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Receptors which induce immune system effector function bear similar intracellular sequences and respond to aggregation through a nonreceptor tyrosine kinase-dependent pathway. The mechanism by which receptor aggregation leads to cell activation is poorly understood, but recent experiments with chimeric receptors and kinases have begun to simplify the analysis.
Time-resolved anisotropy measurements (TRAMS) have been used to study the aggregation of the beta-amyloid (Abeta) peptide which is suspected of playing a central role in the pathogenesis of Alzheimer's Disease (AD). The experiments, which employ small quantities of fluorescently-labelled Abeta, in addition to the untagged peptide, have shown that the sensitive TRAMS technique detects the presence of preformed "seed" particles in freshly prepared solutions of Abeta. More importantly, as 100 microM solutions of Abeta containing tagged Abeta at a concentration level of either 0.5 or 1 microM are incubated, the TRAMS prove capable of detection of the peptide aggregation process through the appearance of a continuously increasing "residual anisotropy" within the time-resolved fluorescence data. The method detects Abeta aggregation in its earliest stages, well before complexation becomes apparent in more conventional methods such as the thioflavin T fluorescence assay. The TRAMS approach promises to provide a most attractive route for establishment of a high-throughput procedure for the early detection of the presence of amyloid aggregates in the screening of biological samples.
The present study aims at optimizing dermal fibroblast seeding and cultivation in Polyactive scaffolds in order to limit the biopsy size needed for autologous treatment of full-thickness skin defects and chronic wounds. Three methods for seeding and cultivation of fibroblasts in porous scaffolds were compared: dynamic seeding followed by static cultivation (DS), static seeding followed by static cultivation (SS) and dynamic seeding followed by dynamic cultivation (DD). Human dermal fibroblasts isolated from cultured explants were seeded in porous PEO/PBT (Polyactive) scaffolds. Samples were taken from 6 h to 21 days post-seeding for both histological analysis (cell distribution and extracellular matrix (ECM) formation), and quantitative cell number assay. The seeding efficiency 24 h post-seeding was 76% (+/-3.6%) for dynamically seeded matrices, whereas it was only 30% (+/-5%) for statically seeded matrices (p<0.001). ECM formation was abundant in DS samples already at day 10, while even after 21 days ECM formation was less pronounced in SS samples. Surprisingly, cells detached from DD samples as aggregates, starting from day 10. Cell numbers as assayed quantitatively correlated with the histological results. At all timepoints cell numbers found for DS samples were significantly higher as compared to SS samples. At day 21, DS samples contained approximately twofold more cells as compared to SS and DD samples and comprised ECM consisting of collagen types I and III. Our results indicate that the combination of dynamic seeding and static cultivation assures efficient utilization of isolated fibroblasts and improved neodermis formation, thereby allowing a reduction in the skin biopsy size needed for the engineering of living skin substitute.
Advances in micropatterning methodologies have made it possible to create structures with precise architecture on the surface of cell culture substrata. We applied these techniques to fabricate microfeatures (15-65 microm wide; 40 microm deep) on the surface of a flexible, biocompatible polysaccharide gel. The micropatterned polymer gels were subsequently applied as scaffolds for chondrocyte culture and proved effective in maintaining key aspects of the chondrogenic phenotype. These were rounded cell morphology and a positive and statistically significant (p < 0.0001) immunofluorescence assay for the production of type II collagen throughout the maximum culture time of 10 days after cell seeding. Further, cells housed within individual surface features were observed to proliferate, while serial application of chondrocytes resulted in the formation of cellular aggregates. These methods represent a novel approach to the problem of engineering reparative cartilage in vitro.
Bone marrow stromal cells isolated from a model of osteogenesis imperfecta (oim) mice, were transduced with a retrovirus (BAG) carrying the LacZ and neor genes after passage 21. The transduced cells retained the ability to express alkaline phosphatase activity in vitro when treated with recombinant human bone morphogenetic protein two (rhBMP-2), formed cartilage in vitro in aggregate cultures and formed bone in ceramic cubes after 6 weeks of implantation in nude mice. X-gal staining of ceramic cubes seeded with the transduced cells demonstrated the presence of LacZ-positive cells on the edges of bone and also in the lacunae of the newly formed bone 6 weeks after implantation. After infusion into femurs of oim mice, the transduced cells were detected in the marrow cavity and on the edges of the trabecular bone of the injected and contralateral femurs by X-gal staining and PCR analysis at 4, 10, 20, 30 and 40 days after injection. The LacZ gene was also detected in the lung and liver of the recipient mice at 4 and 10 days after injection but not at later time-periods. The present findings suggest that long-term cultured bone marrow stromal cells from osteogenesis imperfecta (OI) animals have the potential to traffic through the circulatory system, home to bone, form bone and continue to express exogenous genes. These findings open the possibility of using these cells as vehicles to deliver normal genes to bone as an alternative approach for the treatment of some forms of OI and certain other bone acquired and genetic diseases.
We have purified a group of four proteases (molecular mass 26-33 kilodalton) from germinating seeds of maize (Zea mays L. var W64A) using ammonium sulfate and isoelectric precipitations, anion exchange chromatography, and electroelution from preparative nondenaturing polyacrylamide gels. Their appearance in the endosperm of germinating seeds coincides with the onset of zein degradation. We have shown that these proteases degrade zeins dissolved in alcoholic solution as well as aggregated in protein bodies from developing maize kernels. The apparent molecular weights and net negative charges of each of these proteases are very similar. Additionally, they are inhibited by thiol-blocking agents and activated by reducing compounds. These characteristics suggest that they are a group of cysteine proteases involved in the first steps of storage protein degradation.
AIM: To compare the percentage of apoptotic cells and the cell cycle profile of fibroblasts and macrophages exposed to either ProRoot mineral trioxide aggregate (MTA) mixed with chlorhexidine (CHX), or exposed to ProRoot MTA mixed with sterile water. METHODOLOGY: Mouse gingival fibroblasts or mouse macrophages were seeded in six-well plates and allowed to attach overnight. Freshly mixed or set (allowed to dry for 24 h) specimens of tooth-coloured (white) ProRoot MTA were prepared with 0.12% CHX gluconate (MTA/CHX) or with sterile water (MTA/H2O). The cells were exposed for 24 h to the MTA specimens, which were placed over permeable membrane inserts to avoid direct contact with the cells. Untreated cells served as controls. Propidium iodide staining followed by flow cytometry was used to evaluate the effects of ProRoot MTA on cell apoptosis and cell cycle. Statistical analyses were performed by one-way anova followed by post-hoc tests with the use of the SigmaStat 2.0 software, and significance was determined at P < or = 0.05. RESULTS: MTA specimens containing CHX induced apoptosis of macrophages and fibroblasts (P < 0.05). In contrast, no change in the proportion of apoptotic cells was observed when sterile water was used to prepare the specimens (P > 0.05). Cell cycle analysis showed that exposure to MTA/CHX decreased the percentage of fibroblasts and macrophages in S phase (DNA synthesis) as compared with exposure to MTA/H2O (P < 0.05). CONCLUSION: This in vitro study demonstrated that the substitution of CHX for sterile water in MTA increases its cytotoxicity. This suggests that the potentially beneficial antimicrobial effect of CHX may be accompanied by an increase in the cytotoxicity of the resulting MTA-based material.
Cells isolated from the avian and mammalian central and peripheral nervous system and cultured in vitro provide an opportunity to study in situ properties of neurons and glial cells under relatively simple and carefully controlled conditions. Since Harrison's success in maintaining in vitro embryonic frog spinal cord 80 years ago, neural tissue culture has developed into an important and versatile discipline of neuroscience. The techniques developed in the past fall into four broad classes: Explant cultures, which are explanted from specific neuroanatomic loci to substrates as small tissue fragments. Dissociated cell cultures, which involve the seeding of enzymatically or mechanically dispersed cells on various attachment substrates. Reaggregate cultures, which require re-association of dissociated cells into small aggregates. Purified cell populations, which are prepared by the isolation of different cell types by gradient centrifugation or other separation techniques. These cultures have been utilized in studying various aspects of brain development and function. In this review several areas of significant and stimulating development in neural cell culture have been documented. They include formulation of serum-free medium, effects of growth factors, utilization of cell type-specific markers, and isolation and culture of purified neuronal/glial cells.
The nucleation event in protein crystallization is a part of the process that is poorly controlled. It is generally accepted that the protein should be in the metastable phase for crystal growth, but for nucleation higher levels of saturation are needed. Formation of nuclei in bulk solvent requires interaction of protein molecules until a critical size of aggregate is created. In many crystallization experiments sufficiently high levels of saturation are not reached to allow this critical nucleation event to occur. If an environment can be created that favours a higher local concentration of macromolecules, the energy barrier for nucleation may be lowered. When seeds are introduced at lower levels of saturation in a crystallization experiment, nucleation may be facilitated and crystal growth initiated. In this study, the use of natural materials as stable seeds for nucleation has been investigated. The method makes it possible to introduce seeds into crystallization trials at any stage of the experiment using both microbatch and vapour-diffusion methods.
The authors successfully applied a method to accelerate endothelialization by tissue fragmentation to a small diameter fabric vascular prosthesis. Tissue fragment seeded grafts showed rapid healing of the neointima. The thrombogenicity of the collagen fibrils in the fragments, however, caused major problems when the method was applied to small diameter grafts: the positively charged collagen fibrils aggregated the negatively charged platelets. The authors masked the fibrils electrostatically with heparin molecules, which are negatively charged. A canine jugular vein was resected, minced into tissue fragments, and suspended in the heparin solution; it then was sieved through the wall of a fabric prosthesis. The grafts (4 mm internal diameter and 3.5 cm in length) were implanted into both carotid arteries of six dogs (12 grafts). Tissue fragment seeded grafts without heparin also were implanted into six dogs. As a control, preclotted fabric grafts were implanted into six dogs (12 grafts). These grafts occluded within 1 week, whereas all the masked grafts were patent without thrombi. In vitro examination of heparin release revealed that approximately 92% of heparin in the graft was released during the first 5 hr, but approximately 6% remained after 25 hr. These results indicate that the method is applicable to small diameter arterial grafts.
Bacterial inclusion bodies are major bottlenecks in protein production, narrowing the spectrum of relevant polypeptides obtained by recombinant DNA. While regarded as amorphous deposits formed by passive and rather unspecific precipitation of unfolded chains, we prove here that they are instead organized aggregates sharing important structural and biological features with amyloids. By using an Escherichia coli beta-galactosidase variant, we show that aggregation does not necessarily require unfolded polypeptide chains but rather depends on specific interactions between solvent-exposed hydrophobic stretches in partially structured species. In addition, purified inclusion bodies are efficient and highly selective nucleation seeds, promoting deposition of soluble homologous but not heterologous polypeptides in a dose-dependent manner. Finally, inclusion bodies bind amyloid-diagnostic dyes, which, jointly with Fourier transform infra red spectroscopy data, indicates a high level of organized intermolecular beta-sheet structure. The evidences of amyloid-like structure of bacterial inclusion bodies, irrespective of potential applications in bioprocess engineering, prompts the use of bacterial models to explore the molecular determinants of protein aggregation by means of simple biological systems.
The main hypothesis for prion diseases proposes that the cellular protein (PrP(C)) can be altered into a misfolded, beta-sheet-rich isoform (PrP(Sc)), which in most cases undergoes aggregation. In an organism infected with PrP(Sc), PrP(C) is converted into the beta-sheet form, generating more PrP(Sc). We find that sequence-specific DNA binding to recombinant murine prion protein (mPrP-(23-231)) converts it from an alpha-helical conformation (cellular isoform) into a soluble, beta-sheet isoform similar to that found in the fibrillar state. The recombinant murine prion protein and prion domains bind with high affinity to DNA sequences. Several double-stranded DNA sequences in molar excess above 2:1 (pH 4.0) or 0.5:1 (pH 5.0) completely inhibit aggregation of prion peptides, as measured by light scattering, fluorescence, and circular dichroism spectroscopy. However, at a high concentration, fibers (or peptide aggregates) can rescue the peptide bound to the DNA, converting it to the aggregating form. Our results indicate that a macromolecular complex of prion-DNA may act as an intermediate for the formation of the growing fiber. We propose that host nucleic acid may modulate the delicate balance between the cellular and the misfolded conformations by reducing the protein mobility and by making the protein-protein interactions more likely. In our model, the infectious material would act as a seed to rescue the protein bound to nucleic acid. Accordingly, DNA would act on the one hand as a guardian of the Sc conformation, preventing its propagation, but on the other hand may catalyze Sc conversion and aggregation if a threshold level is exceeded.
Canatoxin (CNTX), a toxic protein isolated from seeds of Canavalia ensiformis, induces Ca2+ influx across the platelet plasma membrane, mobilization of arachidonic acid mediated by phospholipase A2, ATP secretion, and platelet aggregation. All these events depend on the presence of extracellular Ca2+ and are blocked by methoxyverapamil, a calcium-channel blocker. CNTX does not activate phospholipase C, and the intracellular calcium mobilization mediated by IP3 does not play a role in platelet activation by this toxin. Preincubation of rabbit platelets with 8-Br-cGMP inhibited the CNTX-evoked calcium influx, arachidonate release, ATP secretion, and cell aggregation. Our data suggest that the calcium influx is a prior step on platelet activation by CNTX, being modulated by cGMP.
An analysis of electron micrographs of Al5Mn quasicrystals obtained by rapidly cooling a molten alloy with composition Al17Mn and removing the Al matrix by electrosolution, revealing aggregates of 20 microcrystals at the corners of a pentagonal dodecahedron, supports the proposal that these microcrystals are cubic crystals twinned about an icosahedral seed, with each cubic microcrystal sharing a threefold axis and three symmetry planes with the seed.
Owing to the impossibility of reaching the Golgi for secretion or the cytosol for degradation, mutant Ig-mu chains that lack the first constant domain (muDeltaCH1) accumulate as detergent-insoluble aggregates in dilated endoplasmic reticulum cisternae, called Russell bodies. The presence of similar structures hallmarks many ER storage diseases, but their pathogenic role(s) remain obscure. Exploiting inducible cellular systems, we show here that Russell bodies form when the synthesis of muDeltaCH1 exceeds the degradation capacity. Condensation occurs in different sub-cellular locations, depending on the interacting molecules present in the host cell: if Ig light chains are co-expressed, detergent-insoluble muDeltaCH1-light chain oligomers accumulate in large ribosome-coated structures (rough Russell bodies). In absence of light chains, instead, aggregation occurs in smooth tubular vesicles and is controlled by N-glycan-dependent interactions with ER-Golgi intermediate compartment 53 (ERGIC-53). In cells containing smooth Russell bodies, ERGIC-53 co-localizes with muDeltaCH1 aggregates in a Ca2+ -dependent fashion. Our findings identify a novel ERGIC-53 substrate, and indicate that interactions with light chains or ERGIC-53 seed muDeltaCH1 condensation in different stations of the early secretory pathway.
The successful regeneration of complex tooth structures based on tissue-engineering principles was recently reported. The process of this regeneration, however, remains poorly characterized. In this study, we have used histochemistry to examine the regeneration process of tissue engineered teeth in order to determine the cell types that give rise to these engineered tooth structures. Porcine third molar tooth buds were dissociated into single-cell suspensions and seeded onto a biodegradable polyglycolic acid polymer scaffold. Following varying periods of growth in rat hosts, the specimens were evaluated by histology and immunohistochemistry. Aggregates of epithelial cells were first observed 4-6 weeks after implantation. These aggregates assumed three different shapes: a natural tooth germ-like shape, a circular shape, or a bilayer-bundle. Based on the structure of the stellate reticulum in the dental epithelium, the circular and bilayer-bundle aggregates could be clearly classified into two types: one with extensively developed stellate reticulum, and the other with negligible stellate reticulum. The epithelial cells in the circular aggregates differentiated into ameloblasts. The continuous bilayer bundles eventually formed the epithelial sheath, and dentin tissue was evident at the apex of these bundles. Finally, enamel-covered dentin and cementum-covered dentin formed, a process most likely mediated by epithelial-mesenchymal interaction. These results suggest that the development of these engineered teeth closely parallels that of natural odontogenesis derived from the immature epithelial and mesenchymal cells.
Cell seeding of three-dimensional scaffolds is the first step of the cultivation of engineered tissues. The cell seeding density and spatial distribution in a 3-D scaffold are critical to the morphogenetic development of an engineered tissue. In the present work, human fibroblasts were seeded to collagen-chitosan sponges by static seeding, stirred seeding and perfusion seeding. The effects of seeding conditions on the resulting seeding density, the seeding efficiency and the initial cell distribution were studied. The seeding efficiency was relatively high (88.9%) at low inoculation cell density, and decreased rapidly wjth the increasing of inoculation cell density in static seeding. Stirred seeding yielded the lowest seeding efficiency. Nonuniform initial cell distribution was observed in both static and stirred seeding. The perfusion seeding, which has a characteristic of high seeding efficiency (>77%) , high initial cell density and uniform initial cell distribution in 3-D scaffolds, is the optimum method for cell seeding to 3-D scaffold.
The effects of the anticancer drugs Nimustine (ACNU), Aclacinomycin A (ACR), Adriamycin (ADM), Bleomycin (BLM), Cisplatin (CDDP), and 5-Fluorouracil (5-FU) on the multicellular spheroid of a chemically-induced 9L rat glioma was studied. The multicellular spheroid in which cells grow in vitro as three-dimensional aggregates represents a biological model, which is intermediate between monolayer cells in vitro and solid tumors. Spheroids were initiated in bacteriological grade petri dishes seeded with 10(6) 9L rat glioma cells, cultured for four days and thereafter transferred and further developed in a spinner flask. Spheroids of 200-400 micron diameter were sorted and exposed for 24 hours to 5-FU and one hour for other drugs. After treatment both cytotoxic effect and growth delay were analyzed. Following disaggregation using collagenase, pronase and DNAase, cytotoxic effect on multicellular spheroids was measured by colony forming assay and were compared with those effects on 9L monolayer culture cells in the exponential growth. For growth delay assay, multicellular spheroids were individually transferred to 16 mm well containing 0.4 ml agarose base and 2 ml culture medium. Spheroid size was measured twice a week and growth curves were drawn. The growth delay was determined as the treated group vs. control differences in time required to a size four times that of the initial volume. For cells both in the monolayer culture and the multicellular spheroid, the dose response curve for ADM, BLM and 5-FU was "biphasic" and that for ACNU, ACR and CDDP "shoulder-threshold" type.(ABSTRACT TRUNCATED AT 250 WORDS)