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At least 649 records · Page 36Linked to original sources
PubMed1991

Retinoic acid treatment induces type X collagen gene expression in cultured chick chondrocytes.

The vitamin A derivative retinoic acid (RA) is widely thought to be involved in cartilage development, but its precise roles and mechanisms of action in this complex process remain unclear. We have tested the hypothesis that RA is involved in chondrocyte maturation during endochondral ossification and, in particular, is an inducer of maturation-associated traits such as type X collagen and alkaline phosphatase. Immature chondrocytes isolated from the caudal region of Day 19 chick embryo sterna were seeded in secondary monolayer cultures and treated either with a high dose (100 nM) or with physiological doses (10-35 nM) of RA for up to 3 days. We found that after an initial lag of about 24 h, physiological doses of RA indeed induced type X collagen gene expression in the immature cells. This induction was not accompanied by obvious changes in expression of the type II collagen and large aggregating proteoglycan core protein genes. As revealed by immunocytochemistry, 30-35% of the cells in cultures treated with RA for 3 days were engaged in type X collagen production. Interestingly, these cells were relatively similar in size to chondrocytes in which no type X collagen was detected, suggesting that chondrocytes can initiate type X collagen production independent of cell hypertrophy. RA treatment also led to increased alkaline phosphatase activity occurring as early as 24 h after the start of treatment. The data in this study indicate that RA may have a role in endochondral ossification as an inducer/promoter of maturation-associated traits during chondrocyte maturation.

Alkaline Phosphatase↗
PubMed2004

Oleosins of Arabidopsis thaliana: expression in Escherichia coli, purification, and functional properties.

The interfacial behavior of oleosins, the most abundant proteins from seeds oil bodies, was investigated using the pendant drop method at water/oil interfaces and compared to the behavior of beta-casein and lysozyme, proteins with contrasted emulsifying properties. Recombined high (rS3) and low (rS4) molecular weight oleosins comprising N-terminal histidine tags were purified to electrophoretic homogeneity. rS3 decreased the interfacial tension at the oil/water interface better than rS4, oleosins being more efficient than beta-casein. Oleosins formed aggregates when spread on noncompressed phospholipid (PL) films at the air/water interface as observed using a Langmuir-Blodgett balance equipped with a Brewster angle microscope. Oleosin spread at the surface of a compressed PL monolayer (5-20 mN/m) did not aggregate. Pressure increased immediately and proportionally to the amount of protein spread on the monolayer. The results stress the capacity of oleosins to be inserted in oil and in PL monolayers, which is of particular relevancy to their potential uses as water/oil emulsifiers.

Arabidopsis Proteins↗
PubMed1998

Kinetic analysis of integrin-dependent cell adhesion on vitronectin--the inhibitory potential of plasminogen activator inhibitor-1 and RGD peptides.

Distinct binding interactions between cell-surface receptors and extracellular matrix components are characteristic of multifunctional adhesion proteins such as vitronectin. The close proximity of binding sites for alpha(v)-integrins and plasminogen activator inhibitor-1 (PAI-1) on vitronectin may have consequences for cell adhesion and migration, or for the localized inhibition of plasminogen activators. In this study, the kinetics and reversibility of vitronectin-dependent cell adhesion via alpha(v)-integrins was investigated using RGD peptides and PAI-1 as competitors. Active, but not latent or cleaved PAI-1, and RGD peptides were effective in preventing cell adhesion to vitronectin provided the inhibitor was present at the time of cell seeding. In a concentration-dependent manner urokinase or thrombin abrogated the inhibitory effect of PAI-1. Following cell seeding onto a vitronectin substratum, delayed addition of RGD peptides or active PAI-1 (10-20 min post-seeding) resulted in the loss of their inhibitory potential. These data were supported by experiments in a purified system where delayed addition of active PAI-1 could no longer prevent vitronectin binding to immobilized alpha(v)beta3, while a cyclic RGD peptide gave some moderate inhibition. The apparent stabilization of vitronectin-integrin contacts was observed with immobilized native or multimeric vitronectin but not with the more rigid form of denatured, aggregated multimers. These results demonstrate that the cell adhesive properties of vitronectin depend on its conformational flexibility and can be tightly regulated in a spatio-temporal manner through direct competition of cellular integrins by soluble or matrix-bound factors such as PAI-1.

Amino Acid Sequence↗
PubMed2003

Oscillating perfusion of cell suspensions through three-dimensional scaffolds enhances cell seeding efficiency and uniformity.

We developed a bioreactor for automated cell seeding of three-dimensional scaffolds by continuous perfusion of a cell suspension through the scaffold pores in oscillating directions. Using quantitative biochemical and image analysis techniques, we then evaluated the efficiency and uniformity of perfusion seeding of Polyactive foams as compared to conventional static and spinner flask methods. Finally, we assessed the efficacy of the perfusion seeding technique for different scaffolds and cell types. Perfusion seeding of chondrocytes into Polyactive foams resulted in "viable cell seeding efficiencies," defined as the percentages of initially loaded cells that were seeded and remained viable, that were significantly higher (75 +/- 6%) than those by static (57% +/- 5%) and spinner flask seeding (55% +/- 8%). In addition, as compared to static and spinner flask methods, cells seeded by perfusion were respectively 2.6-fold and 3.8-fold more uniformly distributed and formed more homogeneously sized cell clusters. Chondrocytes seeded by perfusion into Hyaff-11 nonwoven meshes were 26% and 63%, respectively, more uniformly distributed than following static and spinner flask seeding. Bone marrow stromal cells seeded by perfusion into ChronOS porous ceramics were homogeneously distributed throughout the scaffold volume, while following the static method, cells were found only near the top surface of the ceramic. In summary, we demonstrated that our cell seeding perfusion bioreactor generated constructs with remarkably uniform cell distributions at high efficiencies, and was effective for a variety of scaffolds and different mesenchymal cell types.

Animals↗
PubMed2006

Structure-specific effects of protein topology on cross-beta assembly: studies of insulin fibrillation.

Systemic amyloidoses, an important class of protein misfolding diseases, are often due to fibrillation of disulfide-cross-linked globular proteins otherwise unrelated in sequence or structure. Although cross-beta assembly is regarded as a universal property of polypeptides, it is not understood how such amyloids accommodate diverse disulfide connectivities. Does amyloidogenicity depend on protein topology? A model is provided by insulin, a two-chain protein containing three disulfide bridges. The importance of chain topology is demonstrated by mini-proinsulin (MP), a single-chain analogue in which the C-terminus of the B chain (residue B30) is tethered to the N-terminus of the A chain (A1). The B30-A1 tether impedes the fiber-specific alpha --> beta transition, leading to slow formation of a structurally nonuniform amorphous precipitate. Conversely, fibrillation is robust to interchange of disulfide bridges. Whereas native insulin exhibits pairings [A6-A11, A7-B7, and A20-B19], metastable isomers with alternative pairings [A6-B7, A7-A11, A20-B19] or [A6-A7, A11-B7, A20-B1] readily undergo fibrillation with essentially identical alpha --> beta transitions. Respective pairing schemes are in each case retained. Isomeric fibrils and the amorphous MP precipitate are each able to seed the fibrillation of wild-type insulin, suggesting a structural correspondence between respective nuclei or modes of assembly. Together, our results demonstrate that effects of polypeptide topology on amyloidogenicity depend on structural context. Although the native structures and stabilities of single-chain insulin analogues are similar to those of wild-type insulin, the interchain tether constrains the extent of conformational distortion at elevated temperature, retards initial non-native aggregation, and is apparently incompatible with the mature structure of an insulin protofilament. We speculate that the general danger of fibrillation has imposed a constraint in protein evolution, selecting for topologies unfavorable to amyloid formation.

Amyloid↗
PubMed2002

Elicitation of quantitative data from a heterogeneous expert panel: formal process and application in animal health.

This paper presents a protocol for a formal expert judgment process using a heterogeneous expert panel aimed at the quantification of continuous variables. The emphasis is on the process's requirements related to the nature of expertise within the panel, in particular the heterogeneity of both substantive and normative expertise. The process provides the opportunity for interaction among the experts so that they fully understand and agree upon the problem at hand, including qualitative aspects relevant to the variables of interest, prior to the actual quantification task. Individual experts' assessments on the variables of interest, cast in the form of subjective probability density functions, are elicited with a minimal demand for normative expertise. The individual experts' assessments are aggregated into a single probability density function per variable, thereby weighting the experts according to their expertise. Elicitation techniques proposed include the Delphi technique for the qualitative assessment task and the ELI method for the actual quantitative assessment task. Appropriately, the Classical model was used to weight the experts' assessments in order to construct a single distribution per variable. Applying this model, the experts' quality typically was based on their performance on seed variables. An application of the proposed protocol in the broad and multidisciplinary field of animal health is presented. Results of this expert judgment process showed that the proposed protocol in combination with the proposed elicitation and analysis techniques resulted in valid data on the (continuous) variables of interest. In conclusion, the proposed protocol for a formal expert judgment process aimed at the elicitation of quantitative data from a heterogeneous expert panel provided satisfactory results. Hence, this protocol might be useful for expert judgment studies in other broad and/or multidisciplinary fields of interest.

Animal Welfare↗
PubMed2004

Bacterial diversity and function of aerobic granules engineered in a sequencing batch reactor for phenol degradation.

Aerobic granules are self-immobilized aggregates of microorganisms and represent a relatively new form of cell immobilization developed for biological wastewater treatment. In this study, both culture-based and culture-independent techniques were used to investigate the bacterial diversity and function in aerobic phenol- degrading granules cultivated in a sequencing batch reactor. Denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA genes demonstrated a major shift in the microbial community as the seed sludge developed into granules. Culture isolation and DGGE assays confirmed the dominance of beta-Proteobacteria and high-G+C gram-positive bacteria in the phenol-degrading aerobic granules. Of the 10 phenol-degrading bacterial strains isolated from the granules, strains PG-01, PG-02, and PG-08 possessed 16S rRNA gene sequences that matched the partial sequences of dominant bands in the DGGE fingerprint belonging to the aerobic granules. The numerical dominance of strain PG-01 was confirmed by isolation, DGGE, and in situ hybridization with a strain-specific probe, and key physiological traits possessed by PG-01 that allowed it to outcompete and dominate other microorganisms within the granules were then identified. This strain could be regarded as a functionally dominant strain and may have contributed significantly to phenol degradation in the granules. On the other hand, strain PG-08 had low specific growth rate and low phenol degradation ability but showed a high propensity to autoaggregate. By analyzing the roles played by these two isolates within the aerobic granules, a functional model of the microbial community within the aerobic granules was proposed. This model has important implications for rationalizing the engineering of ecological systems.

Aerobiosis↗
PubMed2006

Rapid generation of single-tumor spheroids for high-throughput cell function and toxicity analysis.

Spheroids are widely used in biology because they provide an in vitro 3-dimensional (3D) model to study proliferation, cell death, differentiation, and metabolism of cells in tumors and the response of tumors to radiotherapy and chemotherapy. The methods of generating spheroids are limited by size heterogeneity, long cultivation time, or mechanical accessibility for higher throughput fashion. The authors present a rapid method to generate single spheroids in suspension culture in individual wells. A defined number of cells ranging from 1000 to 20,000 were seeded into wells of poly-HEMA-coated, 96-well, round-or conical-bottom plates in standard medium and centrifuged for 10 min at 1000 g. This procedure generates single spheroids in each well within a 24-h culture time with homogeneous sizes, morphologies, and stratification of proliferating cells in the rim and dying cells in the core region. Because a large number of tumor cell lines form only loose aggregates when cultured in 3D, the authors also performed a screen for medium additives to achieve a switch from aggregate to spheroid morphology. Small quantities of the basement membrane extract Matrigel, added to the culture medium prior to centrifugation, most effectively induced compact spheroid formation. The compact spheroid morphology is evident as early as 24 h after centrifugation in a true suspension culture. Twenty tumor cell lines of different lineages have been used to successfully generate compact, single spheroids with homogenous size in 96-well plates and are easily accessible for subsequent functional analysis.

Biocompatible Materials↗
PubMed1998

Ascaris lumbricoides intensity in relation to environmental, socioeconomic, and behavioral determinants of exposure to infection in children from southeast Madagascar.

Ascaris lumbricoides worm counts were examined as the outcome products of exposure proxy variables. A survey of 663 children, 4-10 yr old, living in southeastern Madagascar revealed prevalences of 93% for A. lumbricoides, 55% for Trichuris trichiura, and 27% for hookworm. Worm expulsions were conducted on 428 of these children; the data revealed an overdispersed distribution of A. lumbricoides, with an arithmetic mean of 19.2 worms per child. A concurrent socioeconomic household survey was conducted by visitation and interview. Exposure to infection was assessed by environmental, demographic, behavioral, and socioeconomic indicators. Ascaris lumbricoides aggregations were associated with gender, housing style, ethnicity, and agricultural factors. The results suggest that exposure and infection are ubiquitous in this child population, and that A. lumbricoides intensity is influenced by gender-related behavioral and environmental factors that contribute to exposure.

Animals↗
PubMed2004

Constitution of stable artificial oil bodies with triacylglycerol, phospholipid, and caleosin.

Seed oil bodies are lipid storage organelles of 0.5-2 microm in diameter and comprise a triacylglycerol matrix shielded by a monolayer of phospholipids and proteins. These proteins include abundant structural proteins, oleosins, and at least two minor proteins termed caleosin and steroleosin. This study examined if artificial oil bodies (AOBs) composed of triacylglycerol and phospholipid could be stabilized by oleosin, caleosin, or steroleosin. Our results showed that stabilization effects could be realized by oleosin or caleosin but not by steroleosin. The sizes of the AOBs constituted with oleosin (0.5-2 microm) or caleosin (50-200 nm) were similar to or 10 times smaller than those of the native oil bodies. Recombinant caleosin expressed in Escherichia coli also encapsulated AOBs with a size, topology, and stability comparable to those encapsulated with native caleosin. A proteinase K digestion indicated that caleosin anchored the AOBs via its central hydrophobic domain of approximately 4 kDa. Isoelectrofocusing revealed that the isoelectric point of the caleosin-stabilized AOBs was pH 4.0. Aggregation of AOBs was observed at a pH lower than 4.5; thus, their stability and integrity were presumably contributed by surface caleosin via electronegative repulsion and steric hindrance. The caleosin-stabilized AOBs were thermostable up to 70 degrees C and potentially useful for biotechnological applications.

Calcium-Binding Proteins↗
PubMed1990

Effects of urinary macromolecules on the crystallization of calcium oxalate.

The macromolecular fraction of urine with a molecular weight above 3,000 was isolated by dialysis. In the dialysed urine the rate of calcium oxalate (CaOx) crystallization was reduced both in the presence and absence of CaOx seed crystals. There was a clear relationship between crystallization and the relative concentration of the dialysed urine, with the highest crystallization propensity at the lowest concentration of macromolecules. Dilution of dialysed urine also affected crystal size distribution, with a predominance of small (2.8-4.5 microns) crystals in 100% dialysed urine and of large (5.6-14.0 microns) crystals in 5% dialysed urine. This is consistent with a macromolecular inhibition of both crystal growth and aggregation. Analysis of the crystal size distribution 120 min after supersaturation of whole urine to a level at which approximately 100 crystals in the size interval 3.5-5 microns were detected in a Coulter counter surprisingly disclosed a higher mean crystal volume in urine samples from normal subjects than from stone formers. This gives support to the assumptions that macromolecules might be of importance during the initial phase of CaOx crystallization and that urine from stone formers and normal subjects might be different in this respect.

Calcium Oxalate↗
PubMed2004

Ovarian carcinoma spheroids disaggregate on type I collagen and invade live human mesothelial cell monolayers.

Ovarian carcinoma patients frequently develop malignant ascites containing single and aggregated tumor cells, or spheroids. Spheroids have been shown to be resistant to many therapies, but their contribution to ovarian cancer dissemination remains undetermined. We have previously shown that ascites spheroids adhere to extracellular matrix (ECM) proteins and live human mesothelial cells via beta1 integrin subunits. Here, we assessed the ability of spheroids that were generated from the human ovarian carcinoma cell line NIH:OVCAR5 to disseminate and invade in vitro. Spheroids were seeded on ECM proteins for 24 h. While laminin and type IV collagen stimulated some cell migration, spheroids completely disaggregated on type I collagen substrates. A monoclonal antibody against the beta1 integrin subunit significantly inhibited disaggregation on all proteins tested. To test their invasive ability, spheroids were added to monolayers of live human LP9 mesothelial cells. Within 24 h, the spheroids adhered and disaggregated on top of the monolayers, and within a week had established foci of invasion encompassing a 200-fold larger surface area. Addition of a monoclonal antibody against the beta1 integrin subunit drastically reduced spheroid invasion into the mesothelial cell monolayers. GM 6001, a broad-scale matrix metalloproteinase inhibitor, also significantly blocked spheroid invasion into the mesothelial cell monolayers. Epsilon-amino-N-caproic acid, a serine protease inhibitor, partially inhibited spheroid invasion. Based on their ability to attach to, disaggregate on, and invade into live human mesothelial cell monolayers, spheroids should thus be regarded as potential contributors to the dissemination of ovarian cancer.

Aminocaproates↗
PubMed1993

Prostacyclin secretion by human endothelial cells grown on carbodiimide cross-linked proteins: an assessment of the cytocompatibility of a substratum.

Prostacyclin production by human umbilical vein endothelial cells cultured on carbodiimide cross-linked albumin and/or gelatin was quantified during the exponential growth phase and in confluent cultures as a response to arachidonic acid stimulus. In confluent cultures, basal production of prostacyclin measured by radioimmunoassay of the stable metabolite 6-keto-PGF1 alpha was comparable for both substrates to a control culture. Maximal release of prostacyclin occurred during the first 24 h following cell seeding and these values were significantly higher in media from cultures performed on membranes. In both cases, PGI2 production decreased as cell density increased. After stimulation with 20 microM arachidonic acid for 20 min, media from confluent cells grown on membranes contained slightly greater amounts of PGI2 than control culture medium. These results indicate involvement of substratum in PGI2 Release. Early enhancement of PGI2 secretion could improve biocompatibility of membranes by preventing platelet aggregation.

Albumins↗
PubMedDate not supplied

Loofa sponge as a scaffold for culture of rat hepatocytes.

The dried fruit from Luffa cylindrica (loofa sponge, LS), which represents a new chitinous source material, was used as a 3-D scaffold for the culture of rat hepatocytes. With the macroporous structure and large pore size (ca. 800 microm) of LS, cell loading to the scaffold should be carried out by dynamic seeding with continuous shaking throughout the seeding period. Hepatocytes attach well to the surface of loofa fibers after seeding and maintain their round shapes. The initial ammonia removal and urea-N synthesis rates of hepatocytes immobilized within LS slightly decreased with increasing cell densities, but their metabolic activities were comparable to or better than those in monolayer culture on tissue culture polystyrene control surfaces. Both urea-N synthesis and albumin secretion rates could be maintained up to 7 days for cells immobilized within LS and spheroid-like cell aggregates could be found after the second day.

Animals↗
PubMed2002

Synthesis and properties of water-soluble asterisk molecules.

An asterisk is comprised of six semirigid arms projecting from a benzene nucleus. In the case at hand, asterisks were synthesized with one, two, or three aromatic rings (connected by sulfur atoms) in each of the six arms. A phosphomonoester at the termini of each arm solubilized the asterisks in water. The colloidal properties of these amphiphilic molecules were investigated by UV-vis and fluorescence spectroscopy, calorimetry, light scattering, surface tensiometry, and pulse-gradient spin-echo NMR. Solubility, solubilization, metal binding, and micelle "seeding" experiments were also carried out. Chain-conformation and supramolecular assembly into remarkable molecular "scrolls" were investigated by X-ray analysis and electron microscopy, respectively. One of the more interesting properties of the asterisks is that they remain monomeric in water despite having as many as 19 hydrophobic aromatic rings exposed to the water. The reasons for this behavior, and the possibility of exploiting it for constructing enzyme models free from aggregation equilibria, are discussed.

Journal Article↗
PubMed2001

Conformational change in a single molecular species, beta3, of beta-conglycinin in acidic ethanol solution.

Several physicochemical experiments were done to obtain further information on the conformational changes occurring in beta-conglycinin in acidic-ethanol solution, using a single molecular species of this protein, beta3. By far-UV circular dichroism (CD), a transition from beta-sheet to alpha-helical structure was observed upon addition of acidic-ethanol, and the alpha-helix content was found to reach 76% in 70% ethanol (pH 2). From analyses of near-UV CD and difference absorption spectra, it was found that the tertiary structure of the beta3 species was significantly altered at ethanol concentrations between 10 and 20%. The profiles of binding of 1-anilinonaphthalene-8-sulfonic acid to the beta3 species during acidic-ethanol denaturation were indicative of the existence of intermediate conformers in the molten globule-like denaturation state. By measuring Fourier transform infrared spectra and estimating the Stokes radius by dynamic light scattering, the beta3 molecules were found to aggregate with an increase in ethanol concentration.

Anilino Naphthalenesulfonates↗
PubMed2001

Tubulin seeds alpha-synuclein fibril formation.

Increasing evidence suggests that alpha-synuclein is a common pathogenic molecule in several neurodegenerative diseases, particularly in Parkinson's disease. To understand alpha-synuclein pathology, we investigated molecules that interact with alpha-synuclein in human and rat brains and identified tubulin as an alpha-synuclein binding/associated protein. Tubulin co-localized with alpha-synuclein in Lewy bodies and other alpha-synuclein-positive pathological structures. Tubulin initiated and promoted alpha-synuclein fibril formation under physiological conditions in vitro. These findings suggest that an interaction between tubulin and alpha-synuclein might accelerate alpha-synuclein aggregation in diseased brains, leading to the formation of Lewy bodies.

Amino Acid Sequence↗
PubMedDate not supplied

Influence of dietary fats on blood coagulation and prostaglandin production in the common marmoset.

Common marmosets were fed a standard marmoset diet (REF) or diets supplemented with 12% (wt/wt) sunflower seed oil (SSO) or sheep fat (SF) for a period of 90 weeks. The values for coagulation indices, clotting time, and Russel viper venom time were consistent with decreased thrombotic tendency of platelets from animals on the SSO diet relative to the low fat, REF diet animals, while an increased tendency to thrombosis was observed with SF-fed marmosets. The SSO- and SF-supplemented marmosets showed a significantly reduced thromboxane (TXB2) generation from platelets aggregating to collagen (ASC) relative to the REF group, while at 50 micrograms/ml ASC this difference was maintained only by the SSO group. The SF diet-fed marmosets showed a reduced prostacyclin (measured as 6-keto-PGF1 alpha) generation from incubated aorta relative to the REF or SSO-fed groups, which were not different from each other. A reduced proportion of platelet phospholipid arachidonic acid (20:4, n-6) and increased alpha-tocopherol concentration was consistent with the decreased aggregability and thromboxane generation of platelets from SSO-fed marmosets relative to the REF and SF groups. The SF diet-fed marmosets, on the other hand, showed minimal change in arachidonic acid, alpha-tocopherol or platelet reactivity from the REF group. These differing responses to dietary fats are discussed in relation to the potential for the development of thrombosis and atherosclerosis.

Animals↗