [PSI(+)] propagation depends on this threading and that Hsp104 "breaks" prion aggregates by extracting protein monomers from the amyloid fibers.

Alleles↗
PubMed2005

A long-range attraction between aggregating 3T3 cells mediated by near-infrared light scattering.

At what range can a mammalian cell sense the presence of another cell and through what medium? To approach these questions, the formation of aggregates of a 3T3 cell variant (3T3x cells) grown on solid substrates was studied. Each of the aggregates consisted of cells that, at the time of their seeding, were single and located randomly. Yet somehow they seemed to detect each other within a certain range (R(a)) and move together to form aggregates. The article describes a simple assay to measure the value of R(a). When applied to 3T3x cells with altered intensities of near-infrared light scattering (I(sc)) the assay showed that (i) R(a) was much larger than one cell diameter, and (ii) R(a) was directly related to I(sc). The results suggest that near-infrared light scattering by the cells mediate a long-range attraction between them, which does not require physical contact and enables them to detect each other's presence.

Animals↗
PubMed1997

Models of amyloid seeding in Alzheimer's disease and scrapie: mechanistic truths and physiological consequences of the time-dependent solubility of amyloid proteins.

Ordered protein aggregation in the brain is a hallmark of Alzheimer's disease and scrapie. The disease-specific amyloid fibrils comprise primarily a single protein, amyloid beta, in Alzheimer's disease, and the prion protein in scrapie. These proteins can be induced to form aggregates in vitro that are indistinguishable from brain-derived fibrils. Consequently, much effort has been invested in the development of in vitro model systems to study the details of the aggregation processes and the effects of endogenous molecules that have been implicated in disease. Selected studies of this type are reviewed herein. A simple mechanistic model has emerged for both processes that involves a nucleation-dependent polymerization. This mechanism dictates that aggregation is dependent on protein concentration and time. Furthermore, amyloid formation can be seeded by a preformed fibril. The physiological consequences of this mechanism are discussed.

Alzheimer Disease↗
PubMed1985

Spheroidal aggregate culture of rat liver cells: histotypic reorganization, biomatrix deposition, and maintenance of functional activities.

Liver cells isolated from newborn rats and seeded on a non-adherent plastic substratum were found to spontaneously re-aggregate and to form, within a few days, spheroidal aggregates that eventually reached a plateaued diameter of 150-175 micron. Analyses on frozen sections from these spheroids by immunofluorescence microscopy using antibodies to various cytoskeletal elements and extracellular matrix components revealed a sorting out and a histotypic reorganization of three major cell types. A first type consisted of cells that segregated out on the aggregate surface forming a monolayer cell lining; a second type was identified as hepatocytes that regrouped in small islands often defining a central lumen; and a third group of cells reorganized into bile duct-like structures. This intercellular organization in the aggregates was paralleled by the accumulation of extracellular matrix components (laminin, fibronectin, and collagen) and their deposition following a specific pattern around each cell population structure. Determinations of albumin secretion and tyrosine aminotransferase induction by dexamethasone and glucagon at various times after the initiation of the cultures revealed a maintenance of the hepatocyte-differentiated functions for at least up to 2 mo at the levels measured at 3-5 d. It is concluded that cells dispersed as single cells from newborn rat liver conserve in part the necessary information to reconstruct a proper three-dimensional cyto-architecture and that the microenvironment so generated most likely represents a basic requirement for the optimal functioning of these differentiated cells.

Albumins↗