Nuclear DNA content in differentiated tissues of sunflower (Helianthus annuus L.)

Summary Scanning cytophotometry following Feulgen-staining was used to determine nuclear DNA content in many differentiated tissues of nine cultivars, hybrids or selfed lines ofHelianthus annuus. Apart from such ephemeral tissues as endosperm and anther tapetum, it was found that tissue differentiation in sunflower occurs in the diploid condition, cells being arrested in the DNA presynthetic phase (G₁). In certain cases, however, the nuclear DNA content of differentiated G₁ cells does not exactly match the 2C DNA content found in meristematic cells, but may be either higher or lower. In endosperm and anther tapetum cells, nuclear DNA content may be as high as 24 C and 32 C, respectively. Cytological and autoradiographic analyses after³H-thymidine incorporation reveal that polyploidy in the tapetal cells is due to chromosome endoreduplication. No detectable difference between male-fertile and male-sterile plants exists as far as occurrence and level of cell polyploidy are concerned. The results are discussed in the context of previous investigations on the nuclear condition of differentiatedHelianthus annuus tissue.     

Epithelial differentiation at the edentulous alveolar ridge in man

Summary The epithelial lining of the mucosa of the edentulous, maxillary alveolar ridge was subjected to an ultrastructural and stereological analysis. Four biopsies collected from the non-inflamed crest, i.e., the center over former tooth sockets, in non-denture-wearing female patients 30 to 55 years of age were processed for light and electron microscopy. At the light-microscopic level, epithelial thickness was determined histometrically. Electron micrographs were sampled at two levels of magnification, from five strata in regions of epithelial ridges and from three strata over connective tissue papillae. Standardized stereological pointcounting techniques were employed to analyze a total of 990 electron micrographs. Observations and data revealed that at the alveolar ridge the oral epithelium is truly keratinizing and comprises four strata including a 40±5 m-thick stratum corneum, which displays the oral keratin pattern. The histoand cytodifferentiation were peculiar: (1) Compared to the neighbouring gingival and hard palate epithelium, that of the alveolar crest was markedly thicker, with elongated rete ridges indicating acanthosis. (2) The cytoarchitecture was identical neither to the gingival nor to the hard palate epithelium but revealed a mixture of features typical for either of these two epithelia. Reasons for this are explained on the basis of factors, possible genetic, inherent in epithelial cells that are possibly derived from both the gingival and the palatal environment.     

Disorganization of stroma alters epithelial differentiation of the glandular stomach in adult mice

Summary The response of adult epithelium in contact with heterologous mesenchymes/stromas was studied in three digestive organs (forestomach, glandular stomach, and duodenum). After various tissues were implanted beneath the epithelial layer of adult mice, the epithelial differentiation was examined after sacrifice of animals at intervals up to 24 weeks. In the forestomach and duodenum, the epithelial differentiation was not affected at all by the tissue implantation. In the glandular stomach, in contrast, epithelial cells exhibited altered differentiation in which chief and parietal cells disappeared and were replaced by columnar epithelial cells with PAS-positive granules. These epithelial cells often formed immature villi. Such differentiation-altered columnar epithelium (DACE) was induced by implanting any type of tissue and even by sham operation, indicating that it was induced by disorganization of the tissue-implanted stroma. The size of DACE was significantly influenced by the stage of implanted tissue; 14.5-day fetal mesenchyme induced the largest DACE, and was followed by 16.5-day fetal mesenchyme, adult stroma, and sham operation. These results suggest the importance of stromal organization in maintaining epithelial differentiation in the glandular stomach.     

Continuous requirement of cAMP for pre-spore differentiation in Dictyostelium discoideum

Abstract Using a shaking culture system, we have previously shown that both cell contact and cAMP are required for pre-spore differentiation in Dictyostelium discoideum [2]. In the present study, cAMP was removed from the medium by the use of a hydrolysing enzyme after cells had formed agglomerates. This treatment left the agglomerates unchanged, but caused a rapid decrease in the activity of UDP galactose transferase, a pre-spore-specific enzyme. This result indicates that cAMP is required even after agglomerate formation to maintain pre-spore differentiation.     

Cyclic AMP-induced reversible decrease in cAMP-binding to cell surface receptors of Dictyostelium discoideum

Abstract Adaptation may be the result of a change in affinity and/or number of cAMP-binding sites at the cell surface. To test this possibility we used agip 53, a mutant that does not synthesize cAMP in response to cAMP stimulation. cAMP induced a fast decrease in cAMP-binding to aggregation-competent cells, which reached a maximum at 10–20 s and was reversible with a t _0.5 of about 70 s. The decrease in cAMP-binding involved 46000 sites per cell and was mainly due to a reduction in the apparent affinity for cAMP-binding and to a smaller extent to slowly dissociating cAMP. Our results suggest that under these conditions only a fraction of the cAMP-binding sites at the cell surface are involved in transmembrane signalling, which is indeed observed for many of the physiological responses in Dictyostelium discoideum .     

Changes in the Expression of the αα Form of Enolase During Neuroblastoma Differentiation

Abstract: The relative amounts of the different enolase isozymes present in neuroblastoma cells change during differentiation. When differentiation is induced by low serum in the presence of DMSO (dimethyl sulfoxide), there is a 50% decrease in the concentration of enolase activity associated with the form αα, and an increase in the activity associated with the γ-containing isozymes (αγ plus γγ); in the absence of DMSO, there is no decrease in αα or in total enolase activity. In order to study the mechanism of the changes in αα, cells differentiated with low serum with and without DMSO were compared. Measurements of the concentration of the α antigen by microcomplement fixation and by immunotitration demonstrate that the decreased enolase activity in DMSO cells is due to a decreased concentration of the α antigen. Measurements of the relative rate of synthesis of the antigen show that the decreased concentration of the α antigen is due to a decreased rate of synthesis. Enolase in differentiated cells is sufficiently stable (t_1/2 > 100 h) that a comparison of the relative rates of degradation has not been possible. The decreased synthesis of the α subunit of enolase that occurs under these conditions appears to be a useful model system for studying the de-expression of the α gene that occurs in vivo during neuronal differentiation.     

The differentiation of glial cells and glia limitans in organ cultures of chick spinal cord

Summary Differentiation of glial cells and the glia limitans in organ cultures of chick spinal cord explanted at early neural tube stages, alone or with adjacent tissues, was studied by electron microscopy. Oligodendrocytes and astrocytes comparable to those seen in the chicken in vivo were observed, mainly in areas of good neuronal differentiation. A glia limitans with basal lamina, comparable to that in vivo, was found when spinal cord was bordered by normally adjacent tissues. When it was surrounded by vitelline membrane only, a characteristic limiting layer of glial processes, but no basal lamina, was seen. Contact with a filter membrane (Millipore) elicited excessive differentiation of glial filaments and modified cell fine structure; no glia limitans was formed. Supported by Grant 5 RO 1 NB 0637 from the United States Public Health Service.     

Elektronenmikroskopische Zytomorphologie der männlichen Brustdrüse

Zusammenfassung Im Vergleich zu lichtmikroskopischen Untersuchungen an der Mamma virilis wird anhand von 2 operativ entfernten Brustdrüsen eines 57 und 63 Jahre alten Mannes die elektronenmikroskopisch erfaßbare Zytomorphologie beschrieben. Die Befunde werden den physiologischen Wachstumsimpulsen dieses Organs gegenübergestellt und Fragen der Zelldifferenzierung, der Desquamation und apokrinen Sekretion beantwortet. Elektronenmikroskopisch werden am Drüsenepithel Basalzellen, größere Zellen der oberflächlichen Zellreihen und Myoepithelzellen unterschieden. Diese Zellen entsprechen den Gangepithelien der weiblichen Brustdrüse und besitzen intracytoplasmatische Filamente. Diese stellen ein häufiges Differenzierungsprodukt des Zytoplasmas dar. Mechanismen einer Sekretion waren nicht nachweisbar. In die Drüsenlichtung werden pseudopodienartig vorgewölbte Zytoplasmateile abgeschnürt (Extrasionsvorgang). — Superfiziale Zellen werden desquamiert, wobei die Zytolyse in den marginalen Zytoplasmaschichten erfolgt. Kern und Teile des Zytoplasmas gelangen in die Drüsenlichtung. — Die Befunde zeigen die von Lebensalter und Proliferationsreiz abhängigen Vorgänge eines permanenten Zellersatzes in der männlichen Brustdrüse an.

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Ultrastructure of the mammary gland of the human male

Summary The ultrastructure of two mammary glands obtained operatively from a 57-year old and a 63-year old man was compared to the structure observed in the light microscope, and related to stimuli controlling growth of the gland, cellular differentiation and desquamation, and apocrine secretion. The glandular epithelium, which is analogous to that of the female mammary gland, is differentiated into basic cells, large superficial cells, and myoepithelial cells. The cells have intracytoplasmic filaments, that may be a sign of differentiation. Mechanisms for secretion were not observed, although pseudopodia-like parts of the cytoplasm are extruded into the glandular lumen. Superficial cells are desquamated, followed by cytolysis of their margins. These findings illustrate the replacement of cells due to age and altered stimuli.

Frl. St. Walter, lt. Assistentin des elektronenmikroskopischen Labors, danken wir für Präparationen und Photoarbeiten.     

Human arterial smooth muscle cells in culture: Inverse relationship between proliferation and expression of contractile proteins

Summary Human arterial smooth muscle cells (hASMC) from explants of the inner media of uterine arteries were studied in secondary culture. We had previously found that these cells depend on exogenous platelet-derived growth factor (PDGF) for proliferation in vitro. Deprivation of the serum mitogen(s) by culture in plasma-derived serum or bovine serum albumin (BSA) caused a true growth arrest that was reversible upon reexposure to the mitogen(s). When added to serum-containing medium, heparin caused a reversible growth arrest which could be competed for by increasing concentrations of serum. In the current study we used a set of smooth muscle-specific actin and myosin, antibodies to study the expression of contractile proteins in stress fibers under indirect immunofluorescence on hASMC in culture. Even in sparse culture, grwoth-arrested hASMC expressed stress fibers containing these actin and myosin epitopes. This was true irrespective of whether growth arrest was achieved by culture in media containing only BSA or a combination of heparin and whole blood serum. hASMC proliferating in whole blood serum in sparse culture did not express such strees fibers, as judged by immunofluorescent staining. This was true also for cells that were restimulated to proliferate in serum after a growth arrest. Utilizing a monoclonal antibody against a nuclear antigen expressed in proliferating human cells, we were able to demonstrate an inverse relationship between the expression of this antigen and the SMC-specific contractile proteins, respectively. Under these culture conditions, the reversible transition between defifferentiated and differentiated hASMC was almost complete and terminated about 1 wk after the change in culture condition. We conclude that hASMC in vitro respond, to exogenous PDGF by proliferation and dedifferetiation as a single population of cells. We also conclude that this modulation is reversible, because the cells become uniformly quiescent and differentiated when the mitogenic stimulus is blocked or removed. This study was supported by grants from the Swedish Medical Research Council (Project no. 4531 and 6816), the Swedish Association against Heart and Chest Diseases, the King Gustaf V and Queen Victoria Foundation, the National Institutes of Health, Bethesda, MD (grant HL 29873) and the Swedish National Board for Laboratory Animals.     

Microporosity of the substratum regulates differentiation of MDCK cells in vitro

Summary We have analyzed the ability of the physical substratum to modulate both the ultrastructural and protein synthetic characteristics of the Madin-Darby canine kidney (MDCK) renal cell line. When MDCK cells were seeded on Millipore Millicell CM microporous membrane cell culture inserts they demonstrated a more columnar organization with an increase in cell density sixfold greater than the same cells seeded on conventional plastic substrata. After 1 wk postseeding on the microporous membrane a partial basal lamina was noted, with a contiguous basement membrane being apparent after 2 wk. One-dimensional sodium dodecyl sulfate gel electrophoresis was used to analyze detergent-solubilized proteins from MDCK cells maintained on plastic substrata vs. microporous membranes. When proteins were pulse-labeled with [³⁵S]methionine, a 55 kDa protein was evident in the cytosolic extract of cells grown on collagen, laminin, and nontreated plastic substrata; but this labeled protein was not evident in similar extracts from cells grown on collagen and laminin-coated microporous membranes. To test if the polarized, basement-membrane secreting phenotype of the MDCK cells could be generated on a microporous membrane without pretreatment with any extracellular matrix (ECM) components, cells were seeded on the Millipore Millicell HA (cellulosic) microporous membrane. This type of substrata does not need a coating of ECM components for cell attachment. A partial basement membrane was formed below cells where the basal surface of the cell was planar, but not in areas where the cell formed large cytoplasmic extensions into the filter. This led us to the conclusion that the microporous nature of the substrata can dictate both ultrastructural and protein synthetic activities of MDCK cells. Furthermore, we suggest that both the planar nature of the basal surface and the microporosity of the substrate are corequisites for the deposition of the basement membrane.