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PubMed2005

Biological performances of collagen-based scaffolds for vascular tissue engineering.

Collagen is widely used for biomedical applications and it could represent a valid alternative scaffold material for vascular tissue engineering. In this work, reconstituted collagen films were prepared from neutralized acid-soluble solutions for subsequent haemocompatibility and cell viability performance assays. First, haemoglobin-free, thrombelastography and platelet adhesion tests were performed in order to investigate the blood contact performance. Secondly, specimens were seeded with endothelial cells and smooth muscle cells, and cell viability tests were carried out by MTT and SEM. Results show that neutralized acid-soluble type I collagen films do not enhance blood coagulation, do not alter normal viscoelastic properties of blood and slightly activate platelet adhesion and aggregation. Cell culture shows that the samples are adequate substrates to support the adhesion and proliferation of endothelial and smooth muscle cells.

Biocompatible Materials↗
PubMed2006

Interactions and compatibility of 11 S globulin from Vicia faba seeds and sodium salt of carboxymethylcellulose in an aqueous medium.

This work studies specific interactions and compatibility between a legumin and a linear carboxylated polysaccharide using gel permeation chromatography, sedimentation analysis, SDS gel electrophoresis, viscometry and phase analysis measurements. It uses the system water/11 S globulin/CMC as a model. Carboxymethylcellulose (CMC) molecules are able to cause a partial dissociation of the protein, subsequent formation of soluble interbiopolymeric complexes and partial aggregation of the free non combined protein at room temperature and pH 6.0-6.5. The maximal binding of biopolymers is observed at their equimolar ratio. The decrease in temperature of the mixture from 293 to 277 K leads to formation of the complex coacervate. The increase in pH from 6.0 to 7.6 results in suppression of complex formation and manifestation of the phenomenon of thermodynamic incompatibility if the total concentration of biopolymers in the system exceeds the critical concentration of segregative phase separation.

Carboxymethylcellulose Sodium↗
PubMedDate not supplied

Effects of retinoic acid on cell differentiation and reversion toward normal in human endometrial adenocarcinoma (RL95-2) cells.

BACKGROUND: All-trans retinoic acid is currently used in clinical trials in combination with tamoxifen to treat breast cancer, and 13-cis retinoic acid is used with a-interferon to treat metastatic endometrial cancer. We examined the effects of all-trans retinoic acid and 13-cis RA alone on endometrial adenocarcinoma (RL95-2) cells to investigate the cell biological mechanisms by which retinoic acid may reduce the metastatic phenotype and induce differentiation. METHODS: RL95-2 cells were seeded onto 4-chamber plastic slides and treated with 13-cis retinoic acid or all-trans retinoic at 0.5 microM, 1 microM and 5 microM doses for 90 minutes at 37 degrees C and stained for F-actin. RESULTS: Untreated RL95-2 cells exhibited staining of disrupted aggregates of F-actin only near the cell periphery. Cells treated with the three doses of 13-cis retinoic acid exhibited a dramatic reorganization of F-actin throughout the cells. When cells were treated with 0.5 microM all-trans retinoic acid, actin filaments reorganized. Cells treated with 1 microM all-trans retinoic acid and 5 microM all-trans retinoic acid displayed increased organization of F-actin and cell size increased. The percentage of S-phase cells increased at the high doses of retinoic acid treatment. This effect was apparently transient, since retinoic acid did not significantly affect cell growth. CONCLUSION: An organized cytoskeleton and an increase in cell size are associated with differentiation. We suggest that retinoic acid exerts its effects on these transformed cells by reorganizing actin filaments, and inducing differentiation, thus inducing a more stationary phenotype.

Actins↗
PubMed1993

The endothelium: a key to the future.

The vascular endothelium is a complex modulator of a variety of biological systems and may well be the key to definitive success in the treatment of cardiovascular disorders. Surgically-induced endothelial injury may occur preoperatively during cardiac catheterization and intraoperatively from mechanical manipulation, ischemia, hypothermia, and exposure to cardioplegic solutions. The normal endothelium is antithrombogenic and yet promotes platelet aggregation and coagulation if injured. Vasospasm, occlusive intimal hyperplasia, and accelerated arteriosclerosis can also all occur as a result of endothelial injury. Furthermore, endothelial injury is harmful even in the absence of disruption of its monolayer integrity. Thus, preservation of the endothelium should be an additional objective for all cardiovascular surgeons. Synthetic vascular grafts, cardiac valves, and artificial ventricles do not spontaneously endothelialize and thus usually require some form of anticoagulation to maintain patency. Hence, endothelialization of prosthetic implants became an attractive concept. A number of different methods of obtaining an endothelial lining of prosthetic material has since been developed; these include facilitated endothelial cell migration, and endothelial cell seeding by using either venous or microvascular endothelial cells. Manipulating the endothelium might well provide the next major advancement for therapeutic and preventive measures for cardiovascular disease.

Arteriosclerosis↗
PubMed1995

Skin fibroblasts are the only source of nidogen during early basal lamina formation in vitro.

The purpose of this study was to determine whether nidogen, the linkage protein of the basal lamina, is of epidermal or dermal origin. The development of the basal lamina was studied in an in vitro skin model. Preputial fibroblasts seeded onto a nylon mesh attached, proliferated, and developed a rich extracellular matrix (dermal model). Preputial keratinocytes were added to the dermal model to form a keratinocyte dermal model that ultrastructurally resembled in many respects human skin. Ultrastructural analysis revealed early stages of dermal development, including an incomplete basal lamina, aggregates of dermal filamentous material connecting to the lamina densa, bundles of 10-nm microfibrils, formation of premature hemidesmosomes, anchoring filaments, and anchoring fibrils. The cell origin of nidogen was determined in the dermal model and in the epidermal and dermal components of the keratinocyte dermal model. Specific antibodies and a cDNA probe for nidogen were used for immunofluorescence microscopy, Western and Northern blots, and for in situ hybridization studies. Our data show that fibroblasts are the only source of nidogen during early basal lamina formation. Although fibroblasts can synthesize nidogen and deposit it in the dermal matrix, no basal lamina will form unless they are recombined with keratinocytes. This suggests that the epidermis plays a major regulatory role in the production and assembly of nidogen into the basal lamina.

Basement Membrane↗
PubMed1992

Use of 8-methoxypsoralen and long wavelength ultraviolet radiation for decontamination of platelet concentrates.

Transmission of viral diseases through blood products remains a problem in transfusion medicine. We have developed a photochemical decontamination system (PCD) for platelet concentrates (PC) utilizing treatment with long wavelength ultraviolet radiation (UVA, 320-400 nm) and 8-methoxypsorlan (8-MOP). This system is capable of inactivating 25-30 logs/hour of bacteria E. coli or S. aureus, 6 logs/hour of bacteriophage fd, 0.9 log/hour of bacteriophage R17, and 1.1 logs/hour of feline leukemia virus (FeLV) in PC. Immediately following 6 hours of PCD treatment, platelet integrity and function of PCD-treated and control PC were equivalent. After overnight storage, PCD-treated and control PC platelet properties were equal, but there was a slight reduction in TXB-2 production of PCD-treated PC compared to controls. Following PCD treatment, PC were stored for 48 to 96 hours. Platelet counts, morphology scores, extracellular LDH levels, aggregation response, dense body (db) content, and alpha granule (alpha g) content of PCD-treated and control PC were comparable. We assessed the ability of the PCD technique to inactivate intracellular and extracellular virus, quantified the degree of DNA adduct formation in contaminating lymphocytes, and measured the inhibition of polymerase chain reaction (PCR) mediated amplification of intracellular DNA. High titers of cell-free murine cytomegalovirus added to human platelet concentrates (final concentration 10(6)) were inactivated by PCD within 30 minutes. Cat renal fibroblasts infected at high levels with feline rhinotracheitis virus (FeRTV) were seeded into PC followed by PCD treatment with inactivation of 4.8 logs of FeRTV within 10 minutes. Purified human lymphocytes were seeded into PC and treated with PCD in the presence of 3H 8-MOP. Six hours of PCD treatment resulted in the formation of 9.3 to 12.8 8-MOP adducts per 1000 base pairs (bp) of DNA. PCR amplification of a 242 bp segment at the HLA-DQ alpha locus was examined. Inhibition of PCR DNA amplification was dependent on the numbers of 8-MOP adducts formed, and no amplification was present when greater than 12 adducts per 1000 bp were formed. These studies indicate that PCD can effectively inactivate high titers of cell-associated and cell-free virus seeded into standard human PC. The efficiency of DNA adduct formation can be quantitated, and the level of 8-MOP adduct formation in lymphocytes contaminating PC is comparable to the level of adduct formation in cellular DNA reported in the absence of platelets.

Blood Component Transfusion↗
PubMed1997

Small-angle X-ray scattering and crystallographic studies of arcelin-1: an insecticidal lectin-like glycoprotein from Phaseolus vulgaris L.

Arcelin-1 and alpha-amylase inhibitor are two lectin-like glycoproteins expressed in the seeds of the kidney bean (Phaseolus vulgaris). They display insecticidal activities and protect the seeds from predation by larvae of various bruchids through different biological actions. Solution-state investigations by small-angle X-ray scattering (SAXS) show the dimeric structure of arcelin-1, a requirement for its hemagglutinating properties. Anions were found to have specific properties in their effectiveness to disrupt protein aggregates, affect solubility, and improve crystallizability. The SAXS results were used to improve crystallization conditions, and single crystals diffracting beyond 1.9 A resolution were obtained. X-ray diffraction data analysis shows that noncrystallographic symmetry-related arcelin-1 molecules form a lectin-like dimer and reveals the presence of a solvent-exposed anion binding site on the protein, at a crystal-packing interface. The solution state properties of arcelin-1 and crystal twinning may be explained by the anion specificity of this binding site.

Amino Acid Sequence↗
PubMed2000

Ontogenic emergence of the hematon, a morphogenetic stromal unit that supports multipotential hematopoietic progenitors in mouse bone marrow.

Development of the full repertoire of hematopoietic-lymphopoietic cells from a single stem cell requires specific contacts with stromal cells. The spatio-temporal organization of these cell associations in the bone marrow in ontogeny is, however, not well understood. In the adult, 10% of marrow cells form a cohort of compact aggregates, the hematon. In the hematon mesenchymal cells (Stro-1(+)), perivascular lipocytes (desmin(+)), endothelial cells (CD34(+), Flk-1(+), Sca-1(+)), and macrophages amalgamate with the hematopoietic progenitors long-term culture-initiating cells (LTC-IC), cobblestone area-forming cell (CAFC), high-proliferative-potential colony-forming unit (HPP-CFU), granulocyte-macrophage (GM)-CFU, and burst-forming unit-erythroid (BFU-E). During endochondral ossification of the femur, GM-CFU and day 7 CAFC numbers increased progressively from day 17 of gestation, but primitive, day 35 LTC-IC appeared from postnatal day 2. Unexpectedly, bone marrow (BM) taken between embryonic day 17 and day 5 was unable to support myeloid cell production in long-term cultures or to support day 35 LTC-IC growth. However, a gain in stromal cell competence occurred between days 7 and 10, which was correlated with the emergence of hematon in the BM. Thus, acquisition of hematopoietic competence by BM lags behind for approximately 10 days after the initial hematopoietic cell influx. In the adult, the hematon fraction was 3.7-fold enriched in day 35 LTC-IC over the buffy coat. It produced more GM-CFU and HPP-CFU in myeloid culture and more B cells in lymphopoietic "switch" cultures. It is reported that stromal hematopoietic units named hematons are specific morphogenetic structures that emerge at a well-defined postnatal stage of development in long bones, delineate discrete territories for hematopoietic stem cell seeding and development, embody the most productive hematogenous compartment in the BM, and probably enclose a morphogenetic organizer. (Blood. 2000;96:3763-3771)

Animals↗
PubMed2003

Coordinate expression of multiple bacterial carotenoid genes in canola leading to altered carotenoid production.

Carotenoids have drawn much attention recently because of their potentially positive benefits to human health as well as their utility in both food and animal feed. Previous work in canola (Brassica napus) seed over-expressing the bacterial phytoene synthase gene (crtB) demonstrated a change in carotenoid content, such that the total levels of carotenoids, including phytoene and downstream metabolites like beta-carotene, were elevated 50-fold, with the ratio of beta- to alpha-carotene being 2:1. This result raised the possibility that the composition of metabolites in this pathway could be modified further in conjunction with the increased flux obtained with crtB. Here we report on the expression of additional bacterial genes for the enzymes geranylgeranyl diphosphate synthase (crtE), phytoene desaturase (crtI) and lycopene cyclase (crtY and the plant B. napus lycopene beta-cyclase) engineered in conjunction with phytoene synthase (crtB) in transgenic canola seed. Analysis of the carotenoid levels by HPLC revealed a 90% decrease in phytoene levels for the double construct expressing crtB in conjunction with crtI. The transgenic seed from all the double constructs, including the one expressing the bacterial crtB and the plant lycopene beta-cyclase showed an increase in the levels of total carotenoid similar to that previously observed by expressing crtB alone but minimal effects were observed with respect to the ratio of beta- to alpha-carotene compared to the original construct. However, the beta- to alpha-carotene ratio was increased from 2:1 to 3:1 when a triple construct consisting of the bacterial phytoene synthase, phytoene desaturase and lycopene cyclase genes were expressed together. This result suggests that the bacterial genes may form an aggregate complex that allows in vivo activity of all three proteins through substrate channeling. This finding should allow further manipulation of the carotenoid biosynthetic pathway for downstream products with enhanced agronomic, animal feed and human nutritional values.

Brassica napus↗
PubMed1969

Morphogenetic aspects of multilayering in Petri dish cultures of human fetal lung fibroblasts.

Randomly seeded Petri dish cultures of embryonic human lung fibroblasts generate, in the course of their growth, highly ordered cellular arrangements. Thick, bilaterally symmetrical ridges with an axial polarity and an orthogonal, multilayered internal organization are observed within stationary cultures. The generation of these structures has been investigated. Ridges result from the spontaneous aggregation of cells in postconfluent cultures brought about by directed cell movements. These movements are promoted by the localized production of extracellular matrix sheets containing collagen, which provide new substrates for cellular colonization. Cells that have colonized one matrix substrate may secrete another above themselves, which will in turn be colonized. By a continuation of this cycle, thick stacks consisting of alternate layers of cells and matrix are produced to yield the observed aggregations. The distribution and shape of ridges in a culture imply that matrix substrates are confined to specific locations. The suggested control hypothesis assumes that all the cells in fibroblast cultures are potential producers of a single species of matrix. The serviceability of this matrix as a substrate for cellular colonization, however, is destroyed if the producer cells are motile. Matrix substrates, therefore, are only made by nonmotile cells.

Cell Aggregation↗
PubMedDate not supplied

Microfabricated surface designs for cell culture and diagnosis.

Grooved and holed surfaces with a well fabricated design may serve as microsubstrates for cell culture and microreactors for diagnosis. In this study, the authors prepared chemically treated, micrometer scale grooved and holed glass surfaces by combined surface modification and ultraviolet (UV) excimer laser ablation techniques, as follows. 1) Microcell-culture substrate: Amino group attached glass surfaces, prepared by the treatment with an aminopropylsilane, were condensed with a carboxylated radical initiator. Subsequently, polyacrylamide was grafted by surface initiated radical polymerization to create a very hydrophilic surface layer. Ultraviolet excimer laser beams (KrF: 248 nm) were irradiated through a microscope onto surfaces to create grooves or holes that were 10 and 50 microns in width or diameter, respectively. The depth, depending on the irradiation light strength, ranged from a few to several tenths of a micrometer. On endothelial cell (EC) seeding, ECs adhered and grew on the bottoms of the grooved or holed surface where glass was exposed on ablation. Little cell adhesion was observed on non ablated, grafted surfaces. Endothelial cells aligned along the groove, resulting in very narrow tube like tissue formation, whereas ECs tended to form a multilayered spherical aggregate in a hole. A single cell resided in a 10 microns square hole. 2) Microreactor for diagnosis: The glass surface, treated with a fluorinated silane, was ablated to create round holes. On addition of a few microliters of water, water could be quantitatively transferred into a hole because of the water repellent characteristics of non ablated, fluorinated glass. As a model of a microreactor, enzyme reactions to affect different levels of glucose were carried out in tiny holed surfaces.

Acrylic Resins↗
PubMed1998

Transport of storage proteins to protein storage vacuoles is mediated by large precursor-accumulating vesicles

Novel vesicles that accumulate large amounts of proprotein precursors of storage proteins were purified from maturing pumpkin seeds. These vesicles were designated precursor-accumulating (PAC) vesicles and had diameters of 200 to 400 nm. They contained an electron-dense core of storage proteins surrounded by an electron-translucent layer, and some vesicles also contained small vesicle-like structures. Immunocytochemical analysis revealed numerous electron-dense aggregates of storage proteins within the endoplasmic reticulum. It is likely that these aggregates develop into the electron-dense cores of the PAC vesicles and then leave the endoplasmic reticulum. Immunocytochemical analysis also showed that complex glycans are associated with the peripheral region of PAC vesicles but not the electron-dense cores, indicating that Golgi-derived glycoproteins are incorporated into the PAC vesicles. These results suggest that the unique PAC vesicles might mediate a transport pathway for insoluble aggregates of storage proteins directly to protein storage vacuoles.

Journal Article↗
PubMed2000

Functional tissue engineering of articular cartilage through dynamic loading of chondrocyte-seeded agarose gels.

Due to its avascular nature, articular cartilage exhibits a very limited capacity to regenerate and to repair. Although much of the tissue-engineered cartilage in existence has been successful in mimicking the morphological and biochemical appearance of hyaline cartilage, it is generally mechanically inferior to the natural tissue. In this study, we tested the hypothesis that the application of dynamic deformational loading at physiological strain levels enhances chondrocyte matrix elaboration in cell-seeded agarose scaffolds to produce a more functional engineered tissue construct than in free swelling controls. A custom-designed bioreactor was used to load cell-seeded agarose disks dynamically in unconfined compression with a peak-to-peak compressive strain amplitude of 10 percent, at a frequency of 1 Hz, 3 x (1 hour on, 1 hour off)/day, 5 days/week for 4 weeks. Results demonstrated that dynamically loaded disks yielded a sixfold increase in the equilibrium aggregate modulus over free swelling controls after 28 days of loading (100 +/- 16 kPa versus 15 +/- 8 kPa, p < 0.0001). This represented a 21-fold increase over the equilibrium modulus of day 0 (4.8 +/- 2.3 kPa). Sulfated glycosaminoglycan content and hydroxyproline content was also found to be greater in dynamically loaded disks compared to free swelling controls at day 21 (p < 0.0001 and p = 0.002, respectively).

Animals↗
PubMed1998

Cell adhesion to laminin 1 or 5 induces isoform-specific clustering of integrins and other focal adhesion components.

Laminin 1 (alpha1beta1gamma1) and laminin 5 (alpha3beta3gamma2) induce cell adhesion with different involvement of integrins: both are ligands for the alpha6beta1 integrin, while alpha3beta1 integrin has affinity for laminin 5 only. These two laminin isoforms therefore provide good models to investigate whether alpha3beta1 and alpha6beta1 integrins play different roles in signal transduction and in focal adhesion formation. Laminin 1 or 5 induced adhesion of normal human skin fibroblasts to a similar extent but promoted different overall cell shapes. On laminin 1 the fibroblasts formed mainly filopodia-like structures, while on laminin 5 they developed lamellipodias. Staining of fibrillar actin with fluorescein-phalloidin revealed a similar organisation of the actin cytoskeleton on both substrates. However, integrin subunits and several cytoskeletal linker proteins, including vinculin, talin, and paxillin, showed an isoform-specific arrangement into focal adhesions. On laminin 1 they were recruited into thick and short aggregates localized at the termini of actin stress fibers, while on laminin 5 they appeared as dots or streaks clustered on a long portion of actin microfilaments. To test whether the differing affinity of laminin 1 or 5 for alpha3beta1 integrin would explain the formation of morphologically different focal adhesions, cells were seeded on laminin 1 under conditions in which alpha3beta1 integrins were occupied by a function-blocking antibody. This resulted in the formation of focal adhesions similar to that observed on laminin 5, where the integrin is occupied by its natural ligand. These results provide the first evidence for a cross-talk between alpha3beta1 and alpha6beta1 integrins and indicate that occupancy of alpha3beta1 integrins results in a trans-dominant regulation of alpha6beta1 integrin clustering and of focal adhesions. It suggests that recruitment of integrins and cytoskeletal linker proteins are laminin isoform-specific and that tissue specific expression of laminin isoforms might modulate cell behavior by the activation of distinct sets of integrins and by the induction of distinct molecular assemblies within the cell adhesion signaling complexes.

Breast↗
PubMed1999

Heat-induced aggregation of Phaseolus vulgaris L. Proteins: an electron spin resonance study.

The mechanism of heat-induced aggregation of Phaseolus vulgaris L. proteins and of subunit interactions of importance for susceptibility of proteins to proteolysis was studied by electron spin resonance (ESR) spectroscopy. The mobility of a spin label bound to lysine residues was monitored at two different pH-induced (neutral and alkaline) association states of proteins extracted from raw and cooked common bean. The molecular weight of the protein complexes was assessed by size exclusion-high performance liquid chromatography (SE-HPLC) of labeled proteins. Upon alkaline dissociation, both native and denatured protein subunits underwent a reassociation process to form soluble complexes of molecular weight higher than the species originally present at neutral pH. However, unlike native proteins, impaired mobility of the spin label was observed in the aggregates that are formed after dissociation of subunits of denatured proteins, indicating a reduced accessibility of lysine residues. Trapping of lysine residues inside protein aggregates may explain limited digestibility in the small intestine of proteins in cooked legumes.

Cooking↗
PubMed2001

PFA, a novel mollusk agglutinin, is structurally related to the ribosome-inactivating protein superfamily.

The structural organization of PFA, a novel beta-galactose-specific agglutinin from the snail Pomacea flagellata, was partially characterized. Using mass spectrometry, the molecular weight of this glycoprotein was determined as 32,444 Da (7.4% carbohydrate). The agglutinin was found to form very large aggregates in solution, which were dissociated to monodisperse native-like dimers upon addition of polyethyleneglycol. The identity of the first 38 and the last 11 residues of the polypeptide chain was determined. It was found that PFA and the N-glycosidase subunit of ricin, a heterodimeric cytotoxin isolated from castor bean seeds, are homologous to each other in the N-terminal region. Furthermore, the far-UV circular dichroism spectra of these proteins were found to be nearly superimposable, evidencing that they share common general features in their secondary and tertiary structures. On the basis of these similarities, it can be concluded that PFA is structurally related to the ribosome-inactivating protein superfamily.

Agglutinins↗
PubMed2002

Cell membrane translocation of the N-terminal (1-28) part of the prion protein.

The N-terminal (1-28) part of the mouse prion protein (PrP) is a cell penetrating peptide, capable of transporting large hydrophilic cargoes through a cell membrane. Confocal fluorescence microscopy shows that it transports the protein avidin (67kDa) into several cell lines. The (1-28) peptide has a strong tendency for aggregation and beta-structure formation, particularly in interaction with negatively charged phospholipid membranes. The findings have implications for how prion proteins with uncleaved signal peptides in the N-termini may enter into cells, which is important for infection. The secondary structure conversion into beta-structure may be relevant as a seed for the conversion into the scrapie (PrP(Sc)) form of the protein and its amyloidic transformation.

Amino Acid Sequence↗
PubMed2005

polyglutamine aggregation nucleation: thermodynamics of a highly unfavorable protein folding reaction.

Polyglutamine (polyGln) aggregation is implicated in the disease progression of Huntington's disease and other expanded CAG repeat diseases. PolyGln aggregation in vitro follows a simple nucleated growth polymerization pathway without apparent complications such as populated intermediates, alternative assembly pathways, or secondary nucleation phenomena. Previous analysis of the aggregation of simple polyGln peptides revealed that the critical nucleus (the number of monomeric units involved in the formation of an energetically unfavorable aggregation nucleus) is equal to one, suggesting that polyGln nucleation can be viewed as an unfavorable protein folding reaction. We provide here a method for experimentally determining the number of elongation growth sites per unit weight for any polyGln aggregate preparation, a key parameter required for completion of the nucleation kinetics analysis and determination of the thermodynamics of nucleation. We find that, for the polyGln peptide Q(47), the second-order rate constant for fibril elongation is 11,400 liters/mol per s, whereas K(n*)), the equilibrium constant for nucleation of aggregation, is remarkably small, equal to 2.6 x 10(-9). The latter value corresponds to a free energy of nucleus formation of +12.2 kcal/mol, a value consistent with a highly unfavorable folding reaction. The methods introduced here should allow further analysis of the energetics of polyGln nucleus formation and accurate comparisons of the seeding capabilities of different fibril preparations, a task of increasing importance in the amyloid field.

Amyloid↗