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Assessment of production of endothelium-derived relaxing factor by cultured porcine cerebral microvessel endothelial cells.

To establish an experimental system to assess the production of endothelium-derived relaxing factor (EDRF) by cultured porcine cerebral microvessel endothelial cells (PCMEC), the effect of PCMEC on thrombin-induced platelet aggregation was studied. For the platelet aggregation cuvettes lined with PCMEC were used. The cuvettes were prepared by seeding PCMEC in gelatin-coated cuvettes at a cell density of 2 x 10(5) cells/ml and culturing for 48 hours. Thrombin-induced platelet aggregation was markedly inhibited in the presence of PCMEC. Indomethacin reduced the PCMEC-dependent anti-platelet aggregatory effect. This PCMEC-dependent anti-platelet aggregatory effect was increased by the addition of bradykinin which stimulates the production of EDRF. These findings suggest that this simple experimental system is useful in assessing the production of EDRF by PCMEC and in examining the effects of various chemicals (or agents) on EDRF production.

Animals↗

Apomyoglobin reveals a random-nucleation mechanism in amyloid protofibril formation.

Protofibrils (PFs) represent the earliest fibrillar species that occur in the course of amyloid fibril formation. Using apomyoglobin, we report here that PFs arise from a multi-step reaction and that they are preceded by an ensemble of non-fibrillar particles (NFPs). These intermediate aggregates encompass nascent elements of amyloid structure and can act as seeds in PF formation. Taken together with the observation that PFs often protrude from NFPs, our data suggest that PFs form by a random nucleation mechanism in which the polypeptide chains sample many different aggregated conformations. Once the appropriate structural characteristics are acquired, PFs are formed by addition of further polypeptide chains.

Amyloid↗

Platelet release reaction and aggregation induced by canatoxin, a convulsant protein: evidence for the involvement of the platelet lipoxygenase pathway.

Canatoxin is a toxic protein isolated from Canavalia ensiformis seeds. It induces death preceded by convulsions of spinal cord origin and also produces in vitro aggregation of platelets in rabbit, human and guinea-pig plasma. The aggregating effect is dose-dependent at nanomolar concentrations. Rabbit platelets pretreated with canatoxin became refractory to a second exposure to this protein or to collagen, but were still responsive to ADP, Paf-acether or arachidonic acid. [14C]-5-hydroxytryptamine was released from pre-labelled platelets on stimulation with canatoxin. Washed rabbit platelets, but not thrombin-degranulated ones, aggregated on stimulation with canatoxin provided that fibrinogen was added before the toxin. Canatoxin's pro-aggregating activity was inhibited by mepacrine, EDTA, caffeine, prostacyclin, adenosine monophosphate and also by the ADP scavenger system, creatine phosphokinase/creatine phosphate. Furthermore, 3-amino-1-[m-(trifluoromethyl)-phenyl]-2-pyrazoline (BW 755C), eicosatetraynoic acid (ETYA) and nordihydroguaiaretic acid (NDGA) were potent inhibitors of canatoxin-induced aggregation. In contrast, no inhibition was seen with indomethacin. The data indicate that canatoxin is mainly a release-reaction-promoting agent, being devoid of any direct aggregating activity. Thus the aggregation is totally dependent on the release of ADP. Furthermore, canatoxin-induced platelet activation is probably dependent on platelet phospholipase A2 and lipoxygenase activity but is not dependent on cyclo-oxygenase products or the release of Paf-acether.

5,8,11,14-Eicosatetraynoic Acid↗

A new organotypic model to study cell interactions in the intestinal mucosa.

BACKGROUND: Recently, we demonstrated that freshly elutriated monocytes differentiate into macrophages with a phenotype similar to that of intestinal macrophages in a three-dimensional model of intestinal epithelial cells. Here we describe a more organotypic model to study cell interactions in the intestinal mucosa. METHODS: Primary intestinal fibroblasts and freshly elutriated blood monocytes (ratio 1:1) were embedded in collagen type I gels and cultured for 5 days. At day 5, intestinal epithelial cells (HT-29) were seeded on top of the collagen gels. After another 7 days collagen gels were harvested and fixed for immunohistochemical analysis. Cryosections of the aggregates were prepared and staining for monocyte/macrophage markers and basement membrane compounds was performed. Cell interactions inside the aggregates were examined by electron microscopy. RESULTS: Intestinal fibroblasts contracted the collagen gels which formed stable three-dimensional aggregates within the first 5 days of culture. Intestinal epithelial cells formed a monolayer on top of the gels about 3 days after seeding. Intestinal fibroblasts were distributed randomly over the aggregate. Monocytes inside aggregates were localized in the vicinity to epithelial cells by positive staining for CD68. Typical monocyte/macrophage specific markers such as CD14, CD16, CD11b, CD11c and the co-stimulatory molecules CD80 and CD86 were down-regulated or not detectable on these cells after co-culture in three-dimensional aggregates. Omission of epithelial cells from the model was followed by impaired differentiation of intestinal macrophages. CONCLUSION: In the three-dimensional organotypic cell culture model monocytes differentiate into intestinal-like macrophages when co-cultured with control intestinal fibroblasts and intestinal epithelial cells. Intestinal epithelial cells may be necessary for differentiation of intestinal macrophages.

Cell Communication↗

Prion-inducing domain 2-114 of yeast Sup35 protein transforms in vitro into amyloid-like filaments.

The yeast non-Mendelian genetic factor [PSI], which enhances the efficiency of tRNA-mediated nonsense suppression in Saccharomyces cerevisiae, is thought to be an abnormal cellular isoform of the Sup35 protein. Genetic studies have established that the N-terminal part of the Sup35 protein is sufficient for the genesis as well as the maintenance of [PSI]. Here we demonstrate that the N-terminal polypeptide fragment consisting of residues 2-114 of Sup35p, Sup35pN, spontaneously aggregates to form thin filaments in vitro. The filaments show a beta-sheet-type circular dichroism spectrum, exhibit increased protease resistance, and show amyloid-like optical properties. It is further shown that filament growth in freshly prepared Sup35pN solutions can be induced by seeding with a dilute suspension of preformed filaments. These results suggest that the abnormal cellular isoform of Sup35p is an amyloid-like aggregate and further indicate that seeding might be responsible for the maintenance of the [PSI] element in vivo.

Amyloid↗

Pressure cell assisted solubilization of xyloglucans: tamarind seed polysaccharide and detarium gum.

To improve the solubilization of two water-soluble xyloglucans, tamarind seed polysaccharide and detarium gum, by reducing substantially molecular aggregation, a "pressure cell" heating method was used. Conditions allowing solubilization and chain depolymerization were produced by varying appropriately the pressure, time, and temperature applied. The various MW fractions of solubilized xyloglucans were characterized by capillary viscometry and light scattering techniques in order to extract, with reliability, fundamental macromolecular parameters. Mark-Houwink and Flory exponents were found to be 0.67 +/- 0.04 and 0.51 +/- 0.06, respectively for both xyloglucan data combined, consistent with linear random coil behavior. A detailed analysis of the data seems to suggest that tamarind gum solutions are slightly perturbed by the effect of excluded volume, whereas detarium gum samples are close to the theta state. Chain flexibility parameters such characteristic ratio, C( proportional, variant ), and persistence length, L(p), were calculated for tamarind and detarium using the Burchard-Stockmayer-Fixman (BSF) geometric method. L(p) values of 6-8 nm were estimated for xyloglucans. The seemingly linear structure of tamarind and detarium, as suggested by the value of the Mark-Houwink and Flory exponents obtained, follows from analysis of the data by the classical Zimm method but not when employing the square root or Berry method which suggests a more branched chain profile. This was the approach adopted in our previous work on the characterization of detarium samples.

Glucans↗

Synaptogenesis in reaggregating brain cell culture.

Dissociated cells from embryonic mouse brain reassociate in rotation culture to form highly organized aggregates. Electron microscopic observations of aggregates show the presence of synapses that mature and increase in number during culture. Apparent myelination of axons is observed in the aggregates after 5 weeks of culture. Thus, brain cell aggregates perform two highly specialized morphological events that are characteristic of mouse brain differentiation.

Animals↗

The early events of alpha-synuclein oligomerization revealed by photo-induced cross-linking.

Assembly of alpha-synuclein (alpha-Syn) into neurotoxic oligomers and fibrils is an important pathogenic feature of Parkinson's disease. Studying the early events of alpha-Syn aggregation, such as oligomerization and nucleation, is indispensable to understanding the complicated process. Here, photo-induced cross-linking of unmodified proteins (PICUP) technique is applied to elucidate the early-stage oligomerization of alpha-Syn. Results show that alpha-Syn in solution exhibits a mixture of various species, including at least monomers, dimers and trimers. Aggregation of alpha-Syn probably originates from the dimeric and trimeric seeds. Furthermore, the N-terminal amphipathic region is proposed to be required for the oligomerization (dimerization and trimerization) process. This observation may extend our knowledge on the early events of alpha-Syn aggregation and the neurotoxic aggregation species.

Cross-Linking Reagents↗

Multistep nucleus formation and a separate subunit contribution of the amyloidgenesis of heat-denatured monellin.

Monellin (MN) is a sweet-tasting plant protein known to form fibrous aggregates in the heat-denatured state. Here the amyloid-type aggregation process of MN is extensively characterized. The amyloidgenesis was initiated in a highly denatured state of MN. A seeding effect of skipping a lag phase of the amyloid formation kinetics established a nucleation-dependent aggregation mechanism. A finely controlled experimental protocol revealed an additional prenucleus stage preceding the maturation of the nucleus, indicating that the initial lag phase is composed of multiple conformational events. The results obtained for the aggregation properties of the separate A and B subunit chains of MN and a recombinant single-chain MN suggest that the B chain exclusively contributed to the amyloid-type aggregation. These findings suggest a scheme for the amyloidgenesis of MN and their subunits, and provide a unique model of amyloidgenesis that is regulated by the subunit composition of protein.

Amyloid↗

Detection of single amyloid beta-protein aggregates in the cerebrospinal fluid of Alzheimer's patients by fluorescence correlation spectroscopy.

Alzheimer's disease is associated with the intraparenchymal growth of plaque-like amyloid deposits. Amyloid plaques are formed by the progressive deposition and transformation of soluble amyloid beta-protein monomers into insoluble and fibrillar aggregates that contain amyloid beta-protein in a beta-pleated sheet conformation. This process is described as 'seeded polymerization' of the monomers with slow-nucleation and fast-growth kinetics. Soluble amyloid beta-protein monomers are present in the cortical extracellular space and in the cerebrospinal fluid, whereas insoluble aggregates so far can be found only by the examination of brain tissue by biopsy or autopsy. Here we present a biophysical method that uses the principle of seeded polymerization in combination with fluorescence correlation spectroscopy, which allowed us to detect single amyloid beta-peptide aggregates in the cerebrospinal fluid samples from Alzheimer's patients. All of 15 Alzheimer's samples but none of the 19 age-matched control samples produced large peaks with fluorescence correlation spectroscopy indicating the rapid aggregation of the fluorescent labelled synthetic amyloid beta-protein probe onto the amyloid beta-protein 'seeds' present in the cerebrospinal fluid. Our method could enable easy in vivo detection of the cerebral amyloid beta-protein pathology of Alzheimer's disease and might be of potential value to facilitate its routine diagnosis.

Aged↗

Synthesis of single-crystalline platinum nanorods within a soft crystalline surfactant-Pt(II) complex.

Single-crystalline platinum nanorods, monodisperse in diameter, are synthesized through a simple process at room temperature, in cetyltrimethyl ammonium bromide (CTAB) solution. The complexation of the CTA+ surfactant ion with tetrachloroplatinate in the presence of hexanol leads to the formation of a precipitate with a lamellar crystalline structure. The reduction of Pt(II) metal ions to Pt(0) is carried out using gamma radiolysis. Transmission electron microscopy (TEM) observations of the nanoparticles extracted from the solution, three weeks after radiolysis, revealed single-crystalline Pt nanorods, monodisperse in diameter (3-4 nm) and 20-60 nm long. By following the shape of the nanorods at various stages of the growth, it was found that the single-crystalline nanorods grow by coalescence of spherical seeds 3-4 nm in diameter. This suggests an aggregative mechanism similar to that recently observed for silver particles in solution.

Journal Article↗

Three-dimensional reconstruction of confocal laser microscopy images to study the behaviour of osteoblastic cells grown on biomaterials.

The adhesion, spreading and cytoskeletal organization of osteoblastic cells seeded onto titanium and titania/hydroxyapatite composite coating (TiO2/HA) were studied using images acquired by confocal laser scanning microscopy. The fluorescence staining technique was employed to visualize actin cytoskeletal organization of cells, 2-D images were exhaustive when the cells were seeded at low density (in the first 24 h of incubation), but they were less clear when the cells proliferated and appeared stacked. Since the shareware software were not satisfactory, a new 3-D image reconstruction was developed using ordinary software and a model was obtained directly from the optical section set, in order to achieve a more realistic and faithful vision of morphological structures and to evaluate the behaviour of bone cells grown on materials. The results showed that the cells grown on titanium conform to the irregular substrate surfaces maximizing the contact between the cell membrane and the substrate and proliferate disposing close to each other. On the contrary, the osteoblasts seeded onto TiO2/HA coating develop clusters where the cells aggregated extending processes in order to establish intercellular connections. Cell aggregation is an early and critical event leading to cell differentiation and mineralization process and could be a first signal of the tendency of TiO2/HA coating to stimulate cell differentiation.

Biocompatible Materials↗

Purified first and third trimester placental trophoblasts differ in in vitro hormone secretion.

In vitro studies with cytotrophoblasts obtained from term placentas have shown low levels of placental protein hormone secretion during the first 2 days in culture, followed by a marked increase during days 3 and 4. Since maternal serum placental hormone levels at term and during the first trimester differ, it is conceivable that cytotrophoblasts from first trimester placentas will differ in endocrine function from those derived from term placentas. Therefore, we examined the secretion of hCG, hCG alpha, human placental lactogen (hPL), and progesterone (P) both in the basal state and after exposure to 8-bromo-cAMP or endogenous cAMP stimulation with cholera toxin in cytotrophoblasts purified by enzymatic dispersion and Percoll gradient centrifugation from four first trimester and four third trimester placentas. At the time of seeding, all cells were mononuclear, and the degrees of aggregation and syncytia formation were similar in first and third trimester trophoblasts during the 4 days in culture. First trimester trophoblasts secreted greater quantities of hCG than did term trophoblasts, while basal secretion of hCG alpha, hPL, and progesterone were similar. Qualitative differences in the hormone secretory patterns were apparent. hCG secretion by first trimester trophoblasts decreased over the 4 days in culture, while the amounts secreted by third trimester trophoblasts increased. hCG alpha levels increased for 2-3 days in first trimester trophoblasts and then decreased, while hCG alpha increased in term trophoblast medium over the 4 days. The ratio of hCG alpha to hCG in media from first and third trimester cultures reflected the relative ratios of these hormones in placental tissue and maternal serum at analogous stages of pregnancy. hPL concentrations in the medium declined between days 3-4 in first trimester cultures, while they increased between days 3-4 in third trimester cultures. The secretory pattern of P was somewhat more erratic. cAMP stimulation led to a similar rise in hCG, hCG alpha, and P secretion in first and third trimester trophoblasts, and a variable response for hPL secretion. These results indicate that the functional activity of placental trophoblasts in culture depends in part upon the age of the placenta from which the cells are derived. The differences may represent intrinsic differences in function or the presence of inhibitory or stimulatory factors of maternal, fetal, or trophoblast origin.

Adult↗

[The effect of kanpou medicine on the growth and aggregation of calcium oxalate crystals in vitro].

The effects of the compositional drugs of kanpou medicine on the growth and aggregation of calcium oxalate crystals were investigated in vitro. In the seeded crystal system, the inhibitory activities were calculated from the change of the number (Ia), total volume (Ig) and fractional volume (I) of crystals determined by a Coulter Multisizer. Among the tested drugs (Takusha, Akyou, Chorei, Bukuryou, Kasseki, Kinsensou, Kagosou), only Takusha and Kagosou had strong inhibitory activities on the growth and aggregation at the concentration of 5 micrograms/ml and 10 micrograms/ml (Ia greater than 80%, Ig, I greater than 90%). No significant correlation was observed between inhibitory activities and alcian blue precipitable polyanions (ABPP) of each drug. Thus some naturally existing substances composing the kanpou medicine may help prevent the stone formation in clinical use.

Calcium Oxalate↗

Assessment of production of endothelium-derived relaxing factor (EDRF) by cultured human vascular endothelial cells based on its anti-aggregatory effect on human platelets.

In order to establish an experimental system to assess the production of endothelium-derived relaxing factor (EDRF) by cultured human umbilical vascular endothelial cells (HUVECs), the effect of endothelial cells on thrombin-induced platelet aggregation was examined. Cultured HUVECs were harvested from umbilical veins by collagenase treatment. The platelet aggregation experiments were performed using cuvettes lined with HUVECs. The cuvettes were prepared by seeding HUVECs in gelatin-coated cuvettes at a cell density of 2 x 10(5) cells/ml and culturing for 48 hours. Thrombin-induced platelet aggregation was inhibited in the presence of HUVECs. This HUVEC-dependent anti-platelet aggregatory effect was enhanced by the addition of bradykinin, which stimulates the production of EDRF, and thrombin-induced platelet aggregation was completely inhibited. Indomethacin reduced the HUVEC-dependent anti-platelet aggregatory effect. These findings suggest that this simple, new experimental system is useful in assessing the production of EDRF by HUVECs and in examining the effects of various chemicals (or agents) on EDRF production.

Biological Assay↗

[Study on chondrogenesis of rabbit bone marrow-derived mesenchymal stem cells by centrifuge-tube culture in vitro].

OBJECTIVE: To investigate the feasibility of constructing tissue engineered cartilage by inducing bone marrow-derived mesenchymal stem cells (MSCs) in centrifuge-tube culture. METHOD: By Percoll density gradient centrifuge and monolaryer culture in vitro MSCs were isolated from rabbit bone marrow and were purified. Some MSCs were seeded into centrifuge tube culture and allowed to form three-dimensional aggregates in a chemically defined medium mainly including transforming growth factor- b1 (TGF-beta1, 10 ng/ml), dexamethasone (10-7), ascorbic acid (50 microg/ml), 1% insulin, 1% selenium and 1% transferring. At day 7, 14, 21, aggregates were take out and specific markers of differentiation of cartilage were detected by the appearance of toluiding blue merachromasia and the immunohistochemistry detection of type II collagen and in situ hybridization detection of collagen type II mRNA. RESULT: MSCs seeded into centrifuge tube culture became analogous cartilage tissue after 7-21 days, their specific markers of chondrogeneses were positive in the appearance of toluiding blue merachromasia and the immunohistochemistry detection of type II collagen and in situ hybridization detection of collagen type II mRNA. CONCLUSION: MSCs have the capacity to undergo chondrogenic differentiation.

Animals↗

Spectroscopic analysis of the cytoagglutinating activity of abrin-b isolated from Abrus precatorius seeds against leukemic cells.

The cytoagglutinating activity of abrin-b, a toxic lectin isolated from Abrus precatorius seeds, against cultured cell strains derived from acute lymphoblast leukemia (ALL) was investigated by visible (VIS) spectroscopy. Upon addition of abrin-b, the turbidity at 600 nm of cell suspension decreased and this change could be recorded as the cytoagglutination curve. From this curve, the cytoagglutination velocity (CV) and cytoagglutination intensity (CI) of each cell strain was measured. Each cell strain showed the respective CV and CI values and the cell strains derived from the T cell line were strongly agglutinated by abrin-b compared with those derived from the B cell line. Further, it has become apparent that the cytoagglutinating activity increased with an increase in the order of the differentiation of cell strains.

Abrin↗