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PubMed2004

A three-dimensional finite element model of an adherent eukaryotic cell.

Mechanical stimulation is known to cause alterations in the behaviour of cells adhering to a substrate. The mechanisms by which forces are transduced into biological responses within the cell remain largely unknown. Since cellular deformation is likely involved, further understanding of the biomechanical origins of alterations in cellular response can be aided by the use of computational models in describing cellular structural behaviour and in determining cellular deformation due to imposed loads of various magnitudes. In this paper, a finite element modelling approach that can describe the biomechanical behaviour of adherent eukaryotic cells is presented. It fuses two previous modelling approaches by incorporating, in an idealised geometry, all cellular components considered structurally significant, i.e. prestressed cytoskeleton, cytoplasm, nucleus and membrane components. The aim is to determine if we can use this model to describe the non-linear structural behaviour of an adherent cell and to determine the contribution of the various cellular components to cellular stability. Results obtained by applying forces (in the picoNewton range) to the model membrane nodes suggest a key role for the cytoskeleton in determining cellular stiffness. The model captures non-linear structural behaviours such as strain hardening and prestress effects (in the region of receptor sites), and variable compliance along the cell surface. The role of the cytoskeleton in stiffening a cell during the process of cell spreading is investigated by applying forces to five increasingly spread cell geometries. Parameter studies reveal that material properties of the cytoplasm (elasticity and compressibility) also have a large influence on cellular stiffness. The computational model of a single cell developed here is proposed as one that is sufficiently complex to capture the non-linear behaviours of the cell response to forces whilst not being so complex that the parameters cannot be specified. The model could be very useful in computing cellular structural behaviour in response to various in vitro mechanical stimuli (e.g. fluid flow, substrate strain), or for use in algorithms that attempt to simulate mechanobiological processes.

Actin Cytoskeleton↗
PubMed2004

Crystal structures and cellular automata.

Cellular automata are used as dynamic topological models of crystal structures based upon frameworks consisting of fundamental building blocks. Structure is considered as an automaton that works in accord with a prescribed program. Cellular automata models are constructed for metal sulfide frameworks in minerals and compounds of the pentlandite-djerfisherite-bartonite series, for zeolite ACO and compounds with pharmacosiderite structure, leucophosphite, phosphovanadylite etc.

Journal Article↗
PubMed1976

[Destruction of the aortic allo- and xenovalves of the heart depending on their position and time of transplantation].

The aortic valve, as well as the other valves of the heart, has a cellular structure. Collagenous fibres form the skeleton of the cellular structures, and the plexus of thinner fibres--the "walls". At early periods following the implantation destruction of thin fibres distributed along the periphery of the alveolus takes place. In the course of prolonged functioning of the allo- and xenovalves in a mitral position the large collagenous fibres collapse, defibration thereof develops and slits-vacuums and "cysts" are formed. In the valves which functioned for a long time in the tricuspid position no considerable destructive changes in the connective-tissue carcass take place.

Animals↗
PubMed2006

Occurrence of glucosylsucrose [alpha-D-glucopyranosyl- (1-->2)-alpha-D-glucopyranosyl-(1-->2)-beta-D-fructofuranoside] and glucosylated homologues in cyanobacteria. Structural properties, cellular contents and possible function as thermoprotectants.

Little is known about the structure and function of oligosaccharides in cyanobacteria. In this study, a new class of saccharides from Nostoc was identified by MS and NMR techniques, consisting of alpha-D-glucopyranosyl-(1-->2)-[alpha-D-glucopyranosyl-(1-->2)]n-beta-D-fructofuranosides ranging from the trisaccharide (n = 1) to decasaccharide (n = 8). In Nostoc ellipsosporum the cell content of saccharides increased 10-20-fold after heat stress (1 day, 40 degrees C) or during prolonged cultivation. Under these conditions the abundance of homologues of higher molecular mass (> pentasaccharide) increased and finally exceeded that of homologues of lower molecular mass including sucrose. Total intracellular content of the saccharides after heat stress was 5-10 mg x (g dry weight)(-1) corresponding to intracellular concentrations of 0.25-0.5% (w/v). A possible role of the oligosaccharides identified is in the protection of enzymes against heat inactivation. Whereas amylase from Nostoc was only weakly protected by the decasaccharide, alpha-amylase from porcine pancreas was more efficiently stabilized by the octasaccharide and decasaccharide. Evidence is presented for the widespread occurrence of the newly identified saccharides in cyanobacteria. The results are discussed including previous reports on cyanobacterial oligosaccharides and with respect to possible functions of these compounds in the living cell.

Amylases↗
PubMed2004

Apolipoprotein E: diversity of cellular origins, structural and biophysical properties, and effects in Alzheimer's disease.

Apolipoprotein E4 (apoE4) is a major risk factor for Alzheimer's disease (AD). Several hypotheses have been proposed to explain the association of the APOE epsilon4 allele with AD; however, the mechanisms underlying this association are largely unknown. Initially, apoE was thought to be synthesized primarily by astrocytes but not by neurons in the brain. However, subsequent studies have demonstrated that central nervous system neurons also express apoE under diverse physiological and pathological conditions. Detailed studies of the structure and biophysical properties of apoE isoforms have demonstrated unique properties distinguishing apoE4 from apoE3. Because the structural and biophysical properties of a protein determine how it functions under normal and abnormal conditions, apoE4, with its multiple cellular origins and multiple structural and biophysical properties, might contribute to the pathology of AD through several different mechanisms. Some of these mechanisms might be suitable targets for the development of new treatments for AD.

Alzheimer Disease↗
PubMed2000

Structural and cellular adaptation of duodenal iron uptake in rats maintained on an iron-deficient diet.

Iron deficiency induced in rats maintained on a commercial diet with a low iron content has been used to investigate adaptive mechanisms that enhance duodenal iron uptake. These adaptive changes have been divided into those that result from changes in villus surface area (structural adaptation) and those that reflect changes in the way individual enterocytes express iron transport function (cellular adaptation). Cellular adaptation was assessed by carrying out microdensitometry of autoradiographs prepared from duodenal tissue previously incubated for 5 min in 200 micromol/l 59Fe2+-ascorbate. Structural adaptation was studied by performing image analysis of microdissected and sectioned villi. Cellular adaptation involved increased iron uptake by enterocytes present in the lower villus. Thus iron deficiency resulted in a threefold enhanced expression of uptake in the lower 100 microm villus (3.9+/-2.4 versus 12.6+/-1.5 arbitrary units, P<0.001). Maximal uptake was reached in the upper region of both control and iron-deficient villi, but iron deficiency had no effect on cellular uptake at this part of the villus. Structural adaptation involved the lengthening (+16%, P<0.05) and broadening (+14%) of villi in the duodenum of iron-deficient rats. The resultant expansion in villus area caused a further increase in uptake that was mostly expressed in the upper villus. Maximal uptake corrected for structure occurred in the middle third of villi from control and iron-deficient rats. Cellular plus structural adaptation produced a twofold increase in iron uptake. More than half of this effect was caused by changes in villus structure. [3H]Thymidine labelling experiments revealed a slightly earlier expression of enterocyte iron uptake in iron deficiency.

Adaptation, Physiological↗
PubMed2002

Angular dependent light scattering from multicellular spheroids.

We demonstrate that the effects of cell-cell contact and of changes in cell shape have only a minor effect on the angular distribution of light scattering from mammalian fibroblast cells. This result is important for the development of light scattering as a noninvasive tool for tissue diagnostics such as cancer detection. Changes in cell organization that are not accompanied by changes in internal cellular structure may not be measurable. On the other hand, changes in internal cellular structure should be measurable without interference from changes in overall cellular organization. The second major result of this work is that there are small but significant differences between light scattering of tumorigenic and nontumorigenic cells grown in a three-dimensional culture system. The cause of the differences in light scattering are likely due to the nontumorigenic cells arresting in the G1 phase of the cell cycle, while the tumorigenic cells continue to proliferate.

Animals↗
PubMed1982

[LeuB24]insulin and [AlaB24]insulin: altered structures and cellular processing of B24-substituted insulin analogs.

We have used insulin analogs having leucine or alanine substitutions at positions B24 and B25 to examine the structural basis for insulin binding and insulin metabolism by isolated rat hepatocytes. Apparent receptor binding affinities for the analogs were in the order insulin greater than [LeuB24]insulin greater than [LeuB25]insulin = [AlaB24]insulin. Incubation of the corresponding 125I-labeled peptides with hepatocytes followed by analysis of the cell-associated products showed that [125I]iodoinsulin and [125I]iodo-[LeuB25]insulin were processed to a peptide intermediate which appeared as an ascending shoulder on the peak of cell-associated hormone during gel filtration; similar incubations using [125I]iodo-[LeuB24]insulin or [125I]iodo-[AlaB24]insulin failed to yield detectable amounts of the intermediate. In addition, assessment of the structures of insulin and the three insulin analogs by tyrosine radioiodination showed that [LeuB24]insulin and [AlaB24]insulin maintain similar solution conformations which differ from the conformations taken by insulin and [LeuB25]insulin. We conclude that (a) alterations in side-chain bulk at position B24 result in long-range structural perturbations in the insulin molecule, (b) these structural alterations lead to an altered cellular processing of the two B24 insulin analogs, and (c) the selectivity of this processing arises from events subsequent to ligand-receptor recognition.

Animals↗
PubMed2000

Characterization of the porcine gammadelta T-cell receptor structure and cellular distribution by monoclonal antibody PPT27.

The T-cell receptor (TCR) is the critical structure involved in antigen recognition of T lymphocytes. Although the pig has a large proportion of circulating T lymphocytes bearing the gammadelta TCR, their study has been impeded due to the lack of specific antibodies. Here a monoclonal antibody (mAb) PPT27 directed to gammadelta TCR is described. Flow cytometry analyses showed that the mAb recognized a subset of T lymphocytes of which the majority expressed no CD2, CD4 and CD8 whilst the minority bore CD2 and CD8. The mAb precipitated a protein of 86 000 MW under non-reducing conditions and a doublet of 43 000 MW under reducing conditions from peripheral blood T lymphocytes lysed in nonidet P-40 buffer, whilst it precipitated the CD3-TCR complex from the cells lysed in digitonin. Further analysis revealed that the antibody recognized the majority, but not all, of the gammadelta T cells, suggesting that there may be more isotypes of gammadelta TCR than currently believed. The antibody was unable to stimulate gammadelta T cells to proliferate in vitro, suggesting that these cells are activated by a different activation mechanism from that of alphabeta T cells.

Animals↗
PubMed1995

Common links in the structure and cellular localization of Rhizobium chitolipooligosaccharides and general Rhizobium membrane phospholipid and glycolipid components.

Several common links between the structural chemistry of the chitolipooligosaccharides of Rhizobium and the general rhizobial membrane lipid and lipopolysaccharide chemistry of these bacteria have been uncovered. Aspects of common chemistry include sulfation, methylation, and the position and extent of fatty acyl chain unsaturation. We find that bacteria which are known to synthesize sulfated chitolipooligosaccharides (such as Rhizobium meliloti strains and the broad-host-range Rhizobium species strain NGR234) also have sulfated lipopolysaccharides. Their common origins of sulfation have been demonstrated by using mutants which are known to be impaired in sulfating their chitolipooligosaccharides. In such cases, there is a corresponding diminution or complete lack of sulfation of the lipopolysaccharides. The structural diversity of the fatty acids observed in the chitolipooligosaccharides is also observed in the other membrane lipids. For instance, the doubly unsaturated fatty acids which are known to be predominant components of R. meliloti chitolipooligosaccharides were also found in the usual phospholipids and glycolipids. Also, the known functionalization of the chitolipooligosaccharides of R. sp. NGR234 by O- and N-methylation was also reflected in the lipopolysaccharide of this organism. The common structural features of chitolipooligosaccharides and membrane components are consistent with a substantial degree of biosynthetic overlap and a large degree of cellular, spatial overlap between these molecules. The latter aspect is clearly demonstrated here since we show that the chitolipooligosaccharides are, in fact, normal membrane components of Rhizobium. This increases the importance of understanding the role of the bacterial cell surface chemistry in the Rhizobium/legume symbiosis and developing a comprehensive understanding of the highly integrated membrane lipid and glycolipid chemistry of Rhizobium.

Carbohydrate Conformation↗
PubMed1990

The fate of free autogenous fascial grafts in the rabbit.

Fascial grafts were taken from 34 New Zealand rabbits and implanted above and below the cranial periosteum in the same animal. They were placed as four-layered folded grafts and as single-layered grafts. When harvested from 6 to 14 months after transplantation, the multi-layered grafts and the single-layered grafts on bone had maintained their bulk but consisted histologically of only a retained collagen matrix with no viable cellular structure. The one-layered grafts on periosteum, however, retained their cellular make-up with normal vascularity and normal cellular structure when harvested at approximately the same intervals.

Animals↗
PubMed1986

Fine structure of cellular and vascular reaction in brain tuberculomas: a model for phagocytosis in the CNS.

The fine structure of cells of the mononuclear phagocyte series (MPS) and a few other cells with phagocytic capacity, has been critically evaluated, mainly from an electronmicroscopic examination of the reactive border zone of 11 human brain tuberculomas, which provide ideal material for the study of macrophages. Most of them appeared to be blood monocyte-derived epithelioid cells of various forms and stages. The cytoplasm of these cells showed either more rough ER representing protein synthesising activity; or more frequently, phagosomes, phagolysosomes, dense bodies or empty vacuoles, representing various stages of ingestion and digestion of necrotic material. Often such material, which was more or less osmiophilic, was seen abundantly between the cells. These actively phagocytic cells occasionally undergoing, mitosis, are referred to as "epithelioid macrophages" and were morphologically similar to the "activated microglia" described in other conditions. They also showed a tendency to be closely adjacent to each other and occasionally fuse to form giant cells. There were also a number of lymphocytes and plasma cells. The latter showed various stages of active and granular or depleted and distended rough ER tubules, phagocytic activity and tendency to fuse. Expected vasculitis and small vessel necrosis formed part of this granulomatous reaction. Constituents of oedematous or necrosed brain tissue were seen immediately around the reactive zone of these tuberculomas, the most frequent being reactive astrocytes, many of which showed membrane-bound vacuoles. It is conceivable that the excessive pleomorphic cellular, vascular and necrotic reaction in these brain tuberculomas could have resulted from a delayed type of hypersensitivity to a very small quentity of antigenic tuberculoprotein, which probably initiates the chain of immunologic responses.

Adolescent↗
PubMed2001

Differential expression of multiple genes during articular chondrocyte redifferentiation.

Articular chondrocytes undergo a rapid change in phenotype and gene expression, termed dedifferentiation, when isolated from cartilage tissue and cultured on tissue culture plastic. On the other hand, "redifferentiation" of articular chondrocytes in suspension culture is characterized by decreased cellular proliferation and the reinitiation of synthesis of hyaline articular cartilage extracellular matrix molecules. The molecular triggers for these events have yet to be defined. Subtracted cDNA libraries representing genes involved in the early events of adult human articular chondrocyte redifferentiation were generated from human articular chondrocytes that were first cultured in monolayer, and subsequently transferred to suspension culture at 10(6) cells/ml for redifferentiation. Differential regulation of genes involved in cellular organization, nuclear structure, cellular growth regulation, and extracellular matrix deposition and remodeling were observed within 48 hr of this transfer. Many of these genes had not been previously identified in the chondrocyte differentiation pathway and a number of the isolated cDNAs did not have homologies to sequences in the public data banks. Genes involved in IL-6 signal transduction including acute phase response factor (APRF), Mn superoxide dismutase, and IL-6 itself were up-regulated in suspension culture. Membrane glycoprotein gp130, a component of the IL-6 receptor, was down-regulated. Other genes involved in cell polarity, cell adherence, apoptosis, and possibly TGF-beta signaling were differentially regulated. The differential regulation of the cytokine connective tissue growth factor (CTGF) during the early stages of articular chondrocyte redifferentiation, decreasing within 48 hours of transfer to suspension culture, was particularly interesting given its reported role in the stimulation of cellular proliferation. CTGF was highly expressed in proliferative monolayer culture, and then greatly reduced by redifferentiation in standard high-density suspension culture. When articular chondrocytes were seeded in suspension at low-density (10(4) cells/ml), however, high levels of CTGF were observed along with increased levels of mature articular cartilage extracellular matrix protein RNAs, such as type II collagen and aggrecan. Although the role of CTGF in articular cartilage biology remains to be elucidated, the results described here demonstrate the potential utility of subtractive hybridization in understanding the process of articular chondrocyte redifferentiation.

Adult↗
PubMed1991

Confocal fluorescence microscopy and three-dimensional reconstruction.

Several recent technological advances have considerably improved the field of confocal fluorescence microscopy. Improvements in confocal microscope design, new fluorescent probes and indicators, more sensitive imaging devices, and computer advances which allow for data manipulation and storage provide a convenient method to acquire complex three-dimensional (3-D) architectural details which previously were difficult or impossible to obtain from biological specimens. Applications of the laser scanning and tandem scanning confocal microscopes offer the potential for gaining powerful insights into the complex relationship of cellular structure and function. Confocal microscopy generates optical sections free from out-of-focus blur. With the development of new visualization tools to render and display complex 3-D data, a set of optical sections taken at different focal planes can be three-dimensionally reconstructed to create an animated sequence which can reveal latent features of the specimen. The combination of confocal microscopy and 3-D reconstruction provides a powerful new imaging tool to advance knowledge about structural and functional cellular properties as they occur dynamically in three dimensions.

Animals↗
PubMed1997

Self-organization of microtubules and motors.

Cellular structures are established and maintained through a dynamic interplay between assembly and regulatory processes. Self-organization of molecular components provides a variety of possible spatial structures: the regulatory machinery chooses the most appropriate to express a given cellular function. Here we study the extent and the characteristics of self-organization using microtubules and molecular motors as a model system. These components are known to participate in the formation of many cellular structures, such as the dynamic asters found in mitotic and meiotic spindles. Purified motors and microtubules have previously been observed to form asters in vitro. We have reproduced this result with a simple system consisting solely of multi-headed constructs of the motor protein kinesin and stabilized microtubules. We show that dynamic asters can also be obtained from a homogeneous solution of tubulin and motors. By varying the relative concentrations of the components, we obtain a variety of self-organized structures. Further, by studying this process in a constrained geometry of micro-fabricated glass chambers, we demonstrate that the same final structure can be reached through different assembly 'pathways.

Animals↗
PubMed1998

Drosophila betaHeavy-spectrin is essential for development and contributes to specific cell fates in the eye.

The spectrin membrane skeleton is a ubiquitous cytoskeletal structure with several cellular roles, including the maintenance of cell integrity, determination of cell shape and as a contributor to cell polarity. We have isolated mutations in the gene encoding &bgr ;Heavy-spectrin in Drosophila, and have named this essential locus karst. karst mutant individuals have a pleiotropic phenotype characterized by extensive larval lethality and, in adult escapers, rough eyes, bent wings, tracheal defects and infertility. Within karst mutant eyes, a significant number of ommatidia specifically lack photoreceptor R7 alongside more complex morphological defects. Immunolocalization of betaHeavy-spectrin in wild-type eye-antennal and wing imaginal discs reveals that betaHeavy-spectrin is present in a restricted subdomain of the membrane skeleton that colocalizes with DE-cadherin. We propose a model where normal levels of Sevenless signaling are dependent on tight cell-cell adhesion facilitated by the betaHeavy-spectrin membrane skeleton. Immunolocalization of betaHeavy-spectrin in the adult and larval midgut indicates that it is a terminal web protein, but we see no gross morphological defects in the adult apical brush border in karst mutant flies. Rhodamine phalloidin staining of karst mutant ovaries similarly reveals no conspicuous defect in the actin cytoskeleton or cellular morphology in egg chambers. This is in contrast to mutations in alpha-spectrin, the molecular partner of betaHeavy-spectrin, which affect cellular structure in both the larval gut and adult ovaries. Our results emphasize the fundamental contribution of the spectrin membrane skeleton to normal development and reveals a critical interplay between the integrity of a cell's membrane skeleton, the structure of cell-cell contacts and cell signaling.

Alleles↗
PubMed2004

Loss of chaotic trabecular structure in OPG-deficient juvenile Paget's disease patients indicates a chaogenic role for OPG in nonlinear pattern formation of trabecular bone.

The RANK-RANKL-OPG system of osteoclast regulation may play a key role in determining chaotic structure in trabecular bone. Iliac trabecular bone from juvenile Paget's disease patients deficient in functional OPG shows parallel, anisotropic structure instead of normal chaotic structure. Evidence from experimental systems suggests that RANK-RANKL-OPG controls key nonlinear "chaogenic" parameters, such as friction, forcing frequency, feedback, and boundary forcing. The RANK-RANKL-osteoprotegerin (OPG) system of osteoclast regulation may play a key role in determining chaotic structure in trabecular bone. Iliac trabecular bone from juvenile Paget's disease (JPD) patients deficient in functional OPG shows parallel, anisotropic structure instead of normal chaotic structure. Evidence from experimental systems suggests that RANK-RANKL-OPG controls key nonlinear "chaogenic" parameters, such as friction, forcing frequency, feedback, and boundary forcing. The Belousov-Zhabotinsky reaction-diffusion system, the catalytic oxidation of CO on platinum surfaces, and thermal diffusion in liquid helium allow visualization of nonlinear emergent patterns such as labyrinthine structures, turbulence, and cellular structures, all of which bear some resemblance to trabecular bone. In JPD, the gene for OPG (TNFRSF11B) is subject to an inactivating mutation, leading to increased resorption and accelerated remodeling. Histomorphometric images of iliac crest trabecular bone from teenagers suffering from JPD show a highly unusual array of parallel, regular trabecular plates, instead of the typical chaotic, fractal patterns of normal trabecular bone. Loss of OPG function is associated with a change from chaotic to regular structure, suggesting that the RANK-RANKL-OPG system is controlling key nonlinear "chaogenic" parameters. Looking at trabecular bone from the perspective of nonlinear pattern formation may help understand other phenomena, such as the marked dependence of trabecular bone's architectural and mechanical quality on remodeling rate independent of the trabecular bone mass.

Adolescent↗