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PubMed2001

The natural silk spinning process. A nucleation-dependent aggregation mechanism?

The spinning mechanism of natural silk has been an open issue. In this study, both the conformation transition from random coil to beta sheet and the beta sheet aggregation growth of silk fibroin are identified in the B. mori regenerated silk fibroin aqueous solution by circular dichroism (CD) spectroscopy. A nucleation-dependent aggregation mechanism, similar to that found in prion protein, amyloid beta (Abeta) protein, and alpha-synuclein protein with the conformation transition from a soluble protein to a neurotoxic, insoluble beta sheet containing aggregate, is a novel suggestion for the silk spinning process. We present evidence that two steps are involved in this mechanism: (a) nucleation, a rate-limiting step involving the conversion of the soluble random coil to insoluble beta sheet and subsequently a series of thermodynamically unfavorable association of beta sheet unit, i.e. the formation of a nucleus or seed; (b) once the nucleus forms, further growth of the beta sheet unit becomes thermodynamically favorable, resulting a rapid extension of beta sheet aggregation. The aggregation growth follows a first order kinetic process with respect to the random coil fibroin concentration. The increase of temperature accelerates the beta sheet aggregation growth if the beta sheet seed is introduced into the random coil fibroin solution. This work enhances our understanding of the natural silk spinning process in vivo.

Animals↗
PubMed1989

Role of posttranslational cleavage in glycinin assembly.

Glycinin, like other 11S seed storage proteins, undergoes a complex series of posttranslational events between the time proglycinin precursors are synthesized in endoplasmic reticulum and the mature glycinin subunits are deposited in vacuolar protein bodies. According to the current understanding of this process, proglycinin subunits aggregate into trimers in endoplasmic reticulum, and then the trimers move to the vacuolar protein bodies where a protease cleaves them into acidic and basic polypeptide chains. Stable glycinin hexamers, rather than trimers, are isolated from mature seeds. We used a re-assembly assay in this study to demonstrate that proteolytic cleavage of the proglycinin subunits is required for in vitro assembly of glycinin oligomers beyond the trimer stage. The possibility that the cleavage is a regulatory step and that it triggers the deposition of 11S seed storage proteins as insoluble aggregates in vivo is considered.

Base Sequence↗
PubMed1982

Gut and bronchus associated lymphoid tissue: an overview.

The mucosal lymphoid aggregates in lung and gut contain precursor cells destined to seed mucosal tissues with IgA containing cells. It is likely that both tissues also are sources of both IgE B cell precursors as well as the cells responsible for their regulation (38). Although the traffic of B cells is relatively well understood from the standpoint of IgA, the factors responsible for localization in mucosal tissue are at best unclear. Furthermore, little is known about the migration patterns of helper or suppressor cells of any kind and these may have, if derived from mucosal tissue, a preferential site of action in the mucosa. The mucosal mast cell may well be a cell of different lineage than the mast cell from the peritoneal cavity and may itself have a mucosal localization pattern. Much more work needs to be done to render some of these speculations onto a better factual base, and to harness these systems in provision of better approaches to vaccination and control of disease.

Animals↗
PubMed2005

Study of gelatin-containing artificial skin V: fabrication of gelatin scaffolds using a salt-leaching method.

Porous gelatin scaffolds were prepared using a salt-leaching method and these were compared to scaffolds fabricated using a freeze-drying method. The salt-leached gelatin scaffolds were easily formed into desired shapes with a uniformly distributed and interconnected pore structure with an average pore size of around 350 microm. The mechanical strength and the biodegradation rate of the scaffolds increased with the porosity, and were easily modulated by the addition of salt. After 1 week of in vitro culturing, the fibroblasts in salt-leached scaffolds were mainly attached on the surface of the pores in the scaffold, whereas cells seeded on freeze-dried scaffolds were widely distributed and aggregated on the top and the bottom of the scaffold. After 14 d of culturing, the fibroblasts showed a good affinity to, and proliferation on, the gelatin scaffolds without showing any signs of biodegradation. An in vivo study of cultured artificial dermal substitutes showed that an artificial dermis containing the fibroblasts enhanced the re-epithelialization of a full-thickness skin defect when compared to an acellular scaffold after 1 week.

Animals↗
PubMed1997

The 'assembly-promoting sequence region' of microtubule-associated protein 4 failed to promote microtubule assembly.

In order to study the function of the bovine MAP4 microtubule-binding domain (the assembly-promoting (AP) sequence region), a fragment corresponding to the AP sequence region was prepared using an Escherichia coli expression system. When the fragment was mixed with purified tubulin at 37 degrees C, the fragment caused a time- and dose-dependent turbidity increase, and the fragment bound to tubulin. However, the products were cold-stable, and amorphous aggregates were observed by electron microscopy. Using axonemes as the seeds for microtubule assembly, the microtubule-elongating activity of the fragment was examined. A dose-dependent turbidity increase of the sample was observed, and electron microscopic observation revealed that microtubules were dose-dependently elongated from the axonemes. Consequently, the AP sequence region does not nucleate microtubules, but elongates them.

Adrenal Cortex↗
PubMed1998

Self-oligomerization of NACP, the precursor protein of the non-amyloid beta/A4 protein (A beta) component of Alzheimer's disease amyloid, observed in the presence of a C-terminal A beta fragment (residues 25-35).

NACP, the precursor protein of the non-amyloid beta/A4 protein (A beta) component of Alzheimer's disease (AD) amyloid, also known as alpha-synuclein, was suggested to seed amyloid plaque formation in AD by stimulating A beta aggregation. We have demonstrated that NACP experienced self-oligomerization only in the presence of a modified A beta fragment (A beta25-35) by using dicyclohexylcarbodiimide. This NACP oligomerization, appearing as a discrete ladder on a Tricine SDS-PAGE, was not observed with other A beta peptides such as the reverse peptide A beta35-25 and A beta1-40, indicating this process was specific not only for the C-terminal peptide sequence of the A beta but also for its orientation. It might be, therefore, suggested that the NACP self-oligomers formed only in the presence of a N-terminally truncated A beta peptide could act as a nucleation center for plaque formation during AD development.

Alzheimer Disease↗
PubMed1981

Nitrogen fixation (acetylene reduction) by Klebsiella pneumoniae in association with 'Park' Kentucky bluegrass (Poa pratensis L.).

Turks of 'Park' Kentucky bluegrass reestablished in the greenhouse and inoculated with Klebsiella pneumoniae (W6) showed significantly increased nitrogen fixation (acetylene reduction) compared with control turfs. Mean ethylene production rates per pot were 368 nmol h-1 for K. pneumoniae treated turfs, 55 nmol h-1 for heat-killed K. pneumoniae treated turfs, and 44 nmol h-1 for untreated turfs. Calculated lag periods before activity was observed were generally very short (less than 1 h). When 'Park' Kentucky bluegrass was grown from seed on soil-less medium of Turface, a fired aggregate clay, inoculation with K. pneumoniae (W6) resulted in 9 of 11 turfs showing nitrogenase activity (mean ethylene production rate per pot was 195 nmol h-1). Only 3 of 11 turfs treated with heat-killed K. pneumoniae showed any activity and their mean rate of ethylene production (40 nmol h-1 per pot) was significantly lower than that for turfs treated with K. pneumoniae. Using the 'Park'--Turface soil-less model system it was shown that acetylene reducing activity was (i) root associated, (ii) generally highest at a depth of 1--4 cm below the surface, (iii) enhanced by washing excised roots, and (iv) inhibited by surface sterilization of excised roots. Klebsiella pneumoniae was recovered from Turface and roots showing acetylene reducing activity.

Acetylene↗
PubMed1985

Primary culture of duck salt gland. I. Morphology of confluent cell layers.

Dissociated avian salt gland secretory cells were maintained in primary culture after plating on hydrated collagen gels. When seeded at 3 X 10(6) cells/cm2, confluent cell sheets formed within 2-3 days, whereas cultures seeded at lower densities formed a complex reticulum of cell aggregates, which remained nonconfluent even after 7 days. Scanning electron microscopy showed that the free surface of 3-day confluent cultures consisted of intermixed convex and flattened cell membranes with prominent junctional boundaries and abundant microvilli. Transmission electron microscopy indicated that these cultures were multilayers of 1-4 cells in thickness. The plasma membranes of the superficial cells were polarized into apical and basolateral regions displaying, respectively, microvilli and interdigitating lateral membrane folds. These membrane domains were separated by shallow occluding junctions, which consisted of both single strands and simple net-like arrays in freeze-fracture images. Underlying epithelial cells retained lateral membrane folds and formed desmosomal contacts with superficial and neighboring cells. These cultures, unlike the intact tissue, allow direct access to the apical and basolateral cell surfaces for electrophysiological analysis of transmural active ion transport.

Animals↗
PubMed1977

Dissociation, aggregation and denaturation of sesame alpha-globulin in urea and guanidine hydrochloride solutions.

The effect of urea and GuHCl on the major protein of sesame seed (Sesamum indicum L.), alpha-globulin, has been investigated by turbidity, sedimentation velocity, viscosity, difference spectra and fluorescence spectral measurements. The protein undergoes dissociation, aggregation and denaturation in the presence of the above denaturants. There is a critical concentration of the denaturant where aggregation is maximum. Both denaturation and aggregation are lower in buffers of high ionic strength. Dissociation and aggregation have been explained by considering two types of subunits present in the protein molecule, one leading to smaller sedimenting component and the other producing the aggregate. The amino acid analysis shows that the aggregated fraction is rich in aliphatic amino acid residues. The endothermic nature of the aggregation process has been considered to arise from hydrophobic interaction of aliphatic side chains of the relevant subunits. The protein exists in a more denatured state in GuHCl than in urea solution.

Amino Acids↗
PubMed2002

Exploring protein aggregation and self-propagation using lattice models: phase diagram and kinetics.

Many seemingly unrelated neurodegenerative disorders, such as amyloid and prion diseases, are associated with propagating fibrils whose structures are dramatically different from the native states of the corresponding monomers. This observation, along with the experimental demonstration that any protein can aggregate to form either fibrils or amorphous structures (inclusion bodies) under appropriate external conditions, suggest that there must be general principles that govern aggregation mechanisms. To probe generic aspects of prion-like behavior we use the model of Harrison, Chan, Prusiner, and Cohen. In this model, aggregation of a structure, that is conformationally distinct from the native state of the monomer, occurs by three parallel routes. Kinetic partitioning, which leads to parallel assembly pathways, occurs early in the aggregation process. In all pathways transient unfolding precedes polymerization and self-propagation. Chain polymerization is consistent with templated assembly, with the dimer being the minimal nucleus. The kinetic effciency of R(n-1) + G --> R(n) (R is the aggregation prone state and G is either U, the unfolded state, or N, the native state of the monomer) is increased when polymerization occurs in the presence of a "seed" (a dimer). These results support the seeded nucleated-polymerization model of fibril formation in amyloid peptides. To probe generic aspects of aggregation in two-state proteins, we use lattice models with side chains. The phase diagram in the (T,C) plane (T is the temperature and C is the polypeptide concentration) reveals a bewildering array of "phases" or structures. Explicit computations for dimers show that there are at least six phases including ordered structures and amorphous aggregates. In the ordered region of the phase diagram there are three distinct structures. We find ordered dimers (OD) in which each monomer is in the folded state and the interaction between the monomers occurs via a well-defined interface. In the domain-swapped structures a certain fraction of intrachain contacts are replaced by interchain contacts. In the parallel dimers the interface is stabilized by favorable intermolecular hydrophobic interactions. The kinetics of folding to OD shows that aggregation proceeds directly from U in a dynamically cooperative manner without populating partially structured intermediates. These results support the experimental observation that ordered aggregation in the two-state folders U1A and CI2 takes place from U. The contrasting aggregation processes in the two models suggest that there are several distinct mechanisms for polymerization that depend not only on the polypeptide sequence but also on external conditions (such as C, T, pH, and salt concentration).

Dimerization↗
PubMed2005

The aggregation kinetics of Alzheimer's beta-amyloid peptide is controlled by stochastic nucleation.

We report here a recombinant expression system that allows production of large quantities of Alzheimer's Abeta(1-40) peptide. The material is competent to dissolve in water solutions with "random-coil properties," although its conformation and factual oligomerization state are determined by the physico-chemical solution conditions. When dissolved in 50 mM sodium phosphate buffer (pH 7.4) at 37 degrees C, the peptide is able to undergo a nucleated polymerization reaction. The aggregation profile is characteristically bipartite, consisting of lag and growth phase. From these curves we determined the lag time as well as the rate of aggregation. Both values were found to depend on peptide concentration and addition or formation of seeds. Moreover, they can vary considerably between apparently identical samples. These data imply that the nucleation event is under influence of a stochastic factor that can manifest itself in profound macroscopic differences in the aggregation kinetics of otherwise indistinguishable samples.

Amyloid beta-Peptides↗
PubMed2005

Independent heterologous fibrillation of insulin and its B-chain peptide.

Insulin is very prone to form amyloid fibrils under slightly destabilizing conditions, and the B-chain region plays a critical role in the fibrillation. We show here that the isolated B-chain peptide of bovine insulin also forms fibrils at both acidic and neutral pH. When a mixture of insulin and the B-chain peptide was incubated at either acidic or neutral pH, the formation of fibrils was clearly separated into two phases, with the faster phase corresponding to the formation of homogeneous fibrils from the B-chain and the slower phase corresponding to homogeneous fibrillation of insulin. To further investigate the interaction (or lack thereof) between the two polypeptides, we examined the effects of cross-seeding. The results indicate that seeds of B-chain fibrils accelerate the fibrillation of insulin at pH 1.6 and inhibit the fibrillation at pH 7.5, but seeds of insulin fibrils have little effect on the fibrillation of the B-chain. We conclude that at pH 7.5 simultaneous independent homologous fibrillation occurs, but at low pH, heterologous fibrillation takes place, and with B-chain seeding of insulin, a unique conformation of fibrils is formed. Our results demonstrate that in the co-aggregation of closely related peptides each peptide species may undergo concurrent homogeneous or heterologous polymerization and that fibrils of one species may or may not seed fibrillation of the other. The results demonstrate the significant "species" barrier in amyloid fibril formation between fibrillation induced by different fibrils. A model for the fibrillation of the heterogeneous system of insulin and B-chain insulin is proposed.

Amino Acid Sequence↗
PubMed1993

Patency and morphology of fibrous polyurethane vascular prostheses implanted in the femoral artery of dogs after seeding with subcultivated endothelial cells.

A cell culture line was established from enzymatically-derived canine jugular endothelial cells and further cultured. Whenever sufficient cells were present, fibrous polyurethane vascular prostheses, impregnated with gelatin and coated with fibronectin, were seeded with 4.8 x 10(5)/cm2 cells, sufficient to establish a confluent monolayer, and implanted in the femoral arteries of 16 dogs. A non-seeded prosthesis on the contralateral side served as control. Eight dogs received antiplatelet aggregation medication: 250 mg aspirin together with 25 mg dipyridamole, orally three times daily, starting 2 weeks prior to the implantation operation and continued for the duration of the experiment. Results show that in the non-medicated dogs all control prostheses become occluded within 3 weeks after implantation, whereas five out of eight seeded prostheses remained patent. In the medicated group, two out of eight control prostheses occluded and all seeded prostheses remained patent. Scanning and light microscopy revealed that seeded prostheses were completely lined with endothelial cells (Factor VIII positive stain) week 3 (n = 3) and 12 (n = 3) after implantation, while endothelialisation in control prostheses had advanced only 5 mm into the prostheses in 12 weeks. Two dogs of each group were included in long-term patency studies. We conclude that prostheses seeded with a confluent monolayer of endothelial cells result in superior patency rates for both medicated and non-medicated dogs. No immunological reaction against the (allogeneic) seeded endothelial cells were noted.

Anastomosis, Surgical↗
PubMed2002

Growth of purified lacrimal acinar cells in Matrigel raft cultures.

The objective of this study was to develop a tissue culture system which closely mimics the in situ lacrimal gland for improved study of lacrimal acinar cell physiology. Highly purified preparations of lacrimal acinar cells from adult female New Zealand White rabbits were isolated and grown in suspension culture in the form of Matrigel 'rafts', i.e., aggregates of acinar cells enclosed within a Matrigel coating. The rafts were seeded onto Matrigel-coated culture plates and their growth was followed for up to 28 days. Immunohistochemistry was used to demonstrate the cellular sites of prolactin (PRL), epidermal growth factor (EGF), basic fibroblast growth factor (FGF-2), secretory component (SC) and major histocompatibility complex class-II molecules (MHC-II) within the acinar cells. By 3 days the cultures contained numerous, well-formed acini enclosed within the Matrigel. The acinar epithelial cells demonstrated histotypic polarity, with large, pale-staining, secretory granules aggregated adjacent to the lumen, and exocytotic release of secretory material into the lumen. From 5-10 days the pale-staining secretory granules decreased in number, while the lumenal contents of the acini increased in staining density. Throughout the culturing period as the pale-staining, secretory granules decreased in number, smaller more densely stained, secretory granules increased in number. The number of cells and size of acinar clusters increased steadily throughout the culturing period, and acini frequently achieved dimensions in excess of 0.5 mm. Increases in the size of acinar clusters were often accompanied by an increase in the size of the lumen. Frequently the lumen and its contents bulged asymmetrically towards one edge of the acinus. Immunhistochemistry demonstrated PRL and EGF within the lumens and within the apical cytoplasm of the acinar cells. Acini were strongly immunopositive for SC throughout the 28 day culture period, whereas immunopositivity for MHC-II molecules was strong initially, but diminished dramatically by 21 days. Immunostaining for FGF-2 was most intense on days 1 and 3, with staining throughout the cytoplasm, but became progressively more localized to the periphery of the acini as the culture period lengthened. In cultures of 1-28 days duration, Western blots of cell lysates demonstrated a major band (approximately 40 kDa) for PRL in 3-28 day preparations; a major band (approximately 80 kDa) for SC in 3 day and 7 day preparations that decreased in intensity in 14-28 day preparations; and a major band (approximately 23 kDa) for MHC-II protein in 1-21 day preparations that decreased in intensity in 28 day preparations. Lysosomes increased in number with time in culture, becoming a dominant cytoplasmic feature in 21 and 28 day cultures. Carbachol stimulation of 4 day rafts resulted in increased release of beta-hexosaminidase and SC from the rafts. The authors conclude that Matrigel rafts containing purified lacrimal gland acinar cells offer a highly advantageous system for study of lacrimal acinar cell function and one that correlates well with the in situ gland.

Animals↗
PubMed2002

[Effect of high hydrostatic pressure on water imbibition, cooking times and microstructure of Phaseolus vulgaris].

The effect of high hydrostatic pressure (HHP) on water imbibition, cooking times, and microstructure of cotyledons and seed coats of black beans was evaluated. High pressure treatments at 275, 410, 550 or 690 MPa and 25 degrees C for 5 min increased the rate of water imbibition of black beans. The saturation degree of HHP treated black beans was reached 50% faster than the saturation degree of untreated black beans. High hydrostatic pressure treatments reduced the cooking times (CT) of unsoaked black beans by 25% to 39%, similar to black beans soaked for 3 h. The microstructure of HHP treated beans exhibited protein aggregation in cell protoplasm and swelling of cell walls and starch granules. Seed coats of HHP treated black beans were less smooth than seed coats of untreated black beans. The swelling of palisade, hourglass, and parenchyma cells was also observed in HHP treated black beans such as soaked beans. High hydrostatic treatment is an alternative for reducing the preparation long times of Phaseolus vulgaris.

Absorption↗
PubMed2002

Resistance of multicellular aggregates to pharmorubicin observed in human hepatocarcinoma cells.

The objective of the present study was to investigate the multicellular resistance of human hepatocarcinoma cells BEL-7402 to pharmorubicin. Cells (1 x 10(4)) and 200 microcarrier Cytodex-3 beads were seeded onto a 24-well plate and cultured in RPMI 1640 medium. After the formation of multicellular aggregates, morphology and cell viability were analyzed by scanning electron microscopy, transmission electron microscopy and flow cytometry, respectively. The IC50 was determined by flow cytometry and MTT assay after the cells cultured in aggregates and monolayers were treated with pharmorubicin. The culture products exhibited structural characteristics somewhat similar to those of trabecular hepatocarcinoma in vivo. Among the microcarriers, cells were organized into several layers. Intercellular spaces were 0.5-2.0 microm wide and filled with many microvilli. The percent of viable cells was 87%. The cells cultured as multicellular aggregates were resistant to pharmorubicin with IC50 4.5-fold and 7.7-fold that of monolayer culture as determined by flow cytometry and MTT assay, respectively. This three-dimensional culture model may be used to investigate the mechanisms of multicellular drug resistance of hepatocarcinoma and to screen new anticancer drugs.

Antibiotics, Antineoplastic↗
PubMed1992

A protease responsible for post-translational cleavage of a conserved Asn-Gly linkage in glycinin, the major seed storage protein of soybean.

The assembly of 11S globulin seed storage proteins in plants is regulated in part by the activity of a protease that cleaves between asparagine and glycine residues. Post-translational cleavage of subunit precursors into acidic and basic polypeptides is associated with the ability of subunits in trimers to aggregate into hexamers in vitro. An activity is present in extracts from immature soybean seeds that specifically cleaves immature 11S seed storage proteins of soybean and Vicia faba into the polypeptides of the mature proteins. Sequence microanalysis has been used to demonstrate that proglycinin and prolegumin are cut at the legitimate site when proteins synthesized in vitro are used as substrates. A single amino acid change in the cleavage site renders the substrate uncleavable. The protease responsible for this activity also hydrolyzes a synthetic octapeptide whose sequence reproduces four amino acids on either side of the glycinin subunit G4 cleavage site. This assay permitted the purification and characterization of the protease. It is a glycosylated enzyme with an acidic pH optimum and a molecular mass of about 45 kDa in solution.

Amino Acid Sequence↗
PubMed2005

Culture of isolated single cells from Taxus suspensions for the propagation of superior cell populations.

Single cells isolated from aggregated Taxus cuspidata cultures via enzymatic digestion were grown in suspension culture. High seeding density (4 x 10(5 )cells/ml) and the addition of cell-free conditioned medium were essential for growth. Doubling the concentration of the nutrients [ascorbic acid (150 g/l), glutamine (6.25 mM: ), and citric acid (150 g/l)] had no effect on single cell growth or viability. A specific growth rate of 0.11 days(-1) was achieved, which is similar to the observed growth rate of aggregated Taxus suspensions. The biocide, Plant Preservative Mixture, added at 0.2% (v/v) to all single cell cultures to prevent microbial contamination, had no significant effect on growth or viability. Following cell sorting, single cell cultures can be used to establish new cell lines for biotechnology applications or provide cells for further study.

Biotechnology↗