Secretions of interleukin-1beta and tumor necrosis factor alpha by whole fetal membranes depend on initial interactions of amnion or choriodecidua with lipopolysaccharides or group B streptococci

The present study evaluated the secretions of interleukin (IL)-1beta and tumor necrosis factor (TNF) alpha by fetal membranes stimulated with group B streptococci (GBS) and lipopolysaccharide (LPS). The aim was to evaluate the initial response of full-thickness membranes to the microbial insult using an in vitro experimental model that allowed testing of the individual contributions of amnion and choriodecidua to stimulation. Full-thickness membranes were obtained after delivery by elective cesarean section from women at 37-40 wk of gestation without evidence of active labor. The membranes were mounted in Transwell devices, physically separating the upper and lower chambers. The LPS (500 ng/ml) or GBS (1 x 10(6) colony-forming units/ml) was added to either the amniotic or choriodecidual surface, and accumulation of IL-1beta and TNFalpha were measured in both compartments using a specific ELISA. Fetal membranes followed different patterns of secretion of proinflammatory cytokines that depended on the side to which the stimulus was added or the nature of the stimulus itself. The TNFalpha was secreted by amnion and choriodecidua in the presence of LPS or GBS, and stimulation with GBS induced a greater synthesis of IL-1beta than did stimulation with LPS. Choriodecidual tissue was more responsive than amniotic tissue, and this response tended to be higher even when the stimulation was only on the amniotic side. However, the amnion plays an active role in recognizing LPS or GBS, contributing a significant amount of TNFalpha. Thus, cooperative and bidirectional communications occur between amnion and choriodecidua in response to bacterial products, which include intermembranous cytokine traffic and signaling between tissues.     

Phagocytosis of collagen fibrils by periosteal fibroblasts in long bone explants. Effect of concanavalin A

In an attempt to determine whether phagocytosis of collagen by fibroblasts involves binding of the fibril to the plasma membrane, the effect of the lectin concanavalin A (Con A) was studied in an in vitro model system. Metacarpal bone rudiments from 19-day-old mouse fetuses were incubated with varying concentrations of the lectin. Quantitative electron microscopic analysis indicated that Con A caused a dose-related increase in the amount of phagocytosed collagen fibrils in periosteal fibroblasts, suggesting either an enhanced uptake or a decreased intracellular breakdown of fibrils. Since a Con A-inducible increase was not seen in the combined presence of both the lectin and the proteinase inhibitor leupeptin, which is known to inhibit the intracellular digestion of phagocytosed fibrillar collagen, it is unlikely that Con A stimulated phagocytosis. Based on the finding that Con A interfered with the digestion of a synthetic substrate by the collagenolytic lysosomal enzyme cathepsin B it is suggested that the augmentation of intracellular fibrillar collagen under the influence of the lectin was due to a decreased intracellular digestion. Since Con A did not inhibit the uptake of collagen fibrils by the fibroblasts it is concluded that Con A-inhibitable binding sites for collagen molecules are unlikely to be involved in phagocytosis of collagen fibrils by fibroblasts.     

THE FINE STRUCTURE OF CHONDROCOCCUS COLUMNARIS : III. The Surface Layers of Chondrococcus columnaris

An electron microscope study of the myxobacterium Chondrococcus columnaris has revealed the following structures in the peripheral layers of the cells: (1) a plasma membrane, (2) a single dense layer (probably the mucopeptide component of the cell wall), (3) peripheral fibrils, (4) an outer membrane, and (5) a material coating the surfaces of the cells which could be stained with the dye ruthenium red.The ruthenium red-positive material is probably an acid mucopolysaccharide and may be involved in the adhesive properties of the cells. The outer membrane and plasma membrane both have the appearance of unit membranes: an electron-translucent layer sandwiched between two electron-opaque layers. The peripheral fibrils span the gap between the outer membrane and the mucopeptide layer, a distance of about 100 A, and run parallel to each other along the length of the cell. The fibrils appear to be continuous across the ends of the cells. The location of these fibrillar structures suggests that they may play a role in the gliding motility of these bacteria.     

Ultrastructural study of upper surface layer in rat mandibular condylar cartilage by quick-freezing method

The purpose of the present study is to clarify native ultrastructures of upper surface layers of the rat mandibular condylar cartilage in vivo by a quick-freezing method. The mandibular cartilaginous tissues were removed with their articular discs attached without opening the lower joint cavity. The specimens were processed for light microscopy, transmission or scanning electron microscopy. Deep-etching replica membranes were also prepared after the routine quick-freezing method. The upper surface layer was well preserved by the quick-freezing method. The cartilaginous tissues, which were fixed without opening their articular discs, appeared to keep better morphology than those after opening them. The upper surface layer was thicker than the corresponding layer as reported before. It consisted of atypical extracellular matrices with lots of apparently amorphous components, which were distributed over typical collagen fibrils, by conventional electron microscopy. As revealed with the replica membranes, it also consisted of variously sized filaments and tiny granular components localized on the typical collagen fibrils. A pair of stereo-replica electron micrographs three-dimensionally showed compact filaments within the upper surface layer. The quick-freezing method was useful for keeping native ultrastructures of the fragile upper surface layer in the mandibular condylar cartilage, which may be functionally important to facilitate smooth movement of the temporomandibular joint.     

Reduced collagen and ascorbic acid concentrations and increased proteolytic susceptibility with prelabor fetal membrane rupture in women

Prelabor rupture of the fetal membranes affects approximately 10% of women at term, resulting in an increased risk of maternal and neonatal infection. Evidence suggests that membrane rupture is related to biochemical processes involving the extracellular matrix of the membranes. We tested the hypothesis that prelabor ruptured membranes are characterized by reduced collagen concentrations, altered collagen cross-link profiles, and increased concentrations of biomarkers of oxidative damage. We also set out to determine whether these effects are modulated by ascorbic acid status. In a case-control study, we explored the role that ascorbic acid, oxidative stress, collagen, and collagen cross-links play in determining membrane integrity and developed a functional assay to assess membrane proteolytic susceptibility. Prelabor ruptured membrane had a reduced ascorbic acid concentration in comparison with controls while protein carbonyl and malondialdehyde concentrations were increased. Collagen concentrations were also reduced in prelabor ruptured membrane, and while the concentration of collagen cross-links was not significantly different between prelabor and timely ruptured membrane, there was a regional variation in cross-link ratio within the amniotic sac. Proteolytic resistance in vitro was reduced in prelabor ruptured membrane and also exhibited regional variation within the amniotic sac. Our findings are strongly supportive of a role for the enhanced degradation of membrane collagen in the determination of prelabor rupture of fetal membranes. The formation of the rupture initiation site is a function of a regional variation in collagen cross-link ratio. Tissue ascorbic acid status may be an important mediator of these processes.     

Location and identification of the collagen found in the 14.5-d rat embryo visceral yolk sac

The collagens associated with 14.5-d rat visceral yolk sacs were localized and identified by a variety of procedures. Morphological examination showed that both the visceral epithelium and mesothelium rested upon thin basement membranes, whereas the majority of the extracellular matrix consisted of a stroma containing occasional cells and abundant banded fibrils. Immunohistochemistry at the electron microscope level showed that the basement membranes specifically cross- reacted with antibodies directed against mouse basement membrane components, whereas the stroma specifically cross-reacted with antibodies directed against rat type I collagen. Extractions of acellular visceral yolk sacs and subsequent analyses showed that type I collagen components were prevalent. Furthermore, in vitro biosynthetic studies showed only the presence of type I procollagen components (or their conversion products) and alpha-fetoprotein. These findings, taken together with our previous studies on the 14.5-d rat parietal yolk sac, provide us with protein markers for studying the origin of cells in rat parietovisceral yolk sac carcinomas.     

Fine structure of the fertilization membranes of sea urchin embryos

The fine structure of the fertilization membranes from S. purpuratus embryos has been studied with the electron microscope. Isolated membranes before and after their full development and membranes formed under the influence of 10⁻³% cystine have been observed. The membrane structure was found to be trilamella: a middle layer about 200 Å thick, which originally was the vitelline membrane, and about 175 Å thick peripheral layers organized by the “crystalline material” from the cortical granules. These surface layers were again found to be trilaminated structure composed of a monolayer of parallel, closely packed flat fibrils, about 160 Å wide and 75 Å thick, adhering on both sides to parallel, 40–50 Å thick filaments separated from each other by about 100 Å and intersecting with the fibrils by an angle of about 75 °.     

Collagen in the egg shell membranes of the hen

Collagen-like proteins have been found in the egg shell membranes of the hen. Materials similar to types I and V collagens were detected in each of the two layers of this membrane, the thick outer membrane and the thin inner membrane. Collagen was extracted by acid-pepsin digestion and isolated by differential salt precipitation. Identification of type-specific collagen-like material was established by coelectrophoresis on SDS-polyacrylamide gels using known collagen standards. These bands were susceptible to digestion by bacterial collagenase. From differential staining of the gels it was estimated that the ratio of collagen types I:V was approximately 100:1. Further confirmation of these biochemical results was obtained with immunofluorescence microscopy using type-specific antisera against chicken types I and V collagen with the indirect sandwich technique. Both the inner and outer shell membranes contained the two types of collagen. Within each membrane, the large, coarse 2.5-micron fibers contained predominantly type I collagen-like material, while type V collagen was mainly associated with the delicate narrower fibers of approximately 0.6-micron diameter. These tended to be concentrated in the inner membrane. At the electron microscopic level, both types of fibers were coated with glycoproteins that stained positively with ruthenium red. The deposition of these collagen-like substances by the hen oviduct on to the surface of the developing egg is an additional example of interstitial-type collagen synthesis and secretion by epithelial rather than by mesenchymal cells.     

The structure and composition of the complex that provides the adhesion of the cellular layers during the morphogenesis of the human embryonic adenohypophysis]

The area of contact between adenohypophysis and diencephalon rudiments of human embryos (5-7 weeks of development) was studied using immunohistochemistry and electron microscopy. Basement membranes of Rathke's pouch ectoderm and of diencephalon bottom neuroectoderm are connected by means of a complex consisting of thin fibrous material and collagen-like fibrils. After Ca+2 and Mg+2 ions removal, this complex with basement membranes was separated from epithelial layers. The material in the contact area differed in its composition from the basement membranes covering the adenohypophysis and diencephalon rudiments by the high content of tenascin and the absence of EDB-fibronectin. Other basement membrane components (collagen IV, heparan-sulphate proteoglycans, entactin and laminin) were also present in this area. Tenascin accumulation was also found in Rathke's pouch epithelium and cavity.     

Collagen vitrigel membrane useful for paracrine assays in vitro and drug delivery systems in vivo

We previously succeeded in converting a soft and turbid disk of type-I collagen gel into a strong and transparent vitrigel membrane utilizing a concept for the vitrification of heat-denatured proteins and have demonstrated its protein-permeability and advantage as a scaffold for reconstructing crosstalk models between two different cell types. In this study, we observed the nano-structure of the type-I collagen vitrigel membrane and verified its utility for paracrine assays in vitro and drug delivery systems in vivo. Scanning electron microscopic observation revealed that the vitrigel membrane was a dense network architecture of typical type-I collagen fibrils. In the crosstalk model between PC-12 pheochromocytoma cells and L929 fibroblasts, nerve growth factor (NGF) secreted from L929 cells passed through the collagen vitrigel membrane and induced the neurite outgrowth of PC-12 cells by its paracrine effect. Also, the collagen vitrigel membrane containing vascular endothelial growth factor (VEGF) showed sustained-release of VEGF in vitro and its subcutaneous transplantation into a rat resulted in remarkable angiogenesis. These data suggest that the collagen vitrigel membrane is useful for paracrine assays in vitro and drug delivery systems in vivo.     

Myoblasts are aligned with collagen fibrils in regenerating frog tadpole tails

Myoblasts in the regenerating frog tadpole tail differentiate from mesen chymal cells that lie next to the basement membrane of the epidermis of the tail. As these cells elongate and form myotubes, they orientate uniformly in the longitudinal axis of the tail. The collagen fibrils of the basement membrane adjacent to the myogenic cells are also orientated in the tail axis just prior to and during the time when the myogenic cells are elongating. This has been demonstrated by transmission electron microscopy of thin sections, by differential interference contrast microscopy of isolated basement membranes, and by scanning electron microscopy of the inner surface of the basement membrane. Since elongating myoblasts are in contact with the longitudinally orientated fibrils, the latter could provide directional cues to the elongating myoblasts. This proposition is supported by the finding that isolated basement membranes readily orientate cells that are cultured upon their inner surfaces.     

Hemidesmosomes and anchoring fibril collagen appear synchronously during development and wound healing

Bullous pemphigoid antisera and monoclonal antibodies to type VII collagen were used to localize hemidesmosomes and anchoring fibrils, respectively, in tissues of developing eyes and healing corneal wounds of New Zealand white rabbits. In the 17-day fetal rabbit eye, both antibodies colocalize to the epithelial-stromal junction of the lid and conjunctival region, but neither binds to the cornea, and electron microscopy demonstrates hemidesmosomes only where the antibodies bind. By 20 days of fetal development, the antibodies colocalize in cornea, and, by electron microscopy, hemidesmosomes are shown to be present as well. In healing 7-mm corneal wounds, both antibodies colocalize at the wound periphery within 66 h. By electron microscopy, hemidesmosomes along small segments of basal lamina are also shown to be present at the wound periphery at this time. These demonstrations of the synchronous assembly of hemidesmosomes and anchoring fibrils support the hypothesis of linkage of hemidesmosomes through the basement membrane to anchoring fibrils.     

Observing growth steps of collagen self-assembly by time-lapse high-resolution atomic force microscopy

Insights into molecular mechanisms of collagen assembly are important for understanding countless biological processes and at the same time a prerequisite for many biotechnological and medical applications. In this work, the self-assembly of collagen type I molecules into fibrils could be directly observed using time-lapse atomic force microscopy (AFM). The smallest isolated fibrillar structures initiating fibril growth showed a thickness of approximately 1.5 nm corresponding to that of a single collagen molecule. Fibrils assembled in vitro established an axial D-periodicity of approximately 67 nm such as typically observed for in vivo assembled collagen fibrils from tendon. At given collagen concentrations of the buffer solution the fibrils showed constant lateral and longitudinal growth rates. Single fibrils continuously grew and fused with each other until the supporting surface was completely covered by a nanoscopically well-defined collagen matrix. Their thickness of approximately 3 nm suggests that the fibrils were build from laterally assembled collagen microfibrils. Laterally the fibrils grew in steps of approximately 4 nm, indicating microfibril formation and incorporation. Thus, we suggest collagen fibrils assembling in a two-step process. In a first step, collagen molecules assemble with each other. In the second step, these molecules then rearrange into microfibrils which form the building blocks of collagen fibrils. High-resolution AFM topographs revealed substructural details of the D-band architecture of the fibrils forming the collagen matrix. These substructures correlated well with those revealed from positively stained collagen fibers imaged by transmission electron microscopy.     

Antigenic characteristics of the L forms of Streptococcus group B

Stable L-forms of group B streptococci (GBS) have been obtained and their antigenic features have been studied by the serological methods (the passive hemagglutination test, the aggregate agglutination test, the gel diffusion test), as well as by using ferritin and peroxidase labels with the subsequent electron microscopy. The use of the serological methods has made it possible to reveal the antigenic differences between the stable L-forms of GBS and their bacterial forms. Specific antigenic substances can be found in the supernatant fluid obtained after the sedimentation of the ultrasonically disintegrated cellular mass of streptococcal L-forms and bacterial cultures. The use of ferritin and peroxidase labels has revealed the specificity of GBS L-form antigen and its localization on the cytoplasmic membrane of all L-form structural elements.     

Collagen expression, ultrastructural assembly, and mineralization in cultures of chicken embryo osteoblasts

A newly defined chick calvariae osteoblast culture system that undergoes a temporal sequence of differentiation of the osteoblast phenotype with subsequent mineralization (Gerstenfeld, L. C., S. Chipman, J. Glowacki, and J. B. Lian. 1987. Dev. Biol. 122:49-60) has been examined for the regulation of collagen synthesis, ultrastructural organization of collagen fibrils, and extracellular matrix mineralization. Collagen gene expression, protein synthesis, processing, and accumulation were studied in this system over a 30-d period. Steady state mRNA levels for pro alpha 1(I) and pro alpha 2 collagen and total collagen synthesis increased 1.2- and 1.8-fold, respectively, between days 3 and 12. Thereafter, total collagen synthesis decreased 10-fold while mRNA levels decreased 2.5-fold. In contrast to the decreasing protein synthesis after day 12, total accumulated collagen in the cell layers increased sixfold from day 12 to 30. Examination of the kinetics of procollagen processing demonstrated that there was a sixfold increase in the rate of procollagen conversion to alpha chains from days 3 to 30 and the newly synthesized collagen was more efficiently incorporated into the extracellular matrix at later culture times. The macrostructural assembly of collagen and its relationship to culture mineralization were also examined. High voltage electron microscopy demonstrated that culture cell layers were three to four cells thick. Each cell layer was associated with a layer of well developed collagen fibrils orthogonally arranged with respect to adjacent layers. Fibrils had distinct 64-70-nm periodicity typical of type I collagen. Electron opaque areas found principally associated with the deepest layers of the fibrils consisted of calcium and phosphorus determined by electron probe microanalysis and were identified by electron diffraction as a very poorly crystalline hydroxyapatite mineral phase. These data demonstrate for the first time that cultured osteoblasts are capable of assembling their collagen fibrils into a bone-specific macrostructure which mineralizes in a manner similar to that characterized in vivo. Further, this matrix maturation may influence the processing kinetics of the collagen molecule.     

Tendon development requires regulation of cell condensation and cell shape via cadherin-11-mediated cell-cell junctions

The ability of tendon to transmit forces from muscle to bone is directly attributable to an extracellular matrix (ECM) containing parallel bundles of collagen fibrils. Although the biosynthesis of collagen is well characterized, how cells deposit the fibrils in regular parallel arrays is not understood. Here we show that cells in the tendon mesenchyme are nearly cylindrical and are aligned side by side and end to end along the proximal-distal axis of the limb. Using three-dimensional reconstruction electron microscopy, we show that the cells have deep channels in their plasma membranes and contain bundles of parallel fibrils that are contiguous from one cell to another along the tendon axis. A combination of electron microscopy, microarray analysis, and immunofluorescence suggested that the cells are held together by cadherin-11-containing cell-cell junctions. Using a combination of RNA interference and electron microscopy, we showed that knockdown of cadherin-11 resulted in cell separation, loss of plasma membrane channels, and misalignment of the collagen fibrils in the ECM. Our results show that tendon formation in the developing limb requires precise regulation of cell shape via cadherin-11-mediated cell-cell junctions and coaxial alignment of plasma membrane channels in longitudinally stacked cells.     

Effects of substrata on the polarization of bovine endometrial epithelial cells in vitro

Summary Epithelial-cell function requires cellular polarity in which apical membrane surfaces have unique characteristics and cellular organelles are stratified. Physiological investigations of endometrial, epithelial cells would be enhanced greatly by the ability of a method to polarize cells in culture. This study investigates the effects of different substrata on polarization of cultured bovine endometrial epithelial cells. Fetal bovine endometrial epithelial-cell lines were developed from explant outgrowth. Epithelial monolayers were subcultured onto amniotic membranes, Millicell-HA membranes, or Millicell-CM membranes coated with rat-tail collagen, Matrigel, laminin, Vitrogen,or fibronectin. Cultures on these substrata were maintained at the air/liquid interface. Cells grown on either collagen-coated or uncoated Milli-cell membranes also were maintained submerged in medium. Excellent polarized morphology was attained in cultures grown at the air/liquid interface on amniotic membranes and rat-tail collagen-coated membranes. Lectin-binding patterns, to apical membranes of polarized epithelial cell cultures paralleled patterns of binding to bovine endometrial surfaces in vivo. Cultures on rat-tail collagen were maintained for several weeks. These methods provide a valuable system for studying the endometrium in vitro.     

Supramolecular interactions in the dermo-epidermal junction zone: anchoring fibril-collagen VII tightly binds to banded collagen fibrils

The dermis and the epidermis of normal human skin are functionally separated by a basement membrane but, together, form a stable structural continuum. Anchoring fibrils reinforce this connection by insertion into the basement membrane and by intercalation with banded collagen fibrils of the papillary dermis. Structural abnormalities in collagen VII, the major molecular constituent of anchoring fibrils, lead to a congenital skin fragility condition, dystrophic epidermolysis bullosa, associated with skin blistering. Here, we characterized the molecular basis of the interactions between anchoring fibrils and banded collagen fibrils. Suprastructural fragments of the dermo-epidermal junction zone were generated by mechanical disruption and by separation with magnetic Immunobeads. Anchoring fibrils were tightly attached to banded collagen fibrils. In vitro binding studies demonstrated that a von Willebrand factor A-like motif in collagen VII was essential for binding of anchoring fibrils to reconstituted collagen I fibrils. Since collagen I and VII molecules reportedly undergo only weak interactions, the attachment of anchoring fibrils to collagen fibrils depends on supramolecular organization of their constituents. This complex is stabilized in situ and resists dissociation by strong denaturants.     

Microbial fouling of reverse-osmosis membranes used in advanced wastewater treatment technology: chemical, bacteriological, and ultrastructural analyses.

Biofouling of reverse-osmosis membranes was investigated at an advanced wastewater treatment facility. Cellulose diacetate membranes operated for approximately 4,000 h became uniformly coated with a mucilaginous fouling layer. The fouling material was approximately 93% water by weight, and nearly 90% of the dehydrated residue was organic in composition. Calcium, phosphorous, sulfur, and chlorine were the major inorganic constituents detected. Protein and carbohydrate represented as much as 30 and 17%, respectively, of the dry weight of the biofilm. Bacteriological plate counts indicated up to 5.6 X 10(6) CFU/cm2 of membrane surface. Accumulation of [3H]glucose in the biofilm and measurement of ATP indicated that the fouling bacteria were metabolically active in situ. The genus Acinetobacter and the Flavobacterium-Moraxella group were the major generic groups associated with the feedwater surface of the membrane, whereas species of the generic groups Acinetobacter, Pseudomonas-Alcaligenes, and Bacillus-Lactobacillus predominated on the permeate water surface. Electron microscopy revealed that the biofilm on the feedwater surface of the membrane was 10 to 20 microns thick and was composed of several layers of compacted bacterial cells, many of which were partially or completely autolyzed. The bacteria were firmly attached to the membrane surface by an extensive network of extracellular polymeric fibrils. Polyester (Texlon) support fibers located on the permeate surface of the reverse osmosis membranes were sparsely colonized, suggesting bacterial regrowth in the product water collection system.     

Arrangement of connective tissue components in the walls of seminiferous tubules of man and monkey.

In primates the membrane separating the seminiferous epithelium from the interstitial space is composed of one to three (monkey) or two to six layers (man) of myoid cells associated with one to two layers of fibrocyte-like adventitial cells. All these cells are separated from each other by irregular spaces filled with various connective tissue intercellular components. Subjacent to the elements of the seminiferous epithelium is a continuous, often redundant, basement membrane. A similar basement membrane-like material forms a layer next to and over small areas of the plasma membrane of myoid cells. Collagen fibrils grouped in bundles of various sizes are seen in all connective tissue layers but are particularly abundant in the space between the seminiferous epithelium and the innermost layer of myoid cells. Elastic fibrils demonstrated by the Verhoeff iron hematoxylin technique are also present. Composed of a homogeneous material, the elastic fibrils are short, irregular, branching entities with a diameter comparable to or smaller than that of collagen fibrils. In addition, an abundance of microfibrils with a diameter of 12-15 nm is present in the various connective tissue layers. These microfibrils have a densely stained cortex and a lightly stained core. When seen close to the myoid cells, bundles of micro fibrils appear to insert on well defined areas next to the plasma membrane. These areas commonly face the patches of electron-dense material observed on the inner aspect of the plasma membrane of the myoid cells and in which the actin filaments are inserted. Bundles of microfibrils often span the gap between myoid cells of the same layer as well as those of adjacent layers. Microfibrils are also closely related to the surface of elastic fibrils and are seen intertwining with collagen fibrils. Thus microfibrils appear to bridge and bind together adjacent myoid cells and anchor the surface of these cells to the bundles of elastic and collagen fibrils present in the intercellular spaces of the limiting membrane.