Increasing the toxic effect of pulmonary carcinogens by destruction of epithelial-mesenchymal interaction in explants of embryonic respiratory tract of mice

The explants from 17-day old embryos of A and C57BL mice were used for long-term organ cultures. The following series of explants were studied: 1) the intact control explants; 2) the explants treated by NMU; 3) the explants treated by BP; 4) the explants isolated from mesenchyma (M); 5) the explants isolated from M and treated by NMU; 6) the explants isolated from M and treated by BP. The survival of explants treated by NMU or BP did not differ from the intact control explants (p less than 0.1). The removal of M decreased the survival only in the explants from the distal RT of A mice (p 0.01). The survival of explants isolated from M and treated by NMU or BP was significantly lower than it was in intact explants (p less than 0.01-0.001). Thus, the destruction of epithelial-mesenchymal interaction induced by removal of M can modulate the toxic effect of pulmonotropic carcinogens.     

The role of the mesenchyme in the development of the early primordium of the respiratory epithelium

Development of respiratory epithelium (RE) rudiment was studied in tissue culture after removal of mesenchyme (M) and respiratory tract (RT) in 13 days old embryos of A and C57BL mice. During long-term cultivation of intact RT, organotypic structures (branching bronchioles, alveolus-like cavities) developed. No epithelial organotypic structures developed in the presence of single M cells; explants were represented by layers of cubic epithelium. During long-term cultivation foci of atypical growth consisting of intensively proliferating basophilic cells with high nucleocytoplasmic ratio appeared in these explants. Regions of planocellular metaplasia with or without keratinization could be found in these foci. The frequency of atypical proliferates depended on the strain of donor mice and on the region of the explanted RT.     

代谢抑制剂对有尾类胚胎表皮细胞兴奋性的影响

It has been reported that the cells in atypical epidermis, which developed from the in vitro cultured ectoderm isolated at early gastrula, showed very low excitability or were even non-excitable at 6 V when examined electro-physiologically. If non-excitable explants were treated with energy supplying substances, such as glucose, the action potential (AP) appeared quickly. It indicates that, the excitability of epidermis cells is related to their energy metabolism. In order to verify the above proposition the effects of metabolic inhibitors on the excitability of the epidermis cells were examined using electrophysiological technique. Two kinds of explants were used: explants which developed from the epidermis underlaid with mesoderm isolated at early neurula (epidermis vesicle) and explant which developed from the ectoderm isolated at early gastrula (atypical epidermis). In all experiments explants were stimulated extracellularly and APs were recorded intracellularly. The specimens were stimulated with electric stimulus at 6 V first, and, if they displayed AP, the strength was lowered to determine the stimulus threshold to evoke AP. The duration of stimulus was fixed at 1 ms. The ratio of the resting potential value during treatment to the original value was taken as index of change level of the resting potential (RP). During treatment of epidermis vesicle with 1 mM NaN3 or 1 mM NaCN or 0.1 mM 2,4-dinitrophenol (DNP), the excitability of the epidermis cells was reduced: the stimulus threshold gradually increased and the cells in most explants lost the excitability. The cells became excitable after the drugs were washed out.(ABSTRACT TRUNCATED AT 250 WORDS)     

An antiproliferative heparan sulfate species produced by postconfluent smooth muscle cells

Heparan sulfate was isolated form the cell surface, cell pellet, and culture medium of exponentially growing as well as postconfluent bovine aortic smooth muscle cells (SMCs). After chromatography on DEAE-Sephadex and Sepharose 4B, the various mucopolysaccharides were examined for their ability to cause growth inhibition in a SMC bioassay. The heparan sulfate isolated from the surface of postconfluent SMCs possessed approximately eight times the antiproliferative potency per cell of the heparan sulfate obtained from the surface of exponentially growing SMCs. Heparan sulfate isolated from other fractions of exponentially growing or postconfluent SMCs possesses little growth inhibitory activity. The difference in the antiproliferative activities of heparan sulfate obtained from the surface of SMCs in the two growth states could not be attributed to the synthesis of a greater mass of mucopolysaccharide by postconfluent SMCs. Indeed, heparan sulfate isolated from the surface of the postconfluent SMCs exhibits a specific antiproliferative activity which is 13-fold greater than mucopolysaccharide obtained from the surface of exponentially growing SMCs and more than 40-fold greater than commercially available heparin. In addition, exponentially growing SMCs did not exhibit an enhanced ability to degrade the complex carbohydrate. Furthermore, other investigations indicate that the small amount of growth inhibitory activity intrinsic to heparan sulfate isolated from the surface of exponentially growing SMCs is due to residual, biologically active, mucopolysaccharide produced by the primary postconfluent SMCs from which the exponentially growing SMCs were derived. These studies suggest that bovine aortic SMCs are capable of controlling their own growth by the synthesis of a specific form of heparan sulfate with antiproliferative potency.     

Characteristics of cell structure and proliferative activity of organ cultures of normal embryonic lung tissue of mice resistant (C57BL) and predisposed (A) to lung blastomogenesis

Linear differences were discovered in the quantitative ratio and proliferative activity of the cells of the epithelium and mesenchyma of organ cultures of the normal embryonal pulmonary tissue of mice resistant (C57BL) and predisposed (A) to lung blastomogenesis. In A mouse embryos, the index of the cell labels of the bronchiolar epithelium and fibroblast-like cells of the mesenchyma which migrate on the surface of the membrane filter was several times higher than that in C57BL mice. The number of the mesenchymal cells capable of migration in A mouse explants was essentially greater, that of the mesenchymal cells being in a direct contact with the epithelium of alveoloid and bronchiolar structures was, on the contrary, lower than in C57BL mouse explants. It is suggested that the differences described, particularly epithelial-mesenchymal interactions, are of importance for realization of the genetically determined sensitivity of these mice to spontaneous and induced lung blastomogenesis, which manifests as early as during prenatal ontogenesis.     

Association in organ culture of the hypophysis and glandular tissue of the testis of Gobius niger L.]

Pituitaries of Gobium niger are previously cultured separately for periods of time varying from 6 to 20 days. Therafter, their gonadotropic potency is tested by associating them during three days with explants of glandular tissue from testis as effectors: in each case, a new fish gives a controll pituitary and three testicular explants. The latter are distributed into (1) controll explant, (2) explant associated with controll pituitary, (3) explant associated with a previously cultured pituitary. The results indicate that a gonadotropic potency subsists in the pituitary even after 20 days of isolated culture.     

Thalidomide may inhibit proliferation of mesenchyme in human limb buds

Limb buds from 4- and 4.5-week-old human embryos were cultured on agar medium consisting of Medium 199, chick embryo extract and horse serum for 4 days with or without thalidomide (1-1.5 microgram/ml), and the direct effect of thalidomide was examined morphologically in histological preparations. In the explants treated with thalidomide, mitotic figures of mesenchymal cells were significantly decreased both in overall explant and in mesenchymal cell aggregates, but the extracellular matrix in the mesenchymal cell aggregates was seen in the experimental and control explants. These findings suggest that thalidomide affects undifferentiated and differentiated mesenchymal cell proliferation but not the chondrogenic capacity of the mesenchyme.     

Involvement of plasma membrane glycoproteins in the contact-dependent inhibition of growth of human fibroblasts

The human embryonal lung fibroblasts used in this study showed a pronounced inhibition of growth when reaching a critical cell density. This effect has been mimicked by the addition of glutaraldehyde-fixed human fibroblasts to sparsely seeded growing cells. Inhibition of growth was not observed when glutaraldehyde-fixed cells were pretreated with galactosidase or with galactose-specific lectins, or when glutaraldehyde-fixed human or rabbit erythrocytes were added to the proliferating fibroblasts. In addition, glutaraldehyde-fixed mitotic cells were without effect on the proliferation, while cells prepared from sparse culture had lesser potency than cells prepared from confluent cultures. Plasma membranes, isolated from cells of confluent cultures, when added to growing cultures of human fibroblasts inhibited DNA synthesis in a concentration-dependent manner. On the other hand, plasma membranes isolated from sparsely seeded cells had only minor inhibitory potency. When the plasma membranes were isolated from cells treated previously with tunicamycin, an antibiotic which inhibits the synthesis of the oligosaccharide portion of asparagine-linked glycoproteins, the inhibitory effect was abolished. The same effect was observed when plasma membranes were pretreated with galactosidase. These data indicate that the growth of cells in vitro is regulated by specific cell-cell contacts. They also show that one of the molecular reactants in this process are membrane glycoproteins with asparagine-linked oligosaccharides.     

Neural differentiation of amphibian gastrula ectoderm exposed to phorbol ester

Summary Ectoderm from early gastrula stages of amphibians was isolated and treated with phorbol 12-myristate 13-acetate. The ectoderm formed neural tissue and in a few cases also mesenchyme and melanophores. The control explants formed atypical epidermis. In explants treated with phorbol 12-myristate 13-acetate the mitotic rate was increased.     

Effect of Bis(guanylhydrazones) on Growth and Polyamine Uptake in Plant Cells

In the present work the effect of several bis(guanylhydrazones) on the growth of Helianthus tuberosus tuber explants was studied. Different aliphatic congeners of glyoxal bis(guanylhydrazone) were tested. Most of the compounds displayed an inhibitory effect on growth, and a correlation between the structure of the molecule and the inhibitory activity was observed. Experiments carried out with glyoxal bis(guanylhydrazone) and its congeners methyl-, ethylmethyl-, and methylpropylglyoxal bis(guanylhydrazones) show that as the total number of side chain carbon atoms in the molecule increases, the inhibitory potency also increases. A depletion of spermidine levels was also found in the explants treated with ethylmethylglyoxal bis(guanylhydrazone), which turned out to be one of the most potent growth inhibitors. The addition of spermidine caused a significant reversion of the antiproliferative action of glyoxal bis(guanylhydrazone). The effect of these compounds on spermidine uptake in protoplasts isolated from carrot phloem parenchyma was also investigated. Only a slight competition was found when antagonists were present at concentrations 20 times higher than the polyamine, thus suggesting that bis(guanylhydrazones) do not share, at least at low concentrations, the polyamine transport system in plant cells. Received January 10, 1997; accepted January 22, 1999     

Sensitivity to benzo(a)pyrene of epithelial and mesenchymal cells of embryonic lungs of mice susceptible (line A) and resistant (line C57BL) to lung blastomogenesis

Proliferative activity of alveolar (EA) and bronchial (EB) epithelial cells, mesenchymal cells of explants (ME) and mesenchymal cells migrated on the cellulose filter (MF) was studied autoradiographically in the normal cultures and after treatment with benz(a)pyrene (BP). Labeling index (LI) of cells from the treated explants was higher or lower than in normal cultures, or did not differ from normal values. The effect of the treatment depended on BP dose, mouse strain, and the type of cells. Epithelial cells, especially EB, and ME cells from embryonic lungs of A mice were very sensitive to growth-stimulating effect of BP. In treated cultures from C57BL mice LI increased only in EB cells, however, unlike A mice, the effect inversely correlated with BP doses. Proliferative activity of EA, ME and MF cells was lower than in untreated explants from C57BL, the effect of treatment increasing with higher BP doses. The importance of local cell-tissue factors, namely epithelial-mesenchymal interactions, and the role of lung mesenchyma in the realization of genetically determined sensitivity to spontaneous and induced lung blastomogenesis in C57BL and A mice are discussed.     

Effect of cloning rate on fitness-related traits in two marine hydroids

Hydractinia symbiolongicarpus and Podocoryna carnea are colonial marine hydroids capable of reproducing both sexually and asexually. Asexual reproduction, by colony fragmentation, produces a genetic clone of the parent colony. This study examines the effect of very different cloning rates on colony growth rate, oxygen uptake rate, and colony morphology. Colonies of one clone of each species were maintained for an extended time in two treatments: in a state of constant vegetative growth by repeated cloning, and in a state restricted from vegetative growth (no cloning). For both species, tissue explants taken from the growing colonies grew more slowly than similar explants taken from the restricted colonies. For one species, tissue explants from the growing colonies used oxygen at a higher rate than similar explants from restricted colonies; for the other species, no difference was detected, although the sample size was small. For both species, tissue explants from restricted colonies formed more circular, "sheet-like" shapes, whereas those from their growing counterparts formed more irregular, "runner-like" shapes. After these experiments, in the third winter of treatment, all colonies experienced a severe tissue regression. Within 6 months after this event, the colonies had regrown to their former sizes. A growth assay at this point revealed no difference in growth rate, possibly suggesting an epigenetic basis for these results. Changes in clonal growth rates and morphology correlated with variation in fragmentation rate might affect the ecology of these and other clonal organisms.     

Propagation through alginate encapsulation of axillary buds of Cannabis sativa L. — an important medicinal plant

Cannabis sativa L. (Cannabaceae) is an important medicinal plant well known for its pharmacologic and therapeutic potency. Because of allogamous nature of this species, it is difficult to maintain its potency and efficacy if grown from the seeds. Therefore, chemical profile-based screening, selection of high yielding elite clones and their propagation using biotechnological tools is the most suitable way to maintain their genetic lines. In this regard, we report a simple and efficient method for the in vitro propagation of a screened and selected high yielding drug type variety of Cannabis sativa, MX-1 using synthetic seed technology. Axillary buds of Cannabis sativa isolated from aseptic multiple shoot cultures were successfully encapsulated in calcium alginate beads. The best gel complexation was achieved using 5 % sodium alginate with 50 mM CaCl₂.2H₂O. Regrowth and conversion after encapsulation was evaluated both under in vitro and in vivo conditions on different planting substrates. The addition of antimicrobial substance — Plant Preservative Mixture (PPM) had a positive effect on overall plantlet development. Encapsulated explants exhibited the best regrowth and conversion frequency on Murashige and Skoog medium supplemented with thidiazuron (TDZ 0.5 μM) and PPM (0.075 %) under in vitro conditions. Under in vivo conditions, 100 % conversion of encapsulated explants was obtained on 1:1 potting mix- fertilome with coco natural growth medium, moistened with full strength MS medium without TDZ, supplemented with 3 % sucrose and 0.5 % PPM. Plantlets regenerated from the encapsulated explants were hardened off and successfully transferred to the soil. These plants are selected to be used in mass cultivation for the production of biomass as a starting material for the isolation of THC as a bulk active pharmaceutical.Key words: Encapsulation, Nodal explants, Plant growth regulators, Plant regeneration, Synthetic seeds     

Partial removal of the mesenchyma disrupts the growth and differentiation of the epithelium in respiratory tract explants from strain A and C57BL mouse embryos

Differentiation of respiratory epithelium (E) of 17-day old embryos of A and C57BL mice after partial removal of mesenchyme (M) was studied in organ culture. In control explants, tissue-specific growth and differentiation of epithelium were observed during long-term culturing. Ih both mouse strains, partial removal of mesenchyme prevented the development of alveolar-like structures in explants of distal part of respiratory tract. In most explants of proximal part of respiratory tract (65.1% in A mice and 85.7% in C57BL mice) with partially removed mesenchyme, we found atypical epithelial structures and foci of poorly differentiated cells with high proliferation rate.     

Comparison in sensitized and unsensitized guinea-pigs of tuberculin PPDs RT 23 and PPD-M by skin tests and lymphocyte stimulation tests. Effect of immunization time

The biological activities of tuberculin PPD RT 23 and the International Standard for Purified Protein Derivative of Mammalian Tuberculin (PPD-M) were compared in sensitized and unsensitized guinea-pigs by skin tests and lymphocyte stimulation (LS) tests. Estimates of relative potency (RP) from skin test results were dependent on the dose level, on the immunogen used, and, in guinea-pigs immunized with killed tubercle bacilli in oil, also on the immunization time. Relative potency estimates from LS results were dependent on the source of the lymphocytes and were different from estimates obtained from skin tests. Lymphocyte stimulation dose-response curves for the tuberculins were qualitatively different. In contrast to RT 23, PPD-M gave rise to non-specific skin reaction in unsensitized guinea-pigs. Both tuberculins were mitogenic to lymph node lymphocytes isolated from unsensitized guinea-pigs, PPD-M being the more mitogenic of the two tuberculins. The present results confirm that qualitatively different tuberculins cannot be unambiguously calibrated in identical terms and thus emphasize that the uncritical use of (international) standards should be avoided in tuberculin calibration.     

Topologically restricted appearance in the developing chick retinotectal system of Bravo, a neural surface protein: experimental modulation by environmental cues [published erratum appears in J Cell Biol 1991 Mar;112(5):1049]

A novel neural surface protein, Bravo, shows a pattern of topological restriction in the embryonic chick retinotectal system. Bravo is present on the developing optic fibers in the retina; however, retinal axons in the tectum do not display Bravo. The appearance of Bravo in vitro is modulated by environmental cues. Axons growing out from retinal explants on retinal basal lamina, their natural substrate, express Bravo, whereas such axons growing on collagen do not. Retinal explants provide a valuable system to characterize the mechanism of Bravo restriction, as well as the cellular signals controlling it. Bravo was identified with monoclonal antibodies from a collection generated against exposed molecules isolated by using a selective cell surface biotinylation procedure. The NH2-terminal sequence of Bravo shows similarity with L1, a neural surface molecule which is a member of the immunoglobulin superfamily. This possible relationship to L1, together with its restricted appearance, suggests an involvement of Bravo in axonal growth and guidance.     

In vitro pancreas formation from Xenopus ectoderm treated with activin and retinoic acid

In the present study, isolated presumptive ectoderm from Xenopus blastula was treated with activin and retinoic acid to induce differentiation into pancreas. The presumptive ectoderm region of the blastula consists of undifferentiated cells and is fated to become epidermis and neural tissue in normal development. When the region is isolated and cultured in vitro, it develops into atypical epidermis. Isolated presumptive ectoderm was treated with activin and retinoic acid. The ectoderm frequently differentiated into pancreas-like structures accompanied by an intestinal epithelium-like structure. Sections of the explants viewed using light and electron microscopy showed some cells clustered and forming an acinus-like structure, including secretory granules. The pancreas-specific molecular markers insulin and XIHbox8 were also expressed in the treated explants. The pancreatic hormones, insulin and glucagon, were detected in the explants using immunohistochemistry. Therefore, sequential treatment with activin and retinoic acid can induce presumptive ectoderm to differentiate into a morphological and functional pancreas in vitro. When ectoderm was immediately treated with retinoic acid after treatment with activin, well-differentiated pronephric tubules were seen in a few of the differentiated pancreases. Treatment with retinoic acid 3-5 h after activin treatment induced frequent pancreatic differentiation. When the time lag was longer than 15h, the explants developed into axial mesoderm and pharynx. The present study provides an effective system for analyzing pancreas differentiation in vertebrate development.     

Novel role for insulin as an autocrine growth factor for malignant brain tumour cells

AT/RTs (atypical teratoid/rhabdoid tumours) of the CNS (central nervous system) are childhood malignancies associated with poor survival rates due to resistance to conventional treatments such as chemotherapy. We characterized a panel of human AT/RT and MRT (malignant rhabdoid tumour) cell lines for expression of RTKs (receptor tyrosine kinases) and their involvement in tumour growth and survival. When compared with normal brain tissue, AT/RT cell lines overexpressed the IR (insulin receptor) and the IGFIR (insulin-like growth factor-I receptor). Moreover, insulin was secreted by AT/RT cells grown in serum-free medium. Insulin potently activated Akt (also called protein kinase B) in AT/RT cells, as compared with other growth factors, such as epidermal growth factor. Pharmacological inhibitors, neutralizing antibodies, or RNAi (RNA interference) targeting the IR impaired the growth of AT/RT cell lines and induced apoptosis. Inhibitors of the PI3K (phosphoinositide 3-kinase)/Akt pathway also impaired basal and insulin-stimulated AT/RT cell proliferation. Experiments using RNAi and isoform-specific pharmacological inhibitors established a key role for the class I(A) PI3K p110alpha isoform in AT/RT cell growth and insulin signalling. Taken together, our results reveal a novel role for autocrine signalling by insulin and the IR in growth and survival of malignant human CNS tumour cells via the PI3K/Akt pathway.     

High-frequency regeneration via multiple shoot induction of an elite recalcitrant cotton (Gossypium hirsutum L. cv Narashima) by using embryo apex

Cotton (Gossypium hirsutum L.) is one of the most commercially important fiber crops in the world. Compared with other crops, cotton represents a recalcitrant species for regeneration protocols. The development of efficient and rapid regeneration protocol for elite Indian cotton variety could help improve the quality characteristics and biotic or abiotic stress tolerance. Here we report a novel regeneration protocol in Indian cotton cultivar Narashima. The maximum number of multiple shoots obtained was 16 per explants, performance which has never been achieved in any prior reports. The embryo apex explants were isolated from 2 d old in vitro growing seedlings. Explants were cultured on MS medium containing different plant growth regulator combinations in order to induce multiple shoots. Among the tested combinations, the 2 mg/l of 6-benzylaminopurine (BAP) and 2 mg/l kinetin (KIN) proved to be most suited for achieving the maximum number of multiple shoots. The elongation of multiple shoots was obtained in media supplemented with gibberellic acid (GA3). The regenerated plants were successfully hardened in earthen pots after adequate acclimatization. This method avoids callus tissue, the stage of regeneration which may lead to somaclonal variation. The important feature of the presented method is shortening of regeneration time, as well as the induction of a high number of multiple shoots per explants. The present protocol may provide an efficient and rapid regeneration tool for obtaining more stable transformants from embryo apex explants of Indian cotton cultivar Narashima.     

Differentiation and transdifferentiation of adrenal chromaffin cells of the guinea-pig

Summary Expiants of adrenal medullary tissue taken from newborn guinea pigs were grown in culture for up to two weeks. The explants exhibited sparse outgrowth of neurite-like processes, in contrast to adrenal medullae taken from young postnatal rats or adult guinea pigs that were (i) grown under identical conditions (Unsicker and Chamley 1977) or (ii) transplanted to the anterior chamber of the eye (Unsicker et al. 1981), respectively. Nerve growth factor (10–100 ng/ml, 2.5S NGF) did not enhance formation of processes. However, electron-microscopic investigations revealed the presence of numerous processes within the explants, which extended from chromaffin cells and were characterized by longitudinally oriented cytoskeletal structures, various populations of clear and dense-cored vesicles, varicosities and growth cones. Chromaffin cell bodies largely resembled their in situ-counterparts, but had fewer and smaller storage vesicles than controls.The results are discussed in light of recent findings regarding the potency of NGF and NGF-like growth factors to induce neuronal transdifferentiation of adrenal chromaffin cells.Supported by grants from the Deutsche Forschungsgemeinschaft (SFB 103 and Un 34/6)