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PubMed1986

The effect of monosodium urate on the capacity of urine, chondroitin sulphate and heparin to inhibit calcium oxalate crystal growth and aggregation.

In order to investigate the possibility of interaction between urinary glycosaminoglycans and particulate monosodium urate, growth and aggregation rates of calcium oxalate seed crystals were measured in the presence of heparin, chondroitin sulphate and normal urine after preincubation with and without monosodium urate (0.5 mg./ml.). Rates of crystal aggregation in the presence of heparin and chondroitin sulphate were significantly (p less than 0.0005) increased after pretreatment with sodium urate, indicating a reduction in the inhibitory potency of heparin and chondroitin sulphate by this salt. A similar effect was observed with the rate of crystal growth in the presence of heparin (p less than 0.01). In contrast, the rate of growth in the presence of chondroitin sulphate was unaffected. Pretreatment with sodium urate had an inconsistent effect on the rate of crystal growth in the presence of 10 normal human urine samples, but significantly (p less than 0.001) raised the rate of crystal aggregation. It was concluded that the influence of urate on crystal aggregation in the presence of urine may be a result of its binding to endogenous chondroitin sulphate, but may not be of practical consequence in the pathogenesis of calcium oxalate renal stones.

Calcium Oxalate↗
PubMed1998

Co-culture of two MDCK strains with distinct junctional protein expression: a model for intercellular junction rearrangement and cell sorting.

Distinct epithelial MDCK cell strains displaying extremes in transepithelial electrical resistance (paracellular permeability) have been established in co-culture and the subsequent cellular behaviour and formation of junctional complexes investigated. After high-density seeding, MDCK strain I and II cells in co-culture are initially randomly distributed but subsequently sort themselves out in a time-dependent manner to form separate homotypic aggregates. The final pattern of cell arrangement of homotypic aggregates depends on the relative seeding proportion of each cell type. Immunostaining of established marker proteins for junctional complexes has revealed that MDCK I and II cells differ in the degree of expression of the zonula-adherens-associated protein, E-cadherin, their cytoskeletal architecture and the junctional distribution of a desmosomal protein, and by showing subtle differences in tight junction staining for the zona-occludens-associated proteins, ZO-1 and occludin. The distinct pattern of junctional protein expression is maintained when the two MDCK strains are co-cultured; however, morphologically atypical intercellular junctions between heterotypic cells at the boundary of homotypic cell aggregates have been observed. It has been suggested that cell sorting, a phenomenon yet to be completely understood, is involved in important morphogenetic processes. We propose that co-culture of strains of the well-characterised MDCK cell line may be a novel but well-defined cell system for studying epithelial cell rearrangement and sorting in intact epithelial sheets.

Animals↗
PubMed2003

High density of immobilized galactose ligand enhances hepatocyte attachment and function.

Galactosylated surface is an attractive substrate for hepatocyte culture because of the specific interaction between the galactose ligand and the asialoglycoprotein receptor on hepatocytes. In this study, we described a scheme to achieve high density of immobilized galactose ligands on polyethylene terephthalate (PET) surface by first surface-grafting polyacrylic acid on plasma-pretreated PET film under UV irradiation, followed by conjugation of a galactose derivative (1-O-(6'-aminohexyl)-D-galactopyranoside) to the grafted polyacrylic acid chains. A high galactose density of 513 nmol/cm(2) on the PET surface was used in this study to investigate the behavior of cultured hepatocyte. This engineered substrate showed high affinity to fluorescein isothiocyanate-lectin binding. Primary rat hepatocytes, when seeded at a density of 2 x 10(5) cells/cm(2), attached to the galactosylated PET substrate at a similar efficiency compared with collagen-coated substrate. The hepatocytes spontaneously formed aggregates 1 day after cell seeding and showed better maintenance of albumin secretion and urea synthesis functions than those cultured on collagen-coated surface.

Acrylic Resins↗
PubMed1996

Effects of growth-factor-enhanced culture on a chondrocyte-collagen implant for cartilage repair.

The effects of incubation and addition of growth factors to a chondrocyte-seeded collagen implant for cartilage repair were studied. Type I collagen matrices seeded with lapine articular chondrocytes and unseeded controls cultured in the presence and absence of fibroblast growth factor and insulin for 2, 6, and 9 weeks were subjected to biomechanical, biochemical, and histological analysis. Aggregate modulus of elasticity of seeded implants decreased by half at 6 weeks, then rose by a factor of 10 above initial values. Permeability of seeded implants and their controls decreased steadily. Glycosaminoglycan content peaked at 6 weeks, coinciding with the greatest number of chondrocytes and mitotic activity in seeded implants. Chondrocytes remained phenotypically stable and metabolically active; they incorporated glycosaminoglycan into the extracellular matrix, and formed an organized pericellular environment despite the predicted resorption of the collagen matrix. Adding fibroblast growth factor and insulin tripled the rate of cell turnover and doubled the glycosaminoglycan content of seeded implants, but had no effect on their material properties. In vitro incubation for 6 weeks in the presence of fibroblast growth factor and insulin creates a metabolically and mitotically active chondrocyte-collagen composite for implantation into articular cartilage defects.

Analysis of Variance↗
PubMed1987

In vitro culture and labeling of neural cell aggregates followed by transplantation.

Septal, cortical, or whole brain fetal (E15-17) cells were dissociated and cultured in serum-supplemented Dulbecco minimum essential medium under rotating culture conditions. Preincubation and exposure to cytosine arabinoside was utilized to produce "neuron-rich" cultures. Fluorescent latex microbeads were added to cultures at seeding time or early after aggregate formation. All cell types were found to incorporate the fluorescent beads, although apparently not to the same extent. Two- to five-day-old aggregates tended to attach and grow neurites after their transfer to poly-l-lysine- or Matrigel-coated dishes under stationary conditions. Early aggregates transplanted to the hippocampus of adult rats developed into identifiable grafts, with fluorescent-labeled cells. We conclude that "young" neural cell aggregates maintain their ability to undergo two basic phenomena for cellular interaction, i.e., attachment and neuritic growth. Floating aggregates may provide a convenient cellular condition whenever culturing of neural cells is to be used before grafting them into a host animal.

Animals↗
PubMed2003

The role of osteopontin on calcium oxalate crystal formation.

OBJECTIVE: We evaluated whether osteopontin (OPN) and other proteins with the RGD sequence as in OPN (RGD family proteins) that are present in renal tubular cells (fibronectin [FN], Tamm-Horsfall glycoprotein [THP], vitronectin [VN], and laminin [LN]) inhibit the aggregation and growth of calcium oxalate (CaOx) crystals by a novel seed crystal method using collagen granules (CG) with and without OPN adhered on the surface. We also evaluated the effect of solid phase OPN, FN and THP in which the relationship between their proteins and CaOx crystallization was reported. Moreover, the state and time-course changes in CaOx crystals adhered to CG were observed under scanning electron microscopy (SEM). METHODS: The inhibitory activity (IA) on the aggregation and growth of CaOx crystals was measured in vitro by the conventional seed crystal method using isotopes. In this study, the following nine samples were used: OPN alone; FN alone; THP alone; VN alone; LN alone; CG alone; and CG with OPN, FN, or THP adhered on the surface (OPN/FN/THP-immobilized CG). In addition, the state and time-course changes in CaOx crystals adhered to CG were evaluated by SEM. RESULTS: Using the conventional seed crystal method, the following values of IA were obtained: 91.7% (37.5 micro g/ml) for OPN, 5.0% (100 micro g/ml) for FN, 2.0% (100 micro g/ml) for THP, 3.0% (100 micro g/ml) for VN, and 1.0% (100 micro g/ml) for LN. However, the value of IA obtained by our seed crystal method using CG was 92.1% (180cm(2)/5ml PBS) when CG alone was used. Although the value of IA was decreased by 33.6% when OPN-immobilized CG was used, it did not significantly change when FN/THP-immobilized CG was used. When CG alone was used, the evaluation of CaOx crystallization by SEM demonstrated mild adherence and aggregation of CaOx crystal suspension (seed crystals) on the CG surface, although newly formed crystals only slightly adhered to the CG surface. When OPN-immobilized CG was used, marked adherence and aggregation of seed crystals were observed, in addition to the relatively increased adherence of newly formed crystals. When FN/THP-immobilized CG was used, newly formed crystals only slightly adhered to the CG surface, although the degree of seed crystal adherence and aggregation did not significantly change. CONCLUSIONS: These findings suggest that the immobilization of OPN to the CG surface enhances the adherence and aggregation of seed crystals, as well as enhancing the adherence of newly formed crystals, resulting in decreased IA of CG (overall promotion of crystal deposition). Therefore, the results of this study clarified that OPN enhances the formation and aggregation of CaOx crystals in this experimental system.

Calcium Oxalate↗
PubMed1993

Modified procedure of monolayer culture of human pituitary adenoma cells on microporous membrane coated with extracellular matrix.

This study was designed to establish an in vitro model system in human hormone-producing pituitary adenoma which is analogous to the in vivo cellular environment. Mechanically dispersed cells composed of single cells and aggregated cells from 9 pituitary adenomas (5 GH-producing adenomas and 4 prolactinomas) were cultured on microporous membrane coated with basement membrane extract. Not only single cells but also large aggregated cells which usually float in the medium when seeded on ordinary plastic flasks were morphologically flattened and firmly attached to coated microporous membrane within a few days under the control of seeding medium volume. In both types of adenoma, surviving aggregated adenoma cells revealed well-preserved hormonal activity with regard to both cell morphology and hormone release after 6 m in culture and no undifferentiation of cell characteristics was observed. It is concluded that this culture method may further aid the investigation of in vitro cellular structure and function in human pituitary adenoma under conditions which closely mimic the in vivo cellular environment.

Adenoma↗
PubMed1993

Effect of proteoglycans on hydroxyapatite growth in vitro: the role of hyaluronan.

The effect of cartilage proteoglycans on HA seed crystal growth was studied using a system providing constant supersaturation with respect to HA. The monomers were much less effective than the aggregates in reducing the rate of HA growth, which correlates with their affinity for the HA crystals. Hyaluronan, which is a normal constituent of the proteoglycan aggregates, behaved as a strong inhibitor of HA seed crystal growth and had an affinity constant similar to that of proteoglycan aggregates. The results indicate that inhibition of HA seed crystal growth is mediated through the interaction of hyaluronan with HA crystal surface and that the proteoglycans add to the volume of the adsorbate causing steric hindrance.

Animals↗
PubMed2006

On the seeding and oligomerization of pGlu-amyloid peptides (in vitro).

Oligomerization of amyloid beta (Abeta) peptides is the decisive event in the development of Alzheimer's disease (AD), the most common neurogenerative disorder in developed countries. Recent evidence links this conformation-driven process to primary- and secondary-structure modifications of Abeta. The N and C terminus of deposited Abeta has been shown to possess conspicuous heterogeneity. While the C-terminally longer form of Abeta, i.e., Abeta (42), is considered more amyloidogenic, the role of the N-terminal modifications, e.g., truncation and glutamate cyclization accounting for the majority of the deposited peptides, is less understood. In the present study, we characterized the oligomerization and seeding capacity of pGlu-amyloid peptides using two unrelated techniques based on flow cytometry or flourescence dye binding. Under different conditions and irrespective of the C terminus of Abeta, i.e., Abeta40 or 42, pGlu-modified peptides displayed an up to 250-fold accelerated initial formation of aggregates compared to unmodified Abeta. The accelerated seed formation is accompanied by a change in the oligomerization kinetics because of N-terminal pGlu formation. Furthermore, the formation of mixed aggregates consisting of either pGlu-Abeta (3-42) or ADan or ABri and Abeta (1-42) was investigated by Abeta fluorescence labeling in flow cytometry. The results suggest that pGlu-modified peptides are potential seeding species of aggregate formation in vivo. The data presented here and the abundance of pGlu peptides in amyloidoses, such as FBD and AD, suggest pGlu-amyloid peptides as a species with biophysical characteristics that might be in particular crucial for the initiation of the disease.

Amyloid beta-Peptides↗
PubMed2006

The effects of plant distribution and frugivore density on the scale and shape of dispersal kernels.

For many plant species, seed dispersal is one of the most important spatial demographic processes. We used a diffusion approximation and a spatially explicit simulation model to explore the mechanisms generating seed dispersal kernels for plants dispersed by frugivores. The simulation model combined simple movement and foraging rules with seed gut passage time, plant distribution, and fruit production. A simulation experiment using plant spatial aggregation and frugivore density as factors showed that seed dispersal scale was largely determined by the degree of plant aggregation, whereas kernel shape was mostly dominated by frugivore density. Kernel shapes ranged from fat tailed to thin tailed, but most shapes were between an exponential and that of the solution of a diffusion equation. The proportion of dispersal kernels with fat tails was highest for landscapes with clumped plant distributions and increased with increasing number of dispersers. The diffusion model provides a basis for models including more behavioral details but can also be used to approximate dispersal kernels once a diffusion rate is estimated from animal movement data. Our results suggest that important characteristics of dispersal kernels will depend on the spatial pattern of plant distribution and on disperser density when frugivores mediate seed dispersal.

Animals↗
PubMed2002

Antithrombotic lipid minor constituents from vegetable oils. Comparison between olive oils and others.

Many epidemiological studies suggest that vegetable oils and especially olive oil present a protective effect against atherosclerosis. In this study, total lipids (TL) of Greek olive oils and seed oils of four kinds, namely, soybean, corn, sunflower, and sesame oil, were separated into total polar lipids (TPL) and total neutral lipids (TNL) via a novel extraction procedure. TPL and TNL of olive oil were fractionated by HPLC for further study. Each lipid fraction from HPLC separation along with TL, TPL, and TNL lipid samples from oils were tested in vitro for their capacity to induce or to inhibit washed rabbit platelet aggregation. Comparison between olive and seed oils supports the superiority of olive oil as high levels of platelet activating factor (PAF) antagonists have been detected, mainly in TPL. In addition, the structure of the most active fraction from olive oil was elucidated, as a glycerol-glycolipid. Because it has already been reported that PAF plays a pivotal role in atherogenesis, the existence of PAF agonists and antagonists in vegetable oils may explain their protective role against atherosclerosis.

Acetylation↗
PubMed2006

Kinetic studies of protein L aggregation and disaggregation.

We have investigated the aggregation of protein L in 25% (vol/vol) TFE and 10 mM HCl. Under both conditions, aggregates adopt a fibrillar structure and bind dyes Congo Red and Thioflavin T consistent with the presence of amyloid fibrils. The kinetics of aggregation in 25% TFE suggest a linear-elongation mechanism with critical nucleus size of either two or three monomers. Aggregation kinetics in 10 mM HCl show a prolonged lag phase prior to a rapid increase in aggregation. The lag phase is time-dependent, but the time dependence can be eliminated by the addition of pre-formed seeds. Disaggregation studies show that for aggregates formed in TFE, aggregate stability is a strong function of aggregate age. For example, after 200 min of aggregation, 40% of the aggregation reaction is irreversible, while after 3 days over 60% is irreversible. When the final concentration of the denaturant, TFE, is reduced from 5% to 0, the amount of reversible aggregation doubles. Disaggregation studies of aggregates formed in TFE and 10 mM HCl reveal a complicated effect of pH on aggregate stability.

Amyloidosis↗
PubMed2001

Statistical mechanics of prion diseases.

We present a two-dimensional, lattice based, protein-level statistical mechanical model for prion diseases (e.g., mad cow disease) with concomitant prion protein misfolding and aggregation. Our studies lead us to the hypothesis that the observed broad incubation time distribution in epidemiological data reflect fluctuation dominated growth seeded by a few nanometer scale aggregates, while much narrower incubation time distributions for innoculated lab animals arise from statistical self-averaging. We model "species barriers" to prion infection and assess a related treatment protocol.

Animals↗
PubMed1988

[Inhibitory effect of CG-120 on the formation, growth and aggregation of calcium oxalate crystals in vitro].

The inhibitory effect of CG-120 on the formation, growth and aggregation of calcium oxalate (CaOx) crystals was estimated by the use of Coulter counter TA II in the whole urine system and seed crystal method. In the whole urine system, after determining the metastable limit, the formation and growth of calcium oxalate crystals precipitated in response to a load of sodium oxalate was measured. CG-120 had a strong inhibitory effect on the formation and growth above the concentration of 500 micrograms/ml. In the seeded crystal system, inhibitory activity of aggregation (Ia) and growth (Ig) were calculated from the change in the number and volume of crystals. Above 250 micrograms/ml, CG-120 inhibited the aggregation and growth of added CaOx monohydrate and dihydrate crystals. CG-120 therefore might be useful for treating the recurrent stone formers.

Calcium Oxalate↗
PubMed1989

[Urinary material affecting calcium oxalate monohydrate stone formation].

We previously reported that the low molecular and uronic acid-rich fractions in the urine from calcium oxalate stone formers promoted aggregation of calcium oxalate monohydrate seed crystals. In this study, we have demonstrated that lyophilized material of the fractions contains hyaluronic acid as a sole glycosaminoglycans, as well as acidic amino acid-rich proteins and urinary pigment which is supposed to combine with protein. It is known that hyaluronic acid is present in stone matrix, that calcium containing stones contain proteins rich in acidic amino acids, and that the external color of calcium oxalate monohydrate calculi is usually brownish. These facts correspond with our present results. Therefore, it is suggested that the urinary material promotes the calcium oxalate crystal aggregation, sticks the crystals together and is incorporated into the stone.

Calcium Oxalate↗
PubMed2004

Characterization of long-term mouse brain aggregating cultures: evidence for maintenance of neural precursor cells.

An extensive characterization of fetal mouse brain cell aggregates has been performed using immunohistochemical and stereological methods. Single cell suspensions from mechanically dissociated cortex and hippocampus were cultured in serum-free, B27-supplemented medium under constant gyratory agitation for up to 56 days. Three-dimensional aggregates started to form immediately after seeding and reached a final average size of 500 microm in diameter. Among the cell types identified, neurons were the most abundant cells in the aggregates, followed by astrocytes, microglia, and oligodendrocytes. Western blotting for synaptophysin and immunostaining for neurotransmitter-related molecules indicated the presence of well-defined phenotypic characteristics of the neurons in this culture system, suggesting functionality. Proliferating cells, many with neural precursor cell properties, were seen throughout the culture period and could be isolated from the aggregates even after 2 months in culture. Neural precursor cells were isolated from the aggregates after more than 1 month in culture; these cells were successfully differentiated into neurons, astrocytes, and oligodendrocytes. The aggregate culture system may provide a versatile tool for molecular dissection of processes identified in mouse models, including transgenic animals and manipulation of neural precursor cells.

Animals↗
PubMed1970

Viral aggregation resulting in the failure to correctly identify an unknown rhinovirus.

A seed lot of strain SF 1684 of rhinovirus type 2 prepared in human embryonic lung cells (WI-38) contained aggregates which interfered with its neutralization by homotypic or homologous antisera. The same virus showed no evidence of aggregation at five other passage levels studied. Virus in the seed lot was not identified correctly, and the titer of homologous antiserum was mistakenly considered to be low as a result of neutralization tests conducted with the aggregated virus. Filtration and a more easily effected treatment with sodium deoxycholate (1%) disaggregated the virus and restored its susceptibility to neutralization by homologous and homotypic antiserum.

Animals↗