Myoepithelial molecular markers in human breast carcinoma PMC42-LA cells are induced by extracellular matrix and stromal cells

Summary The microenvironment plays a key role in the cellular differentiation of the two main cell lineages of the human breast, luminal epithelial, and myoepithelial. It is not clear, however, how the components of the microenvironment control the development of these cell lineages. To investigate how lineage development is regulated by 3-D culture and microenvironment components, we used the PMC42-LA human breast carcinoma cell line, which possesses stem cell characteristics. When cultured on a two-dimensional glass substrate, PMC42-LA cells formed a monolayer and expressed predominantly luminal epithelial markers, including cytokeratins 8, 18, and 19; E-cadherin; and sialomucin. The key myoepithelial-specific proteins α-smooth muscle actin and cytokeratin 14 were not expressed. When cultured within Engelbreth-Holm-Swarm sarcoma-derived basement membrane matrix (EHS matrix), PMC42-LA cells formed organoids in which the expression of luminal markers was reduced and the expression of other myoepithelial-specific markers (cytokeratin 17 and P-cadherin) was promoted. The presence of primary human mammary gland fibroblasts within the EHS matrix induced expression of the key myoepithelial-specific markers, α-smooth muscle actin and cytokeratin 14. Immortalized human skin fibroblasts were less effective in inducing expression of these key myoepithelial-specific markers. Confocal dual-labeling showed that individual cells expressed luminal or myoepithelial proteins, but not both. Conditioned medium from the mammary fibroblasts was equally effective in inducing myoepithelial marker expression. The results indicate that the myoepithelial lineage is promoted by the extracellular matrix, in conjunction with products secreted by breast-specific fibroblasts. Our results demonstrate a key role for the breast microenvironment in the regulation of breast lineage development.     

Hydroxyl free radicals generated by vanadyl[IV] induce cell blebbing in mitotic human Chang liver cells

Vanadium has recently been reported to induce interphase and M-phase (mitotic) programmed cell death via the generation of hydroxyl free radicals (OH*). In this paper, the effects of antioxidants on: (a) vanadyl[IV]-generated OH* free radical levels; and (b) cellular glutathione in vanadyl [IV]-treated Chang liver cells were evaluated. The surface morphology of vanadyl-treated mitotic cells was studied by confocal and scanning microscopy. The free radical scavengers zinc chloride, glucose and thiourea reduced the levels of vanadyl-induced OH* free radicals and partially prevented the depletion of cellular glutathione. Concurrent with OH* free radical production, vanadyl-treated telophase cells exhibited excessive cell blebbing and cell shrinkage. The morphological features demonstrated in vanadyl-induced mitotic programmed cell death as a consequence of oxidative stress is novel.     

Direct plant regeneration from leaf explants of Drosera rotundifolia cultured in vitro

Shoot regeneration was obtained from isolated leaves of Drosera rotundifolia L. cultured on MS media with various concentrations of 6-benzyladenine (BA) and -naphthaleneacetic acid (NAA). The best direct shoot organogenesis was obtained on growth regulator-free medium or medium supplemented with 10^-8 M NAA. Liquid culture medium significantly increased regeneration capacity of leaf tissue. Histological and scanning electron microscopy investigations verify direct plant regeneration without intermediate callus formation. Leaf epidermal cells showed the highest regeneration potential leading to the regeneration of buds. Young shoots with three to seven leaflets rooted spontaneously on the growth regulator-free medium within 38 days of culture and isolated mature plants produced fertile seeds.Abbreviations BA 6-benzyladenine - FAA 40% formalin (5%) +90% acetic acid (5%) +70% ethanol (90%) - ME Murashige and Skoog's (1962) medium - NAA -naphthaleneacetic acid - plumbagin 5-hydroxy-2-methyl-1,4-naphthoquinone - 7-methyljuglone 7-methyl-5-hydroxy-1,4-naphthoquinone - SEM scanning electron microscopy - TEM transmission electron microscopy - PPF photosynthetic photon flux     

Time course of vasopressin-induced formation of microvilli in granular cells of toad urinary bladder

Summary Vasopressin-induced transformation of ridges to microvilli on the surface of granular cells of toad urinary bladder occurs in conjunction with induced alterations in the water permeability of the luminal membrane. This study was designed to establish the relationship between the time course for induction of microvilli and the time course for induction of increased water permeability after vasopressin stimulation. Hemibladders were examined at 2.5, 5, 10, 20 and 30 min following exposure to 20 mU/ml of vasopressin and at 5, 10, 20, 30, 40, 50 and 60 min after washout of vasopressin. Within 2.5 min, vasopressin initiated complete transformation of ridges to microvilli on approximately 13% of the granular cells, while osmotic water flow (Jv) was 0.31±0.10 l·min^–1·cm^–2. Five minutes following vasopressin stimulation, microvilli were present on approximately 30% of granular cells andJv was 2.27±0.13 l·min^–1·cm^–2. At 10 minJv was maximum at 4.03±0.15 l·min^–1·cm^–2 and 50% of the granular cells were covered with microvilli. This percentage increased to 70% at 20 min and was maintained at 30 min, althoughJv decreased to 3.9±0.35 l·min^–1·cm^–2 at 30 min. Five minutes following vasopressin washout, ridges interspersed with microvilli reappeared asJv fell to 1.10±0.30 l·min^–1·cm^–2. At 10 min after vasopressin washout,Jv approached basal levels, but the reversal of microvilli to ridges remained incomplete. At 60 min after vasopressin washout, the granular cells had regained their original ridgelike surface structures. Thus, these studies establish a temporal relationship between the induction and reversibility of vasopressin-induced microvillous formation and alterations in the osmotic water permeability of the apical plasmalemma.     

Contributions to the pollen morphology and taxonomy of the Liliaceae

Pollen of 34 species from 7 genera of the Liliaceae were examined by LM and SEM with respect to the taxonomy of the family. Detailed pollen­morphological characteristics are given for all genera in the family on the basis of the results presented here together with data from the literature. The genera Tulipa and Lilium are heterogeneous in both aperture type and exine ornamentation. Pollen of Tulipa is monosulcate, 3-aperturate or inaperturate, with a microreticulate-striate, reticulate-implecto-striate, scabrate, perforate-rugulate, perforate-striate exine surface. Pollen of Lilium is monosulcate and 3-porate with a macroreticulate exine. The other genera are homogeneous in possessing of single longitudinal aperture (type monosulcate). The pattern of exine ornamentation and the structure of the aperture and its membrane are peculiar features for species and genera. Pollen of Erythronium and Tulipa are occasionally operculate, while in other representatives of the Liliaceae an operculum is lacking. Pollen morphological data support the division of the family into 3 tribes, namely Lloydieae, Lilieae, and Tulipeae.     

Seed morphology and taxonomy of the North African species of Sideritis L. (Lamiaceae)

REJDALI, M., 1990. Seed morphology and taxonomy of the North African species of Sideritis L. (Lamiaceae). Seed morphology data are shown to support data from general morphology and palynology; they can be used for taxonomy at the sectional and specific levels. At times clear differences are apparent at the infraspecific level. Seed sculpturing was found to be of great value for separating taxa at all levels of the hierarchy.     

Cell surface changes during mitosis and cytokinesis of epithelial cells

Summary PtK2 cells were studied with scanning electron microscopy to record changes on the cell surface during mitosis and cytokinesis. During prophase, prometaphase and metaphase, the cells remain very flat with few microvilli on their surfaces. In anaphase cells, there is a marked increase in the number of microvilli, most of which are clumped over the separating chromosomes and polar regions of the mitotic spindle leaving the surface of the interzonal spindle region relatively smooth. Microvilli appear over the interzonal spindle region in telophase and the cells also increase in height. At the beginning of cleavage, the distribution of microvilli is roughly uniform over the surface but it becomes asymmetric at the completion of cleav-age when the daughter cells begin to spread. At this time most microvilli are over the daughter nuclei and the surfaces that border the former cleavage furrow. The regions of the daughter cells distal to the furrow are the first to spread and their surfaces have very few microvilli. When chromosome movement is inhibited by either Nocodazole or Taxol, microvilli formation is inhibited on the arrested cells. Nevertheless cell rounding still takes place in the normal time period. It is concluded from these observations that the signal for the onset of chromosome movement in anaphase is accompanied by a signal for the formation of microvilli. It is suggested that there is also a separate signal for the cell-rounding event in mitosis and that microvilli do not play a role in this contractile process.     

TGF-beta induces formation of F-actin cores and matrix degradation in human breast cancer cells via distinct signaling pathways

Transforming growth factor beta regulates many biological processes including cell motility and invasion. Podosomes are specialized F-actin rich structures found in normal cells, such as osteoclasts and macrophages. Tumor cells often form related structures called invadopodia that are thought to promote invasion and metastasis. Here we show that human breast cancer cells organize F-actin rich structures in response to transforming growth factor beta that colocalize with areas of extracellular matrix degradation. We further show that organizing the complex of proteins needed to form these structures requires signaling through phosphatidylinositide 3-kinase and Src kinase, while activating the proteases involved in degradation of extracellular matrix requires extracellular signal-regulated kinase signaling, and that each of these pathways is activated by transforming growth factor beta in CA1D human breast cancer cells.     

Pollen morphology of the family Cistaceae in Egypt and its systematic significance

The pollen morphology of 11 species (including two subspecies and two varieties) belonging to two genera (Helianthemum and Fumana) of the family Cistaceae in Egypt was studied using light and scanning electron microscopy.Pollen grains of the studied taxa were found to be radially symmetrical and tricolporate.Pollen size,shape,apertures,and exine ornamentation characteristics were valuable parameters among the studied taxa.The largest pollen size was recorded in H.salicifolium and the smallest one observed in H.kahiricum subsp,schweinfurthii.Pollen shape in the Egyptian taxa varied from (sub-)prolate to prolate spheroidal,but F.arabica is different in having sub-oblate grains.The pollen data confirm that H.lippii and H.sessiliflorum are very closely related species.Pollen sculpture was useful in distinguishing between H.vesicarium var.vesicarium and H.vesicarium var.ciliatum.Three main pollen types of exine ornamentation were recognized:retipilate; reticulate to verrucate; and striate.Based on palynological data,a key for the studied taxa is suggested.     

Correlative 3D imaging of whole mammalian cells with light and electron microscopy

We report methodological advances that extend the current capabilities of ion-abrasion scanning electron microscopy (IA-SEM), also known as focused ion beam scanning electron microscopy, a newly emerging technology for high resolution imaging of large biological specimens in 3D. We establish protocols that enable the routine generation of 3D image stacks of entire plastic-embedded mammalian cells by IA-SEM at resolutions of ∼10–20 nm at high contrast and with minimal artifacts from the focused ion beam. We build on these advances by describing a detailed approach for carrying out correlative live confocal microscopy and IA-SEM on the same cells. Finally, we demonstrate that by combining correlative imaging with newly developed tools for automated image processing, small 100 nm-sized entities such as HIV-1 or gold beads can be localized in SEM image stacks of whole mammalian cells. We anticipate that these methods will add to the arsenal of tools available for investigating mechanisms underlying host-pathogen interactions, and more generally, the 3D subcellular architecture of mammalian cells and tissues.     

Rapid regulation of neuronal growth cone shape and surface morphology by nerve growth factor

Scanning electron microscopy was used to study regulation of growth cone shape and surface morphology by nerve growth factor (NGF). The growth cones of cultured rat sympathetic neurons and neuronally-differentiated PC12 cells were observed under conditions of continuous NGF exposure, NGF withdrawal, and NGF readdition. Growth cones of cells cultured in the continuous presence of NGF were mostly spread in shape and about 60% possessed surface ruffles. Ruffles appeared to be largely restricted to growth cones in that few were observed on cell bodies and neurites. Withdrawal of NGF for 4–5 hr caused most of the growth cones to take on a non-spread or contracted appearance and to lose their ruffles. Readdition of NGF promoted rapid changes in growth cone properties. Within 30 sec, ruffling was again evident on the growth cones and remained prominent there throughout the course of treatment (up to 5 hr). This was in contrast to cell bodies on which, as previously reported, ruffling also occurred following NGF readdition, but only transiently (for less than 15 min). Respreading of growth cones also occurred under these conditions. This was evident within 1 min of NGF readdition and reached the levels observed in continuously-treated cultures within 1–2 hr. Neurites were also examined. Ruffles were only rarely present in the continuous presence of NGF and were absent after NGF withdrawal. NGF readdition elicited ruffling along neurites within 30 sec; the prevalence of such ruffles diminished to that seen in continuously-treated cultures within about an hour. As evidence of the specificity of these NGF effects, epidermal growth factor and dibutyryl cAMP, agents that elicit responses in PC12 cells, but do not promote their neuronal differentiation, had no observable effect on NGF-deprived growth cones. These findings demonstrate that NGF exerts very rapid effects on growth cone shape and surface morphology. Such actions may play roles in regulation of growth cone movement and guidance by NGF.Special Issue dedicated to Dr. E. M. Shooter and Dr. S. Varon.     

Thyrotropin induces changes in the morphology and the organization of microfilament structures in cultured thyroid cells

Thyrotropin (TSH) induces morphological changes in cultures of normal rat thyroid cell lines and in primary bovine thyroid cells. It also induces a specific reorganization of the microfilaments of the thyroid cells. Both effects are fully reversible and are mimicked by 8-bromo-cAMP. These results indicate that the trophic response of TSH involves changes in the organization of the actin-containing filaments, probably mediated through cAMP, followed by changes in cell shape.     

Role of physiologic autoantibody in the removal of senescent human red cells

The mechanism by which mononuclear phagocytes distinguish mature “self” from senescent “self” was investigated. Evidence is presented indicating that human mononuclear phagocytes distinguish senescent RBC from mature RBC on the basis of selective Ig attachment to the membranes of senescent cells. This Ig, eluted from senescent human RBC, was shown to be IgG and free of other Igs by immunodiffusion, immunoelectrophoresis, and polyacrylamide gel electrophoresis. The IgG was polyclonal with respect to light chains. The eluted IgG reattaches to homologous stored RBC, but not to mature autologous or allogeneic RBC, via the Fab region. It then initiates phagocytosis of these stored RBC by mononuclear phagocytes. Evidence suggests that the IgG is directed against altered membrane receptors. Thus, this IgG may be a “physiologic” autoantibody and contribute to the maintenance of homeostasis by performing regulatory functions.     

The metareticulate pollen morphology of Alternanthera Forssk. (Gomphrenoideae,Amaranthaceae) and its taxonomic implications

Alternanthera (Amaranthaceae) is a diverse genus largely restricted to the American Tropics that belongs to the alternantheroid clade containing C₄ and C₃–C₄ intermediate species. This research focuses on the study of pollen characters by studying 13 species, representatives of the two major clades and subclades of Alternanthera. General palynological comparisons were conducted with light microscopy (LM), scanning electron microscopy (SEM) and with confocal laser scanning microscopy (CLSM) for exine ultrastructure. Twenty-five characters were measured and described for Alternanthera and among these, 14 pollen characters were used to discriminate pollen groups using cluster analysis and canonical analysis of principal coordinates (CAP). Pollen form and ornamentation, pores number, spines length, number of ektexinous bodies and nanospines on the ektexinous bodies on pore membranes, arrangement of nanopores and spines on structural elements, and metareticula form were taxonomically important and therefore used to construct the first palynological key to the alternantheroid clade species. Our study indicates that the seemingly subtle morphological variation of pollen is useful for recognising three main pollen types within Alternanthera. The much needed palynological terminology for describing the mesoporium in the metareticulate pollen of Amaranthaceae is provided.     

Seed morphology of Rotala L., Ammannia L., Nesaea Kunth and Hionanthera Fernandes & Diniz (Lythraceae)

PANIGRAHI, S. G., 1986. Seed morphology of Rotala L., Ammannia L., Nesaea Kunth and Hionanthera Fernandes & Diniz (Lythraceae) . Seed-surface characteristics of Ammonia lalifolia L., Rotala verticillaris L., Nesaea triflora Comm. ex Kunth and Hionanthera garciae Fernandes & Dinz were studied using the SEM. Seeds from herbarium specimens moistened in water produce cracks along the intercellular walls and distinctive types of invaginating 'hairs'. These 'hairs' are mucilaginous in nature and diagnostic at the species level. They are released in profusion from the upper convex surface of the testa. Viable seeds of Ammonia baccifera L., similarly treated, exude, in addition to the 'hairs', mucilaginous globules from the lower concave surface during germination. The invaginating 'hairs' arise diagonally from the roof of the epidermal cells of the testa and protrude to the lumen of the epidermal cells. A sac of mucilage is observed swelling out on the inner flat surface of the developing seeds. The biological functions of these 'hairs' and mucilage globules in germinating seeds in relation to seed dispersal and regulation of germination in appropriate habitats is discussed.     

Larval ontogeny and morphology of giant trahira Hoplias lacerdae

In the present study, the morphology and behaviour of giant trahira Hoplias lacerdae larvae were investigated, from hatching to complete absorption of the yolk sac, under laboratory conditions. In the first day post‐hatching (dph), the larvae presented a big ovoid‐shaped yolk sac that underwent regression during larval ontogeny. The mouth opened 3 dph, when the pectoral fins were evident. From this day, the larvae were able to perform sudden bursts of activity and appear to be able to swim a few centimetres before sinking again. The branchial apparatus was defined at 5 dph, and by 6 dph the operculum was formed. The internal organs such as intestine, liver, kidney and external sensorial structures were present at 7 dph. The yolk sac remained until 7 dph.     

Steroid modulation of aromatase activity in human cultured breast carcinoma cells

Cortisol and steroids with progestational or androgenic activity were studied to determine the effects of these steroids on the conversion of androstenedione (A) to estrone (E1) in human cultured breast carcinoma cells. Cortisol (10(-6) M) stimulated aromatase activity in two estrogen unresponsive cell lines (MD, DM) and in an estrogen responsive cell line (MCF7) with the maximum stimulation occurring during confluence. Cortisol inhibited the replication of MCF7 cells but not MD and DM. Dihydrotestosterone, androsterone and 5 alpha-androstanedione (10(-6) M) inhibited the conversion of A to E1 by greater than 90% under basal and cortisol stimulated conditions. Progesterone (10(-6) M) had no effect on aromatase activity while the progestational agent R5020 (10(-6) M) produced a 30% inhibition. The anabolic steroids 19-nortestosterone and 19-norandrostenedione which also have progestational activity inhibited the conversion of A to E1 in a dose dependent manner with 90% inhibition at 10(-6) M. Danazol (10(-6) M) a drug with both androgenic and progestational activity inhibited E1 formation by 30%. Under the same conditions, the known inhibitor of aromatase, 4-hydroxyandrostenedione (10(-6) M) decreased E1 formation by more than 90% and aminoglutethimide (10(-6) M) caused only 25% inhibition. These studies demonstrate that endogenous and exogenous steroids may have significant effects in modulating the local formation of estrogens from androgen precursors in cultured breast carcinoma cells. This effect on estrogen formation may be a factor in the biological response of breast tissue.     

Three-dimensional organization of the extracellular matrix secreted by cultured rat smooth muscle cells

Summary Specific interactions between cells and the extracellular matrix (ECM) in which they are embedded play a vital role in tissue organization. In recent years, many of the individual components of the extracellular matrix have been isolated and their molecular structures elucidated, but the detailed topography of most extracellular matrices, as they are deposited by cells, is still largely unknown. In this study, the insoluble extracellular matrix produced by cultured rat vascular smooth muscle cells has been characterized morphologically using high-resolution electron microscopy of rotary platinum replicas. These cells grew as flat sheets in culture, secreting their matrix laterally and basally. The matrix was composed of a cross-linked fibrillar meshwork. Some fine fibers (10 to 15 nm in diameter) were naked, but most of the filamentous mesh was covered with coarse granular material. Limited digestion with trypsin or pancreatic elastase removed most of this coating, indicating that the granules were glycoproteins and proteoglycans. Another subset of matrix fibrils (20 to 40 nm in diameter) was identified as type I collagen by direct comparison with purified bovine skin collagen. In addition to exposing the underlying filamentous substructure of the matrix, protease treatment also revealed large, straight fiber bundles and globules of amorphous material suspended in the filamentous web. This novel view of a complex matrix promises to provide spatial information that will be useful in future studies of cell interactions with the ECM. These studies were supported in part by NIH Biomedical Research Support grant S07-RR-05684.