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PubMed1979

On the reduced intercellular adhesiveness of virally transformed BHK21 cells.

Baby hamster kidney fibroblasts (BHK21 cells) transformed by polyoma or Rous sarcoma viruses aggregate less than the untransformed parental cells when incubated in growth medium in a gyratory shaker for 18-24 h. This difference can be measured by electronic particle counting, or by filtering aggregated suspensions of 32P-labelled cells through bolting fabric. The aggregation of transformed derivatives is not enhanced by the presence, during aggregation of epsilon-amino caproic acid, an inhibitor of plasmin activation. Some lines of transformed BHK21 cells do not appear less adhesive than untransformed cells in a short-term aggregation assay, and none adheres markedly less well when seeded onto homotypic cell sheets. The decreased aggregation of transformed cells is consistent with suggestions that LETS protein is involved in intercellular adhesion of fibroblasts as well as in attachment of cells to non-cellular substrates. If so, the short-term aggregation of freshly trypsinized cells may depend on secretion of LETS from an intracellular pool.

Aminocaproates↗

PubMed2001

Schwann cell response to micropatterned laminin surfaces.

In the peripheral nervous system, Schwann cells are closely associated with, and play key roles in, the development, maintenance, and regeneration of peripheral neurons. Following injury, Schwann cell orientation may also play a role in guiding regenerating axons. To aid in the investigation of these interactions between Schwann cells and growing neurites, we have developed a method of controlling Schwann cell placement and orientation in vitro by using microlithographically patterned laminin substrates, alternating 20-microm regions of laminin with bovine serum albumin (BSA) stripes. The Schwann cells predominantly attached and elongated on the laminin stripes and organized into multicellular aggregates that were oriented with the micropattern. A detailed analysis of Schwann cell aggregate orientation and shape demonstrated a strong dependence on time. At 1 h after seeding the cells, 70% of the aggregates were oriented with respect to the micropattern; 94% were oriented at 24 h. Variations in laminin concentration and seeding density were also investigated. The only significant differences in Schwann cell response occurred 1 h after seeding (the earliest time point the cultures were observed), and the main factor controlling the cellular orientation appeared to be the presence of the laminin-BSA interface. This ability to control cell orientation and placement provides a tool for future investigations of Schwann cell-neuronal interactions in vitro.

Animals↗

PubMed2006

Shear flow induces amyloid fibril formation.

Shear flow is indirectly implicated in amyloid formation in vitro. Despite the association between amyloid fibrils and disease, and the prevalence of flow in physiological systems, the effect of this parameter is uncharacterized. We designed a novel Couette cell to quantitatively investigate shear exposure during fibrillogenesis. Amyloid formation by beta-lactoglobulin was monitored in situ with real-time fluorescence measurements across a range of shear rates. We demonstrate shear-induced aggregation of spheroidal seed-like species. These seeds enhance fibril formation in native beta-lactoglobulin, thereby demonstrating that shear flow generates an amyloidogenic precursor. Furthermore, preformed fibrils are degraded by exposure to high shear rates. Our results have implications for the mechanism of amyloid formation in physiological flow conditions.

Amyloid↗

PubMed2001

Microscopic observation of aerobic granulation in sequential aerobic sludge blanket reactor.

AIMS: This paper attempts to provide visual evidence of how aerobic granulation evolves in sequential aerobic sludge blanket reactors. METHODS AND RESULTS: A series of experiments were conducted in two column-type sequential aerobic sludge reactors fed with glucose and acetate as sole carbon source, respectively. The evolution of aerobic granulation was monitored using image analysis and optical and scanning electron microscopy. The results indicated that the formation of aerobic granules was a gradual process from seed sludge to compact aggregates, further to granular sludge and finally to mature granules with the sequential operation proceeding. Glucose- and acetate-fed granules have comparable characteristics in terms of settling velocity, size, shape, biomass density and microbial activity. However, the microbial diversity of the granules was associated with the carbon source supplied. In this work, an important aerobic starvation phase was identified during sequential operation cycles. It was found that periodical aerobic starvation was an effective trigger for microbial aggregation in the reactor and further strengthened cell-cell interaction to form dense aggregates, which was an essential step of granulation. The periodical starvation-induced aggregates would finally be shaped to granules by hydrodynamic shear and flow. CONCLUSION: Aerobic granules can be formed within 3 weeks in the systems. The periodical starvation and hydrodynamic conditions would play a crucial role in the granulation process. SIGNIFICANCE AND IMPACT OF THE STUDY: Aerobic granules have excellent physical characteristics as compared with conventional activated sludge flocs. This research could be helpful for the development of an aerobic granule-based novel type of reactor for handling high strength organic wastewater.

Aerobiosis↗

PubMed2003

Chemical modification of insulin in amyloid fibrils.

We have investigated the chemical modification of insulin under conditions that promote the conversion of the soluble protein into amyloid fibrils. The modifications that are incorporated into the fibrils include deamidation of Asn A21, Asn B3, and Gln B4. In order to prepare fibrils with minimal deamidation of these residues, the kinetics of aggregation were accelerated by seeding with aliquots of a solution containing preformed fibrils. The resulting fibrils were then reincubated to determine the extent to which chemical modification occurs in the fibril itself. The deamidation of Asn A21 in particular could be followed in detail. Deamidation of this residue in the fibrillar form of insulin was found to occur in only 52 +/- 5% of molecules. This result indicates that there are at least two different packing environments of insulin molecules in the fibrils and suggests that the characterization of chemical modifications may be a useful probe of the environment of polypeptide chains within amyloid fibrils.

Amino Acid Sequence↗

PubMed2005

Hepatocyte function in a radial-flow bioreactor using a perfluorocarbon oxygen carrier.

UNLABELLED: The aims of this study were, first, to indicate the metabolic activity of hepatocytes in a radial-flow polyurethane foam matrix bioreactor relative to monocultures, and second, to evaluate the effect on the hepatocytes of including a synthetic perfluorocarbon (PFC) oxygen carrier to the recirculating medium. The efficient O2-carrying ability of PFCs may be beneficial to bioreactors employed in stressed cellular environments. Thus, they may also be useful in the treatment of an acute liver failure patient with a bioartificial liver support system (BALSS). Data on the function of three-dimensional (3-D) hepatocyte cultures exposed to emulsified PFCs are lacking. RESULTS: the metabolic functions of the 3-D hepatocyte cultures were improved relative to monocultures. Three-dimensional cultures with and without PFC behaved similarly, and no adverse effects could be detected when PFC was included in the recirculating medium. The addition of PFC significantly improved lidocaine clearance possibly due to the presence of higher O2 tension in the medium. Imaging indicated that large aggregates formed and that seeding had followed flow through the matrix. Simulations indicated first, that the cell numbers used in this study had been insufficient to challenge the bioreactor O2 supply explaining the similarity in performance of the 3-D cultures, and second, that the benefit of adding PFC would be more pronounced at the cell densities likely to be used in a BALSS bioreactor.

Animals↗

PubMed2001

Key factors in Alzheimer's disease: beta-amyloid precursor protein processing, metabolism and intraneuronal transport.

During the last years it has become evident that the beta-amyloid (Abeta) component of senile plaques may be the key molecule in the pathology of Alzheimer's disease (AD). The source and place of the neurotoxic action of Abeta, however, is still a matter of controversy. The precursor of the beta-amyloid peptide is the predominantly neuronal beta-amyloid precursor protein. We, and others, hypothesize that intraneuronal misregulation of APP leads to an accumulation of Abeta peptides in intracellular compartments. This accumulation impairs APP trafficking, which starts a cascade of pathological changes and causes the pyramidal neurons to degenerate. Enhanced Abeta secretion as a function of stressed neurons and remnants of degenerated neurons provide seeds for extracellular Abeta aggregates, which induce secondary degenerative events involving neighboring cells such as neurons, astroglia and macrophages/microglia. Beta-amyloid precursor protein has a pivotal role in Alzheimer's disease.

Alzheimer Disease↗

PubMed2005

The heterogeneous murine corneal stromal cell populations in vitro.

PURPOSE: To demonstrate that the murine corneal stroma is inhabited by heterogeneous cell populations that include cells expressing nestin. METHODS: Collagenase-isolated corneal stroma cells obtained from newborn and adult mice (2nd and 12th postnatal weeks, respectively), were seeded at low (5 cells/mm2), intermediate (50 cells/mm2), and high (500 cells/mm2) densities in DMEM/F12 containing insulin, transferrin, selenium, and 1% nonessential amino acids. Corneal stroma cells cultured at 500 cells/mm2 were treated with 10 ng/mL human recombinant transforming growth factor (TGF)-beta1 for 5 days. Cell morphology and expression of alpha-smooth muscle actin, choline acetyltransferase, CD45, glial fibrillary acidic protein (GFAP), keratocan, nestin, neurofilaments, protein gene product 9.5, tyrosine hydroxylase, and vimentin were examined. RESULTS: Phase-contrast microscopy demonstrated that freshly isolated corneal stromal cells are heterogeneous in morphology and include dendritic, stellate, neuronal, and small polyhedral cells. Immunostaining of primary cultures of 2- and 12-week-old mice, 24 hours after seeding at the intermediate density, showed that 100% of cells expressed vimentin and 97.7% +/- 2.7% expressed keratocan. alpha-Smooth muscle actin was expressed by 0.2% +/- 0.05% of cells in the 2-week-old group and 0.1% +/- 0.07% in 12-week-old group. Neurofilament was expressed by 0.5% +/- 0.03% and 0.7% +/- 0.03% of cells in the 2- and 12-week-old groups, respectively. No cell expressed GFAP or nestin. After 5 days in culture, cells seeded at high density aggregated as clusters that were immunoreactive to nestin in both groups. Cell clusters and migrating cells reacted to pgp 9.5, and migrating cells, but not the cell clusters, reacted to tyrosine hydroxylase. Cell cluster formation and nestin expression were abolished by culturing in the presence of TGF-beta1. CONCLUSIONS: Normal murine corneal stroma contains heterogeneous cell populations including cells with the potential to form clusters and express the progenitor marker nestin. This potential is disrupted by the addition of TGF-beta1 to the culture medium.

Actins↗

PubMed2000

Progress in desmin-related myopathies.

Desmin-related myopathies are sporadic and familial neuromuscular conditions of considerable clinical heterogeneity uniformly marked by the pathologic accretion of desmin, often in a filamentous fashion. A large variety of other proteins, some of them cytoskeletal, also accrue. Morphologically, two types may be distinguished, one characterized by inclusions such as cytoplasmic and spheroid bodies or desmin-dystrophin plaques and another marked by granulofilamentous material. The genetic spectrum of desmin-related myopathies is quite diverse in that missense mutations and deletions in the desmin gene and a missense mutation in the alpha-B crystallin gene have been detected and several genes on other chromosomes have been mapped; the encoded protein products of these genes, however, are unknown. Accumulation of desmin and other proteins appears to be due to impaired nonlysosomal proteolysis. Mutant desmin that appears to be hyperphosphorylated seems to act as a seed protein for filament aggregation, inducing formation of inclusions and granulofilamentous material in these conditions. This condition is part of the group of disorders known as "surplus protein myopathies."

Crystallins↗

PubMed1981

Human prolactin secreting adenoma cells maintained on extracellular matrix.

In vitro maintenance of human prolactin secreting adenoma cells on a extracellular matrix (ECM) produced by bovine corneal endothelial cells is associated with the adoption of morphological properties that are not expressed when the cells are seeded on plastic. Cells seeded on ECM were firmly attached and highly flattened, whereas cells seeded on plastic formed aggregates which were loosely attached or floated in the medium. Moreover, prolactin production from cells on ECM is frequently stable for several weeks. Both tumor cells and normal cells are more likely to resemble their in vivo counterparts when maintained on extracellular matrix than on plastic, and therefore, cells cultured on ECM may provide a better system for oncogenic and endocrine studies.

Adenoma↗

PubMed1988

Mitogenic effects of thyrotropin and adenosine 3',5'-monophosphate in differentiated normal human thyroid cells in vitro.

Previous studies of human thyroid cells in culture (mostly from pathological tissues) failed to demonstrate a mitogenic effect of TSH, leading to the proposal that the growth effect of TSH in vivo might be indirect. To reexamine the influence of TSH on DNA synthesis and cell proliferation, we established primary cultures of normal thyroid tissue from nine subjects. When seeded in a 1% serum-supplemented medium, thyroid follicles released by collagenase/dispase digestion developed as a cell monolayer that responded to TSH by rounding up and by cytoplasmic retraction. When seeded in serum-free medium, the cells remained associated in dense aggregates surrounded by few slowly spreading cells. In the latter condition, the cells responded to TSH and other stimulators of cAMP production, such as cholera toxin and forskolin, by displaying very high iodide-trapping levels. Exposure to serum irreversibly abolished this differentiated function. TSH stimulated the proliferation (as shown by DNA content per culture dish) of 1% serum cultured cells (doubling times were reduced from 106 to 76 h) and increased by 100% the [3H]thymidine labeling indices. In serum-free cultured cells (dense aggregates or cell monolayers after initial seeding with serum), control levels of DNA synthesis were lower, and up to 8-fold stimulation of DNA synthesis occurred in response to 100 mU/L TSH (stimulation was consistently detected with 20 mU/L), based on measurements of [3H]thymidine incorporation into acid-precipitable material and counts of labeled nuclei on autoradiographs (up to 40% labeled nuclei within 24 h). The mitogenic effect of TSH required a high insulin concentration (8.3 X 10(-7) mol/L) or a low insulin-like growth factor I concentration. The mitogenic effects of TSH were mimicked in part by cholera toxin, forskolin, and dibutyryl cAMP. Epidermal growth factor and phorbol myristate ester also stimulated thyroid cell proliferation and DNA synthesis, but they potently inhibited TSH-stimulated iodide transport. We conclude that TSH, acting at least in part through cAMP, is a potent growth factor for human thyroid cells and thus provide an experimental basis in vitro for the well established in vivo goitrogenic action of TSH.

Adenosine Monophosphate↗

PubMedDate not supplied

Collagen microcarrier spinner culture promotes osteoblast proliferation and synthesis of matrix proteins.

In vitro propagation of osteoblasts in three-dimensional culture has been explored as a means of cell line expansion and tissue engineering purposes. Studies investigating optimal culture conditions are being conducted to produce bone-like material. This study demonstrates the use of collagen microcarrier beads as a substrate for three-dimensional cell culture. We have earlier reported that microcarriers consisting of cross-linked type I collagen support chondrocyte proliferation and synthesis of extracellular matrix. In this study, we investigated the use of collagen microcarriers to propagate human trabecular bone-derived osteoblasts. Aggregation of cell-seeded microcarriers and production of extracellular matrix-like material were observed after 5 d in culture. Expression of extracellular matrix proteins osteocalcin, osteopontin, and type I collagen was confirmed by messenger ribonucleic acid analysis, radioimmunoassay, and Western blot analysis. The efficient recovery of viable cells was achieved by collagenase digestion of the cell-seeded microcarriers. The collagen microcarrier spinner culture system provides an efficient method to amplify large numbers of healthy functional cells that can be subsequently used for further in vitro or transplantation studies.

Analysis of Variance↗

PubMed2006

Mutagenic exploration of the cross-seeding and fibrillation propensity of Alzheimer's beta-amyloid peptide variants.

Amyloid formation is a nucleation-dependent process that is accelerated dramatically in vivo and in vitro upon addition of appropriate fibril seeds. A potent species barrier can be effective in this reaction if donor and recipient come from different biological species. This species barrier is thought to reflect differences in the amino acid sequence between seed and target polypeptide. Here we present an in vitro mutagenic cross-seeding analysis of Alzheimer's Abeta(1-40) peptide in which we mapped out the effect of systematically varied amino acid replacements on the propensity of seed-dependent amyloid fibril formation. We find that the susceptibility of different peptides toward cross-seeding relates to the intrinsic aggregation propensity of the respective polypeptide chain and, therefore, to properties such as beta-sheet propensity and hydrophobicity. These data imply that the seed-dependent formation of amyloid-like fibrils is affected by the intrinsic properties of the polypeptide chain in a manner that is similar to what has been described previously for aggregation reactions in general. Hence, the nucleus acts in this case as a catalyst that promotes the fibrillation of different polypeptide chains according to their intrinsic structural predilection.

Amyloid↗

PubMed2000

Hepatocyte behavior within three-dimensional porous alginate scaffolds.

A potential approach to facilitate the performance of implanted hepatocytes is to enable their aggregation and re-expression of their differentiated function prior to implantation. Here we examined the behavior of freshly isolated rat adult hepatocytes seeded within a novel three-dimensional (3-D) scaffold based on alginate. The attractive features of this scaffold include a highly porous structure (sponge-like) with interconnecting pores, and pore sizes with diameters of 100-150 microm. Due to their hydrophilic nature, seeding hepatocytes onto the alginate sponges was efficient. DNA measurements showed that the total cell number within the sponges did not change over 2 weeks, indicating that hepatocytes do not proliferate under these culture conditions. Nearly all seeded cells maintained viability, according to the MTT assay. Within 24 h post-seeding, small clusters of viable cells, were seen scattered within the sponge. More than 90% of the seeded cells participated in the aggregation; the high efficiency is attributed to the non-adherent nature of alginate. The spheroids had smooth boundaries and by day 4 in culture reached an average diameter of 100 microm, which is at the same magnitude of the sponge pore size. The cells appeared to synthesize fibronectin which was deposited on the spheroids. No laminin or collagen type IV were detected in the deposit. The 3-D arrangement of hepatocytes within the alginate sponges promoted their functional expression; within a week the cells secreted the maximal albumin secretion rate of 60 microg albumin/10(6) cells/day. Urea secretion rate did not depend on cell aggregation and was similar to that obtained when hepatocytes were cultured on collagen type I coated dishes (100 microg/10(6) cells/day). Our studies show that alginate sponges can provide a conducive environment to facilitate the performance of cultured hepatocytes by enhancing their aggregation.

Albumins↗

PubMed2004

The role of pre-existing aggregates in Hsp104-dependent polyglutamine aggregate formation and epigenetic change of yeast prions.

Amyloid-like protein aggregates have been implicated in various diseases and in the protein-based inheritance of yeast prions. The molecular chaperone Hsp104 has been shown to be necessary for the aggregate formation of polyglutamine in yeast, and for the maintenance of several yeast prion phenotypes through the formation of self-propagating aggregates. In this paper, we show that the polyglutamine aggregates that are formed independently of Hsp104, are required for Hsp104 to efficiently produce more aggregates. Similarly, in the yeast prion [PSI+] system, Hsp104-dependent epigenetic changes to the [PSI+] prion phenotype require the presence of prion aggregates in the normal [psi-] state. We also show that the co-localization of different prion aggregates suggests that cross-seeding by different yeast prions increases the probability of Hsp104-dependent epigenetic change. These findings highlight the role of pre-existing aggregates in chaperone-dependent establishment of the epigenetic trait in yeast prions, and possibly in the pathology of several neurodegenerative diseases.

Epigenesis, Genetic↗

PubMed1993

The effect of pumpkin seeds snack on inhibitors and promoters of urolithiasis in Thai adolescents.

Supplementation of basal mixture or pumpkin seeds snack gave a higher level of inhibitor of crystal formation or aggregation than the control period. Moreover, pumpkin seeds snack showed inhibition effect to a greater extent than basal mixture. In the light of this study, it is suggested that pumpkin seeds snack, a high nutritive mixture, is satisfactory to improve the nutrients and increase the level of inhibitors of crystal formation or aggregation which will subsequently reduce the risk of bladder stone disease in Thailand.

Adolescent↗

PubMed2001

Interaction of metal ions with lupin seed conglutin gamma.

Various metal ions were capable of aggregating and precipitating conglutin gamma, an oligomeric glycoprotein purified from Lupinus albus seeds, at neutral pH values. The most effective metal ions, at 60-fold molar excess to the protein, were Zn2+, Hg2+ and Cu2+; a lower influence on the physical status of conglutin gamma was observed with Cr3+, Fe3+, Co2+, Ni2+, Cd2+, Sn2+, and Pb2+, while Mg2+, Ca2+ and Mn2+ had no effect at all. The insolubilisation of the protein with Zn2+, which is fully reversible, strictly depended on both metal concentration and pH. with middle points of the sharp transitions at three-fold molar excess and pH 6.5, respectively. Conglutin gamma is also fully retained on a metal affinity chromatography column at which Zn2+ and Ni2+ were complexed. A drop of pH below 6.0 and the use of chelating agents, such as EDTA and imidazole, fully desorbed the protein. A slightly lower binding to immobilised Cu2+ and Co2+ and no binding with Mg2+, Cd2+ and Mn2+ were observed. The role of the numerous histidine residues of conglutin gamma in the binding of Zn2+ is discussed.

Cations↗

PubMed2006

Secondary structure and interfacial aggregation of amyloid-beta(1-40) on sodium dodecyl sulfate micelles.

Alzheimer's disease (AD) is characterized by the presence of large numbers of fibrillar amyloid deposits in the form of senile plaques in the brain. The fibrils in senile plaques are composed of 40- and 42-residue amyloid-beta (Abeta) peptides. Several lines of evidence indicate that fibrillar Abeta and especially soluble Abeta aggregates are important in the pathogenesis of AD, and many laboratories have investigated soluble Abeta aggregates generated from monomeric Abeta in vitro. Of these in vitro aggregates, the best characterized are called protofibrils. They are composed of globules and short rods, show primarily beta-structure by circular dichroism (CD), enhance the fluorescence of bound thioflavin T, and readily seed the growth of long fibrils. However, one difficulty in correlating soluble Abeta aggregates formed in vitro with those in vivo is the high probability that cellular interfaces affect the aggregation rates and even the aggregate structures. Reports that focus on the features of interfaces that are important in Abeta aggregation have found that amphiphilic interactions and micellar-like Abeta structures may play a role. We previously described the formation of Abeta(1-40) aggregates at polar-nonpolar interfaces, including those generated at microdroplets formed in dilute hexafluoro-2-propanol (HFIP). Here we compared the Abeta(1-40) aggregates produced on sodium dodecyl sulfate (SDS) micelles, which may be a better model of biological membranes with phospholipids that have anionic headgroups. At both HFIP and SDS interfaces, changes in peptide secondary structure were observed by CD immediately when Abeta(1-40) was introduced. With HFIP, the change involved an increase in predominant beta-structure content and in fluorescence with thioflavin T, while with SDS, a partial alpha-helical conformation was adopted that gave no fluorescence. However, in both systems, initial amorphous clustered aggregates progressed to soluble fibers rich in beta-structure over a roughly 2 day period. Fiber formation was much faster than in the absence of an interface, presumably because of the close intermolecular proximity of peptides at the interfaces. While these fibers resembled protofibrils, they failed to seed the aggregation of Abeta(1-40) monomers effectively.

Amyloid beta-Peptides↗