Biochemical characterization of chromatin fractions isolated from induced and uninduced friend erythroleukemia cells

Summary Chromatin fractions from Friend erythroleukemia cells after induction of differentiation by dimethylsulfoxide (DMSO) were compared in their biochemical characteristics to fractions from uninduced cells. Fractions were prepared by extracting chromatin from nuclei after mild micrococcal nuclease treatment with increasing concentrations of NaCl according to Sanders [1]. This procedure has been found to release chromatin containing hyperacetylated histones preferentially [2]. The fractions obtained by this procedure were analysed in respect to the amount of chromatin released, the amount of histone H1, the degree of acetylation of histone H4, the presence of non-histone proteins and the concentration of transcribed and non-transcribed sequences. It was found that the fractions differ in the amount of histone H1 present, in several non-histone proteins and in the acetylation of histonie H4, regardless whether induced or uninduced cells were analysed. The distribution of transcribed sequences versus non-transcribed sequences among the fractions was the same, demonstrating that this fractionation procedure, although leading to fractions with biochemical differences, is not able to discriminate functional states of chromatin and that the biochemical characteristics of the fractions may be common to both, active as well as inactive states of chromatin.     

Interaction of (Na + K + 2 Cl) cotransport and the Na/K pump in cultured chick cardiac myocytes

We have recently reported the presence of an electroneutral (Na + K + 2 Cl) cotransport mechanism that is bumetanide-sensitive and maintains Cl_i above its electrochemical equilibrium in cultured chick heart cells. In steady state, (Na + K + 2 Cl) cotransport is inwardly directed and so contributes to the Na influx that must be counterbalanced by the activity of the Na/K pump to maintain Na_i homeostasis. We now show that manipulating (Na + K + 2 Cl) cotransport by restoring Cl_o to a Cl-free solution indirectly influences Na/K pump activity because the bumetanide-sensitive recovery of a _infNa ^supi to its control level and the accompanying hyperpolarization could be blocked by 10^–4M ouabain. In another protocol, when the Na/K pump was reactivated by restoring K_o (from 0.5 mM to 5.4 mM) and removing ouabain, the recovery of a_Na was attenuated by 10^–4M bumetanide. The relatively slow rate of ouabain dissociation coupled with the activation of Na influx by (Na + K + 2 Cl) cotransport clearly establishes the interaction of these transport mechanisms in regulating Na_i. Although (Na + K + 2 Cl) cotransport is electroneutral, secondary consequences of its activity can indirectly affect the electrophysiological properties of cardiac cells.     

Effects of salinity on chloride cells in the euryhaline cyprinodontid fish Rivulus marmoratus

Summary The ultrastructure and density of chloride cells in the gill, opercular epithelium, and opercular skin of the euryhaline self-fertilizing fish Rivulus marmoratus (Cyprinodontidae) were studied with electron and fluorescence microscopy. R. marmoratus raised from birth in 1, 50, 100, and 200% seawater were compared. Chloride cells from fish raised in each of the four salinities exhibited an invaginated pit structure at the apical crypt. Multicellular complexes were present in the 1% seawater group and in those fish raised in higher salinities where elaborate interdigitations were seen between cells. Chloride cells from gills of fish raised in 200% seawater had a significantly higher percentage of their cytoplasmic volume composed of mitochondria than did those from fish raised in 1% seawater (69.9% vs 37.4%). The opercular skin and opercular epithelium had the same density of chloride cells (4.2×10⁴-4.5×10⁴ chloride cells/cm²), and this number did not vary significantly with increased salinity. The opercular skin thus appears far more responsive to environmental salinity than the opercular epithelium. Chloride cells from the opercular epithelium of fish raised in 200% seawater were found to be 39% larger than those from fish raised in 1% seawater, whereas the chloride cells from the opercular skin of the 200% seawater group were 107% larger than those from the 1% seawater group.     

Binding and internalization of gold-conjugated somatostatin and growth hormone-releasing hormone in cultured rat somatotropes

Summary The synthetic peptides somatostatin (SRIF) and growth hormone-releasing hormone (GRH) were coupled directly to colloidal gold of different particle sizes. Both conjugates were biologically active in displacing the corresponding radiolabeled hormones from high affinity binding sites in pituitary membranes. Release of growth hormone (GH) from cultured anterior pituitary cells was modulated by both conjugates alone or in combination. Ultrastructural studies were performed with cells incubated at 4° C (2 h) and 37° C (2 min-2 h) with one of the labeled peptides or their combination. Somatotropes were identified by immunostaining with anti-rGH followed by protein A-ferritin, thus obtaining a triple labeling. Both hormone conjugates were internalized in different vesicles in the beginning but accumulated during longer incubation times in the same compartment. The secretory vesicles and the nucleus were not labeled by any hormone conjugate. In contrast to SRIF-gold, the uptake of GRH-gold conjugate decreased with longer incubation times. This effect could be neutralized by simulatenous incubation of the somatotropes with both regulating hormones. Hence, whereas the binding and internalization of SRIF by somatotropes do not seem to be influenced by GRH, the corresponding processes for GRH are stimulated by the presence of SRIF.     

Leydig cell mitoses in human testes bearing early germ cell tumors

Summary Numerous mitoses were noted in testicular tissue from adult men with early germ cell tumors. More than 15 Leydig cells undergoing mitosis were found in the interstitial compartment. The presence of specific crystalline intracytoplasmatic inclusions demonstrated for the first time that differentiated Leydig cells are capable of proliferation. Occasionally cells are difficult to discriminate during mitosis. To establish reference criteria, the light- and electron-microscopic features of the following mitotic cells were examined: Leydig cells, fibroblasts, perivascular cells, peritubular cells, and lymphocytes. Supplementary mitoses in germ cell tumors and in a case of Leydig cell tumor were investigated. In the literature, only single reports of mitoses in Leydig cells are available. The frequent incidence of Leydig cell mitosis in early germ cell tumors may be due to the presence of growth-promoting factors in the testicular tissue.     

The effect of low- and high-density lipoprotein cholesterol on steroid hormone production and ACTH-induced differentiation of rat adrenocortical cells in primary culture

Summary The choroid plexus consists of the choroidal epithelium, a derivative of the neural tube, and the choroidal stroma, which originates from the embryonic head mesenchyme. This study deals with epithelio-mesenchymal interactions of these two components leading to the formation of the organ. Grafting experiments of the prospective components have been performed using the quail-chicken marker technique. Prospective choroidal epithelium of quail embryos, forced to interact with mesenchyme of the body wall of chicken embryos, gives rise to a choroid plexus showing normal morphogenesis and differentiation. The choroidal epithelium induces the differentiation of organtypical fenestrated capillaries, which are highly permeable to intravenously injected horseradish peroxidase. The choroidal epithelium of the grafts constitutes a blood-cerebrospinal fluid barrier. On top of the choroidal epithelium, there are epiplexus cells displaying a typical ultrastructure. The experimental results show that these cells do not originate from the transplanted neural epithelium. Prospective choroidal stroma of chicken embryos does not exert a choroid plexus-inducing influence upon a quail embryo's neural epithelium isolated from parts of the brain that normally do not develop a choroid plexus. The experiments show that the choroidal epithelial cells are determined at least three days before the first organ anlage is detectable.This work was supported by the Deutsche Forschungsgemeinschaft (grant Ch 44/7-1)     

Ultrastructure of carotenoid deprivation in photoreceptors of Manduca sexta: myeloid bodies and intracellular microvilli

Summary The ultrastructural effects of carotenoid (vitamin A) deprivation were studied in the adult photoreceptors of the tobacco hornworm moth Manduca sexta. Moths were reared on a deprived diet, which lacked the carotenoid sources of the photopigment chromophore, 3-hydroxy retinal, or on a control, fortified diet, containing ample carotenoid. The latter supported normal levels of visual function, whereas visual pigment and sensitivity were reduced by at least 3 log units in moths reared on the deprived diet. Myeloid bodies, massed cisternae of hypertrophied smooth endomembrane, filled the cytoplasm in the receptors of deprived animals. The myeloid bodies assumed various configurations that included lamellate stacks of parallel cisternae, and tubular networks in a paracrystalline form. Freeze-fracture preparations of myeloid membranes revealed a high density of P-face particles. Vacuoles containing microvilli similar to those of the rhabdomere were also present in deprived photoreceptors. We suggest that the elaboration of smooth endoplasmic reticulum as myeloid bodies in chromophore-deprived photoreceptors may stem from the hypertrophy of a biochemical system for processing the chromophore or the interruption of the intracellular pathway that normally carries visual pigment to the rhabdomere.     

Vasoactive intestinal polypeptide-like immunoreactivity in the bovine heart: high degree of coexistence with neuropeptide Y-like immunoreactivity

Summary Recent studies have accomplished the establishment of a collagenous fiber-fringe matrix upon dental root surfaces in vitro. The present study was undertaken to follow the development of such a matrix in vitro and to test the possible effects of root surface treatments upon this matrix. Periodontal ligament cells, 0.1 to 0.2-mm thick dental root discs, and alveolar bone cells were derived after extraction from four partially erupted third molars and the accompanying interradicular bony septa of 1 male patient. Autologous serum was obtained by venipuncture. Cultures were initiated by delivering a 1-ml suspension of 50000 tritiated thymidine-labeled periodontal ligament cells and 50000 alveolar bone cells onto each of 42 culture sets. The following day, demineralized or non-demineralized root discs treated with autologous serum, fibronectin or complete medium were placed in pairs, separated by a 0.1–1.0 mm gap, upon the initial cell layer. Representative cultures were terminated after 2, 3, 4, 5 and 6 weeks, and processed for light- and electron microscopy, morphometric analysis and autoradiography. An outstanding feature of the early cultures (2, 3 and 4 weeks) was a patchwise, random distribution of matrix making a precise developmental study impossible, although collagen fibrils were produced within the first 2 weeks. Some 3-week cultures already demonstrated a mature fiber-fringe characterized electron-microscopically as oriented, densely packed collagen fibrils closely abutting the cementum-lined root discs. The treatments (including autologous serum) used in this study had no appreciable morphologic or morphometric effect upon the fiber-fringe formed. Because none of the cultures in the present or past studies have demonstrated a true cementoid matrix, this model may not be suitable for the in-vitro study of cementum formation.     

Control of intracellular glutathione and its effect on ultraviolet radiation-induced K+ efflux in cultured rose cells

Abstract Modification of the ‘intracellular concentration of reduced glutathione’ (IC-GSH) affected the response of cultured rose cells (Rosa damascena) to ultraviolet radiation (UV)-induced leakage of K⁺. High IC-GSH induced by incubation of cells in 10 mol m^?3 GSH (IC-GSH increased linearly with time from 20 to about 600 μmol g^?1 in 61.2 ks) caused cells to become significantly less sensitive to UV. Low IC-GSH induced by treatment with 1 mol m^?3 buthionine sulphoximine (BSO) plus 1 mol m^?3 diethylmaleate (DEM) (IC-GSH decreased from 20 to about 3 μg g^?1 in 61.2 ks) reduced, rather than increased, the UV-sensitivity of the cells. However, treatment with DEM also induced a large transient K⁺ leakage; and treatment with BSO induced a slight leakage. The K⁺ leaked was recovered by 3.24 ks. Following K⁺ recovery, the DEM-treated cells showed almost complete insensitivity to UV, and BSO-treated cells showed a slightly reduced sensitivity to UV. These results are in agreement with our previous findings that other treatments (heat, cycloheximide, UV), which also cause a transient leakage of K⁺, also reduce the induction of K⁺ leakage by a subsequent UV treatment. We conclude that high IC-GSH may play a role in protecting plant cells from UV-induced K⁺ leakage. Increased UV-sensitivity with low ICGSH was not observed, we believe, because of the transient K⁺ leakage, though the mechanism of reduced sensitivity to UV induced by transient leakage of K⁺ is not known at this time. Treatment with UV did not reduce the IC-GSH, showing that this is not the mechanism by which UV induces K⁺ leakage.