Membrane-like Layer Research Articles

Non-equilibrium steady states as saddle points and EDP-convergence for slow-fast gradient systems

The theory of slow-fast gradient systems leads in a natural way to non-equilibrium steady states, because on the slow time scale the fast subsystem stays in steady states that are controlled by the interaction with the slow system. Using the theory of convergence of gradient systems depending on a small parameter ɛ (here the ratio between the slow and the fast time scale) in the sense of the energy-dissipation principle shows that there is a natural characterization of these non-equilibrium steady states as saddle points of a so-called B-function where the slow variables are fixed. We give applications to slow-fast reaction-diffusion systems based on the so-called cosh-type gradient structure for reactions. It is shown that two binary reactions give rise to a ternary reaction with a state-dependent reaction coefficient. Moreover, we show that a reaction-diffusion equation with a thin membrane-like layer convergences to a transmission condition, where the formerly quadratic dissipation potential for diffusion convergences to a cosh-type dissipation potential for the transmission in the membrane limit.

Engineering the Coacervate Microdroplet Interface via Polyelectrolyte and Surfactant Complexation.

Complex coacervate microdroplets, which are formed via spontaneous liquid-liquid phase separation by mixing two oppositely charged polyelectrolytes in water, have emerged as a new paradigm in the fields of origin of life, membraneless subcellular compartmentalization, bioreactors, and drug delivery. However, how to further improve its stability and enhance its selectivity in one particular coacervate system remains a challenge. By generating a membrane-like layer at the surface of coacervate microdroplets via electrostatic interactions between oppositely charged surfactants and polyelectrolytes, we here achieve tunable permeability and enhanced stability of the coacervates at the same time. Depending on the surfactants used, membrane-like layer-coated coacervate microdroplets exhibit different selectivity over solute sequestration and can promote or inhibit DNA hybridization. Our approach provides a practical tool to engineer functional bioinspired microcompartments with potential applications in the fields of controlled drug release and microreactor technology.

In vivo engraftment into the cornea endothelium using extracellular matrix shrink-wrapped cells

Cell injection is a common clinical approach for therapeutic delivery into diseased and damaged tissues in order to achieve regeneration. However, cell retention, viability, and engraftment at the injection site have generally been poor, driving the need for improved approaches. Here, we developed a technique to shrink-wrap micropatterned islands of corneal endothelial cells in a basement membrane-like layer of extracellular matrix that enables the cells to maintain their cell-cell junctions and cytoskeletal structure while in suspension. These μMonolayers exhibited the ability to rapidly engraft into intact, high-density corneal endothelial monolayers in both in vitro and in vivo model systems. Importantly, the engrafted μMonolayers increased local cell density, something that the clinical-standard single cells in suspension failed to do. These results show that shrink-wrapping cells in extracellular matrix dramatically improves engraftment and provides a potential alternative to cornea transplant when low endothelial cell density is the cause of corneal blindness.

Effects of microsurface structure of bioactive nanoparticles on dentinal tubules as a dentin desensitizer.

In this in vitro study, spherical mesoporous bioactive glass nanoparticle (MBGN) and non-porous bioactive glass nanoparticle (BGN) were fabricated. The impact of mesopores on dentinal tubule occlusion and bioactivity was compared to examine the potential of these materials in alleviating dentine hypersensitivity (DH). MBGN, dense BGN were synthesized by sol-gel methods and characterized. Bioactivity and ion dissolution ability were analyzed. Twenty-four simulated sensitive dentin discs were prepared and randomly divided into three groups (n = 8 each); Group 1, no treatment; Group 2, Dense BGN; Group 3, MBGN. Then, four discs per group were treated with 6wt.% citric acid challenge to determine the acidic resistance. The effects on dentinal tubule occlusion were observed by FESEM. The microtensile bond strength (MTBS) was also measured. Cytotoxicity was examined using the MTT assay. According to the results, dense BGN without mesopore and MBGN with mesopore were successfully fabricated. Dense BGN and MBGN occluded the dentinal tubule before and after acid challenge. However, only MBGN formed a membrane-like layer and showed hydroxyapatite formation after soaking SBF solution. There were no significant differences in MTBS among dense BGN, MBGN (P>0.05). The cell viability was above 72% of both materials. The higher bioactivity of MBGN compared with that of dense BGN arises from the structural difference and it is anticipated to facilitate dentin remineralization by inducing hydroxyapatite deposition within the dentinal tubule.

Identification of new components of the RipC-FtsEX cell separation pathway of Corynebacterineae.

Several important human pathogens are represented in the Corynebacterineae suborder, including Mycobacterium tuberculosis and Corynebacterium diphtheriae. These bacteria are surrounded by a multilayered cell envelope composed of a cytoplasmic membrane, a peptidoglycan (PG) cell wall, a second polysaccharide layer called the arabinogalactan (AG), and finally an outer membrane-like layer made of mycolic acids. Several anti-tuberculosis drugs target the biogenesis of this complex envelope, but their efficacy is declining due to resistance. New therapies are therefore needed to treat diseases caused by these organisms, and a better understanding of the mechanisms of envelope assembly should aid in their discovery. To this end, we generated the first high-density library of transposon insertion mutants in the model organism C. glutamicum. Transposon-sequencing was then used to define its essential gene set and identify loci that, when inactivated, confer hypersensitivity to ethambutol (EMB), a drug that targets AG biogenesis. Among the EMBs loci were genes encoding RipC and the FtsEX complex, a PG cleaving enzyme required for proper cell division and its predicted regulator, respectively. Inactivation of the conserved steAB genes (cgp_1603–1604) was also found to confer EMB hypersensitivity and cell division defects. A combination of quantitative microscopy, mutational analysis, and interaction studies indicate that SteA and SteB form a complex that localizes to the cytokinetic ring to promote cell separation by RipC-FtsEX and may coordinate its PG remodeling activity with the biogenesis of other envelope layers during cell division.

Oviduct, Egg, and Egg Sac Structures in the Siberian Salamander, <i>Salamandrella keyserlingii</i> (Caudata, Hynobiidae): a Histological and Histochemical Study

Unraveling morphological and chemical features of the eggs’ jelly layers and other clutch elements, formed by the oviduct of a female, is necessary for the understanding of reproductive adaptation in amphibians. Our study is the first microstructural and histochemical descriptions about the complexity of oviduct, ovisac, jelly layer of the ovum, and egg sac in hynobiid salamanders. We examined female Salamandrella keyserlingii in preovulatory (gravid) and ovulatory (spent) conditions using anatomical, histological, and histochemical methods. Each ovum was covered with two jelly layers. All ova from one oviduct fell into one egg sac. Inner space of the oviduct, in which ova were passed through, was filled with jelly-like substances designated as «intermediate fluids». We named the membrane-like outer layer of the egg sac as «theca». Jelly layers surrounding the ovum were formed by the oviducal tubular glands of the pars recta and pars convoluta of the oviduct, consisting of acidic and neutral glycoproteins. In the ovisac (i.e., homologous uterus), there were three glandular regions and one aglandular dilatable ovisac. Of the ovisac, glands A, B, and C secreted acidic and neutral glycoproteins for the intermediate fluids, neutral glycoproteins for the theca, and mucus-like materials with a neutral glycoprotein nature, respectively. We suggest that these mucus-like materials are essential to attaching the adhesive tips of the egg sacs to some substrates during oviposition.

Effect of different sizes of bioactive glass-coated mesoporous silica nanoparticles on dentinal tubule occlusion and mineralization.

To synthesize two different sizes of bioactive glass-coated mesoporous silica nanoparticles (BGN@MSNs) and to investigate their effects on dentinal tubule occlusion and remineralization. Two different sizes of mesoporous silica nanoparticles (MSNs) were synthesized using the Stöber method (368A, 1840A) and coated with bioactive glass nanoparticles (BGNs) using a modified quick alkali-mediated sol-gel method (368B, 1840B). Sensitive tooth disc models were prepared and divided into six groups and the following treatments were applied: group 1-no treatment, group 2-bioglass, group 3-368A, group 4-368B, group 5-1840A, and group 6-1840B. Then, five discs were selected from each group and soaked in 6wt% citric acid to test acid resistance. Dentinal tubule occlusion and occlusion ratio were observed using field-emission scanning electron microscopy. In vitro mineralization tests using simulated body fluid solution were performed to evaluate the remineralization effect of the treatment. All samples effectively occluded the dentinal tubule and formed a membrane-like layer. After acid treatment, 1840B (group 6) exhibited the highest rate of dentinal tubule occlusion. Remineralization was observed in 368B and 1840B, and 1840B exhibited the fastest remineralization. Dentinal tubule remineralization induced by the BGN@MSN biocomposite can be used to stabilize long-term prognosis in dentin hypersensitivity. The 1840B induced the most efficient remineralization, and its smaller size and larger surface area were effective for remineralization. The BGN@MSN biocomposite with its smaller size and larger surface area was more effective for remineralization and dentinal tubule sealing.

Pathological changes of the nasolacrimal duct in rabbit models of chronic dacryocystitis: correlation with lacrimal endoscopic findings.

The aim of this study was to explore the pathological changes of the nasolacrimal duct in rabbits with experimentally induced obstructive dacryocystitis in correlation with lacrimal endoscopic findings. The rabbit model of obstructive dacryocystitis was created by injecting 0.15ml of self-curing resin into the lacrimal duct. The control group received 0.15ml of normal saline. Within 16weeks after the obstructive, lacrimal endoscopy and pathological examination of the nasolacrimal duct were conducted at different time points of 1, 2, 4, 8, and 16weeks. In the control group, lacrimal endoscopy revealed pink and smooth mucosa; and the pathological analysis revealed an epithelial layer that was composed of superficial columnar cells and a deep basal epithelial layer. The experimental rabbits showed clinical manifestations of obstructive dacryocystitis a week after the injection of self-curing resin. At weeks 1 and 2, the lacrimal endoscopy showed mucosal hyperemia and hemorrhagic spots on the nasolacrimal duct; and the pathological features included epithelial cell swelling and inflammatory cell infiltration. At weeks 4 and 8, the experimental group showed alternatively red and white mucosa under the lacrimal endoscopy, and the pathological features included proliferative epithelium accompanied by papillary hyperplasia. At week 16, the experimental group showed pale and coarse mucosa and white membrane-like layer covering the mucosal surface, and the pathological features included epithelial necrosis, squamous metaplasia, and sub-epithelial fibrosis. The mucosa of the nasolacrimal duct showed different pathological features at different time points after lacrimal duct obstruction, which was well correlated with the endoscopic findings. It is possible to predict the pathological stages by the endoscopic observation in NLOD patients.

Dentin sealing and antibacterial effects of silver-doped bioactive glass/mesoporous silica nanocomposite: an in vitro study.

To synthesize a silver-doped bioactive glass/mesoporous silica nanoparticle (Ag-BGN@MSN), as well as to investigate its effects on dentinal tubule occlusion, microtensile bond strength (MTBS), and antibacterial activity. Ag-BGN@MSN was synthesized using a modified "quick alkali-mediated sol-gel" method. Demineralized tooth disc models were made and divided into four groups; the following treatments were then applied: group 1-no treatment, group 2-bioglass, group 3-MSN, group 4-Ag-BGN@MSN. Next, four discs were selected from each group and soaked into 6wt% citric acid to test acid-resistant stability. Dentinal tubule occlusion, as well as the occlusion ratio, was observed using field-emission scanning electron microscopy. The MTBS was also measured to evaluate the desensitizing effect of the treatments. Cytotoxicity was examined using the MTT assay. Antibacterial activity was detected against Lactobacillus casei, and ion dissolution was evaluated using inductively coupled plasma optical emission spectrometry. Ag-BGN@MSN effectively occluded the dentinal tubule and formed a membrane-like layer. After the acid challenge, Ag-BGN@MSN had the highest rate of dentinal tubule occlusion. There were no significant differences in MTBS among the four groups (P > 0.05). All concentrations of Ag-BGN@MSN used had a relative cell viability above 72%. Ag-BGN@MSN was successfully fabricated using a modified sol-gel method. The Ag-BGN@MSN biocomposite effectively occluded dentinal with acid-resistant stability, did not decrease bond strength in self-etch adhesive system, had low cytotoxicity, and antibacterial effect. Dentinal tubule sealing induced by Ag-BGN@MSN biocomposite with antibacterial effect is likely to increase long-term stability in DH.

Synthesis of Zeolite LTN-(SOD) Self-Supporting Membranes from SiO2-Rich Industrial Waste

Synthesis of zeolite LTN (“Linde Type N”) was investigated under insertion of a SiO2-rich filtration residue (FR) from waste water cleaning of the silane production. A new synthesis procedure was therefore developed applying a flotation mechanism with the aim to grow LTN in form of thin membrane like sheets. Preparation starts with preactivation of FR by slurrying first in alkaline solution, followed by an addition of aluminate solution and citric acid. The latter was added as suitable chelating agent for the initiation of the flotation process. In the course of these experiments, we succeeded in synthesizing zeo-lite LTN with more or less zeolite SOD as byproduct in the form of a stable compact membrane-like layer at low temperature of 60℃. The crystallization was performed under isotherm static conditions in an open reaction system without addition of organic templates as structure directing agents (OSDA’s). FR was utilized as a total substitute of sodium silicate in all experiments and an expansive pre-treatment procedure like calcinations was not needed. Furthermore, membrane formation with LTN of usual synthesis needs chemically functionalized supports. In contrast self-supporting membranous LTN layers were grown for the first time in the present study.

Abstract A58: Cell motility in a basement membrane gel concentrates ECM around breast epithelial cells, a feature lost in malignant cells

Abstract Introduction: The basement membrane (BM) underlying epithelial cells plays a key role in regulating cell function and suppressing malignancy; loss of this layer of ECM proteins is a hallmark of invasive cancer. Using 3D extracellular matrix (ECM) gels, we sought to understand how this structure develops by comparing nonmalignant breast epithelial cells (BEC), which develop into polarized, growth arrested structures to malignant breast cancer cells (BCC), which form disorganized masses under the same conditions. These two cell types show different movement patterns in the ECM gels: BEC rotate around their center of mass in a process termed coherent angular motion, whereas BCC migrate randomly, with unknown consequences for assembly of ECM into a basement membrane-like layer. We investigated whether coherent angular motion could concentrate exogenous ECM into a BM like structure, and whether these changes were due to the physical forces applied by the cells on their microenvironment. Methods: To determine if cells undergoing coherent angular motion were better able to concentrate exogenous ECM at their surface compared to malignant cells, single human BEC and BCC from the HMT3522 breast cancer progression series were cultured in fluorescent laminin rich ECM gels (LrECM; using BD Matrigel), and ECM density at the cell surface was measured during growth with confocal microscopy. To determine how motility translated into forces on the microenvironment, cells were embedded in lrECM labeled with a dilute suspension of fluorescent microbeads. Confocal microscopy was performed over the first several days of culture, and traction force microscopy was used to determine the magnitude and spatial pattern of forces applied by the cells. After 5-6 days of culture, cells stained to determine whether cells had developed into polarized structures. Results: Over the first several days of culture, non-malignant BEC rotated as a coherent mass. At 24 hours, labeled ECM proteins were observed to concentrate in a spherical shell on surface of rotating clusters of BEC, whereas malignant, non-rotating cell masses had lower levels of ECM at the surface of the mass and higher levels of ECM proteins between the cells. Traction patterns between rotating and non-rotating cell masses were observed to differ- cells undergoing random motility applied strains to the ECM along the line of invasion, whereas cells undergoing coherent angular motion showed shear strains at the cell surface and even inward tractions. Conclusions: Coherent angular motion appears to affect concentration of exogenous ECM by physically pulling ECM proteins into an organized shell at the cell surface as an early aspect of formation of a basement membrane-like ECM structure in mammary epithelial cells in culture whereas this process is defective in malignant cells undergoing random motility. Further investigation will determine whether this process of motility, physically altering ECM density, is seen during normal development. Citation Format: Claire Robertson, Mina J. Bissell. Cell motility in a basement membrane gel concentrates ECM around breast epithelial cells, a feature lost in malignant cells. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A58.

Haem-assisted dityrosine-cross-linking of fibrinogen under non-thermal plasma exposure: one important mechanism of facilitated blood coagulation.

Although blood coagulation facilitated by non-thermal plasma has been reported several years ago, the insight to the involved mechanisms is still rather limited. In this work, we report our discovery of a new mechanism for the haem-promoted blood-coagulation caused by non-thermal plasma treatment. The reason for the haem role is due to that its oxidized form, namely, hematin, can promote the dityrosine cross-linking of fibrinogen, the most important coagulation protein, to form a membrane-like layer on the surface of the treated blood with plasma exposure. Both haem and non-thermal-plasma generated hydrogen peroxide are requisite for the cross-linking process. We confirmed that fibrinogen can coordinate with the haem iron to form a protein-haem complex which shows pseudo-peroxidase activity, and in the presence of hydrogen peroxide, the complex can induce the dityrosine formation between fibrinogen molecules, leading to the fibrin network necessary for the blood coagulation. Understanding of such an underlying mechanism can be useful to guide more efficient application of non-thermal plasma in the management of hemostasis, thrombosis and etc.

Formation of stromal collagen fibrils and proteoglycans in the developing zebrafish cornea

Collagen fibrils and proteoglycans are the main components of the corneal extracellular matrix and corneal transparency depends crucially on their proper arrangement. In the present study, we investigated the formation of collagen fibrils and proteoglycans in the developing cornea of the zebrafish, a model organism used to study vertebrate embryonic development and genetic disease. We employed thin-section electron microscopy to investigate the ultrastructure of the zebrafish cornea at different developmental stages. The layering of the zebrafish cornea into an epithelium, a Bowman's layer, stroma and endothelium was observed starting at 72 hr post-fertilization. At this stage, the stroma contained orthogonally arranged collagen fibrils and small proteoglycans. The density of proteoglycans increased gradually throughout subsequent development of the cornea. In the stroma of 2-week-old larvae, the collagen fibrils were organized into thin lamellae and were separated by very large, randomly distributed proteoglycans. At 4 weeks, a regular arrangement of proteoglycans in relation to the collagen fibrils was observed for the first time and the lamellae were also thickened. The present study, for the first time, provides ultrastructural details of collagen fibril and proteoglycan development in the zebrafish cornea. Furthermore, it directly correlates the collagen fibril and proteoglycan composition of the zebrafish cornea with that of the human cornea. The similarities between the two species suggest that the zebrafish could serve as a model for investigating the genetics of human corneal development and diseases.

The Crystal Structure of the Outer Membrane Protein VceC from the Bacterial Pathogen Vibrio cholerae at 1.8 Å Resolution

Multidrug resistance in Gram-negative bacteria arises in part from the activities of tripartite drug efflux pumps. In the pathogen Vibrio cholerae, one such pump comprises the inner membrane proton antiporter VceB, the periplasmic adaptor VceA, and the outer membrane channel VceC. Here, we report the crystal structure of VceC at 1.8 A resolution. The trimeric VceC is organized in the crystal lattice within laminar arrays that resemble membranes. A well resolved detergent molecule within this array interacts with the transmembrane beta-barrel domain in a fashion that may mimic protein-lipopolysaccharide contacts. Our analyses of the external surfaces of VceC and other channel proteins suggest that different classes of efflux pumps have distinct architectures. We discuss the implications of these findings for mechanisms of drug and protein export.

Laminin alpha-chain expression and basement membrane formation by MLE-15 respiratory epithelial cells.

Basement membranes have a critical role in alveolar structure and function. Alveolar type II cells make basement membrane constituents, including laminin, but relatively little is known about the production of basement membrane proteins by murine alveolar type II cells and a convenient system is not available to study basement membrane production by murine alveolar type II cells. To facilitate study of basement membrane production, with particular focus on laminin chains, we examined transformed murine distal respiratory epithelial cells (MLE-15), which have many structural and biochemical features of alveolar type II cells. We found that MLE-15 cells produce laminin-alpha5, a trace amount of laminin-alpha3, laminins-beta1 and -gamma1, type IV collagen, and perlecan. Transforming growth factor-beta1 significantly induces expression of laminin-alpha1. When grown on a fibroblast-embedded collagen gel, MLE-15 cells assemble a basement membrane-like layer containing laminin-alpha5. These findings indicate that MLE-15 cells will be useful in modeling basement membrane production and assembly by alveolar type II cells.

Lipase immobilization into porous chitoxan beads: activities in aqueous and organic media and lipase localization.

Lipases were noncovalently immobilized in Chitoxan, a polyionic hydrogel obtained by complexation between chitosan and xanthan. The properties of free and immobilized lipases have been compared. In the aqueous medium, the activity was twice as high for immobilized lipases as for free lipases. Immobilized lipases in chitoxan were able to hydrolyze triacylglycerols in three distinct organic solvent media. At the microstructural level, lipases were not distributed uniformly in the chitoxan beads. Higher concentrations of lipase were found in the outer membrane-like layer of the beads, as compared with lower concentrations in the inner part of the beads.

A New Tanarctid Arthrotardigrade with Buoyant Bodies

Four cruises have revealed a very rich tardigrade fauna inhabiting the shell gravel at the Faroe Bank and Bill Bailey Bank in the North Atlantic. This study presents the description of Tanarctus bubulubus sp. n., which is one of the many new tanarctids from the Faroe Bank. The new species is found from 104 m to 200 m and is always in clean carbonate sand to shell gravel. Currently 10 species of Tanarctus are described from various localities around the world. At the Faroe Bank 9 species new to science have been found together with T. gracilis Renaud-Mornant, 1980 and T. heterodactylus Renaud-Mornant, 1980 originally described from the Western Atlantic. T. bubulubus sp. n. (‘the balloon animal’) has been investigated using LM, SEM and TEM and is characterised by sense organs on the fourth pair of legs (P4) which terminate in nine to ten balloons each. The surface of the balloons is adhesive and in whole-mount preparations the balloons attach strongly to the coverslip. The epicuticle of the balloons consists of only a very thin membrane-like layer, which may be folded or smooth, depending of the degree of extension of the balloon. The stalk of P4 is hollow and seems to lack sensory structures. The internal space of the balloon is enclosed with a secondary membrane (inner trilaminary layer?), to which some osmiophilic granulae are attached. The balloon is filled with liquid that may be different from seawater. It is hypothesised that the balloons are used as buoyancy devices, which allow the animal to move easily in the detritus layer on top of the sediment. Live animals are observed upside-down in the water column floating with the head downwards. The dorsal surface of the animal is often covered with coccoliths of the species Emiliania huxleyi, camouflaging the animal in the sediment.

Interferon beta-1b inhibits gelatinase secretion and in vitro migration of human T cells: a possible mechanism for treatment efficacy in multiple sclerosis.

Treatment with interferon beta-1b has substantial clinical benefit in the demyelinating disease multiple sclerosis, yet the mechanism of action in the disease remains largely unknown. Gelatinase A (matrix metalloproteinase-2, 72-kd gelatinase) and B (matrix metalloproteinase-9, 92-kd gelatinase) are matrix metalloproteinases capable of enzymatic digestion of subendothelial basement membrane constituents. In human T cells, interleukin-2 induces gelatinase secretion and enhances gelatinase-dependent migration across an artificial basement membrane-like layer in vitro. Pretreatment of T cells with interferon beta-1b for 48 hours decreased interleukin-2-induced gelatinase production and secretion as determined by zymography. In parallel to the downregulation of gelatinase secretion, pretreatment with interferon beta-1b inhibited T-cell migration across the basement membrane in vitro by up to 90%, but had only a minor impact on cell locomotion per se. For both gelatinase secretion and T-cell migration, the inhibitory effect mediated by exposure to interferon beta-1b was dose dependent. Fluorescence-activated cell sorter analysis also showed that interferon beta-1b downregulates the interleukin-2 receptor alpha-chain and lowered the affinity of interleukin-2 to the cell surface by 30%, which may represent an additional mechanism for the observed effects of interferon beta-1b. The dramatic effects of interferon beta-1b on gelatinase expression and migration raise the possibility that its beneficial effects in multiple sclerosis may result from interference with the capacity of activated T cells to traverse the basement membrane and migrate to the central nervous system.

Description of Lanatospora tubulifera sp. n. (Microspora, Tuzetiidae) with Emended Diagnosis and New Systematic Position for the Genus Lanatospora

Summary Lanatospora tubulifera , a new microsporidian parasite of the ostracod Acanthocyclops vernalis is described based on light microscopic and ultrastructural characteristics. The diagnosis of the genus is emended and a new family position is suggested. The microsporidium has isolated nuclei at all stages of the life cycle. Merogony gives rise to rounded plasmodia with at least four nuclei. Sporogony produces at least 8 sporoblasts by rosette-like budding. Spores are pyriform, uninucleate and measure 3.5–4 × 2 µm when fixed and stained. The exospore consists of four layers: an electron dense layer, a double membrane-like layer, a moderately dense layer and a thin, electron dense surface layer. The polar filament is isofilar and arranged in 7–9 coils close to the spore wall in the posterior half of the spore. The polaroplast is tripartite, consisting of two lamellar parts and a posterior tubular part. The spores are surrounded by individual sporophorous vesicles, connected to the exospore by tubuli

Bcl-2 overexpression inhibits cell death and promotes the morphogenesis, but not tumorigenesis of human mammary epithelial cells [published erratum appears in J Cell Biol 1995 Nov;131(4):following 1121

Overexpression of the B cell leukemia/lymphoma-2 (bcl-2) gene has been shown to confer a survival advantage on cells by inhibiting apoptosis. In epithelia, the bcl-2 gene is also related to development and differentiation, and the protein is strongly expressed in the embryo in the epithelial cells of the developing mammary gland. To investigate directly the effect of bcl-2 on human epithelial cells, we used an amphotropic recombinant retrovirus to introduce the gene into nontumorigenic cell lines developed from luminal epithelial cells cultured from milk. Here we demonstrate that while bcl-2 overexpression does not directly induce the tumorigenic phenotype, it provides a survival advantage to the mammary epithelial cells by inhibiting cell death at confluence or under conditions of serum starvation, bcl-2 can also affect the phenotype of the original epithelial cells, and promote epithelial-mesenchymal conversion, accompanied by loss of the cell adhesion molecules E-cadherin and alpha 2 beta 1 integrin. The extent of the epithelial-mesenchymal conversion varies with small differences in the phenotype of the parental line and with the level of expression of Bcl-2 and in some cases cell lines emerge with a mixed phenotype. The increased survival of Bcl-2-expressing cells at confluence results in multilayering, and the development of three- dimensional structures. Where a mixed phenotype is observed these structures consist of an outer layer of polarized epithelial cells separated by a basement membrane-like layer from an inner mass of fibroblastoid cells. Branching morphogenesis of bcl-2 transfectants is also observed in collagen gels (in the absence of fibroblast growth factors). The results strongly indicate that by increasing their survival under restrictive growth conditions, and by modifying the epithelial phenotype, bcl-2 can influence the specific morphogenetic behavior of mammary epithelial cells.