Connective tissue is involved in adult epithelial development of the small intestine during anuran metamorphosis in vitro

Summary The role of connective tissue in metamorphic changes of the small intestinal epithelium inXenopus laevis tadpoles was investigated by using organ culture techniques and electron microscopy. Tissue fragments isolated from various parts of the small intestine at stage 57 were cultivated. Larval cell death of the epithelium was induced by thyroid hormone in all fragments, whereas adult epithelial development was observed only in fragments isolated from the anterior intestinal region containing the typhlosole where most of the larval connective tissue was localized. The epithelium was then cultivated in recombination with homologous or heterologous non-epithelial components. The adult epithelium developed only in recombinants containing a thick connective tissue layer from the typhlosole. There was no regional difference in the developmental potency of the epithelium itself. In all explants where adult epithelium developed, the connective tissue increased in cell density just beneath the epithelium, which was rapidly proliferating and forming typical islets. At the same time, fibroblasts possessing well-developed rough endoplasmic reticulum differentiated close to epithelial cells and often made contact with them. These results indicate that the connective tissue originating from the typhlosole plays an important role in adult epithelial development of the anuran small intestine, probably via direct cell-to-cell contacts or some factor(s) synthesized by the fibroblasts.     

Autolysis and heterolysis of the epidermal cells in anuran tadpole tail regression

Autolysis and heterolysis of the degenerating epidermis of the tail fin of Rana japonica tadpoles during spontaneous metamorphosis were observed by transmission and scanning electron microscopy. In the early climactic stages of metamorphosis (st. 19–20), the outermost epidermal cells developed vacuoles that were acid phosphatase positive and showed apparent breakdown of the cell membrane. The cells shrunk, perhaps due to the rupture of the cell membrane, and sloughed off without typical cornification. As tail resorption proceeded, autolysis of the epidermal cells spread towards the inner layers, in which some epidermal cells lost desmosomal junctions. They also displayed atrophic figures with condensed cytoplasm, breakdown of the cell membrane, and pycnotic nuclei. Lymphocytes, neutrophils and macrophages were already present in the basal layers of the premetamorphic epidermis (st. 10). Based on ultrastructural observation, blood cells could be distinguished from autolysing epidermal cells. Only a few blood cells were found in the early climactic stages of metamorphosis (st. 19–20), but the number of the blood cells, especially macrophages, greatly increased during the final stages of metamorphosis (st. 23–24). During the final stages, many macrophages were observed to phagocytose the autolysing epidermal cells by projecting slender pseudopodia into the inner epidermis. Macrophages also were observed to pass through the degraded basal lamella. These results suggest that not only autophagy but also heterophagy of the epidermal cells by the macrophages is a major process in the regression of the tail fin epidermis.     

Induction of metamorphosis by thyroid hormone in anuran small intestine cultured organotypically in vitro

Summary We have developed an organ culture system of the anuran small intestine to reproduce in vitro the transition from larval to adult epithelial form which occurs during spontaneous metamorphosis. Tubular fragments isolated from the small intestine ofXenopus laevis tadpoles were slit open and placed on membrane filters in culture dishes. In 60% Leibovitz 15 medium supplemented with 10% charcoal-treated serum, the explants were maintained in good condition for at least 10 days without any morphologic changes. Addition of triiodothyronine (T₃) at a concentration higher than 10⁻⁹ M to the medium could induce cell death of larval epithelial cells, but T₃ alone was not sufficient for proliferation and differentiation of adult epithelial cells. When insulin (5 μg/ml) and cortisol (0.5 μg/ml) besides T₃ were added, the adult cells proliferated and differentiated just as during spontaneous metamorphosis. On Day 5 of cultivation, the adult cells rapidly proliferated to form typical islets, whereas the larval ones rapidly degenerated. At the same time, the connective tissue beneath the epithelium suddenly increased in cell density. These changes correspond to those occurring at the onset of metamorphic climax. By Day 10, the adult cells differentiated into a simple columnar epithelium which possessed the brush border and showed the adult-type lectin-binding pattern. Therefore, the larval epithelium of the small intestine responded to the hormones and transformed into the adult one. This organ culture system may be useful for clarifying the mechanism of the epithelial transition from larval to adult type during metamorphosis.     

Characterization of macrophage-like cells in the external layers of human small and large intestine

Summary In the external layers of human small and large intestine macrophage-like cells were characterized by immunohistochemical, histochemical and electronmicroscopical methods. Using immunohistochemistry and a number of monoclonal antibodies, the presence and distribution of phenotypic subpopulations of macrophages were evaluated. In all locations macrophage-like cells were identified with antibody EBM11, which recognizes CD68 antigen, C3bi which recognizes CD11b, and partly with an antibody which recognizes protein 150,95 (CD11c). Macrophage-like cells in the external muscle layer were HLA-DR-positive (expressing the MHC class-II antigen), in contrast to macrophage-like cells in the subserosa and submucosa. Macrophage-like cells in the external muscle layer were mostly acid phosphatase-negative, and at the electron-microscopic level they were found to have features of macrophages: primary lysosomes, coated vesicles and pits. However, very few secondary lysosomes were present. Birbeck granules were not observed. It is concluded that in the external muscle layer of human small and large intestine numerous macrophages of a special type are present. It is discussed whether this cell type plays a role in gastrointestinal motility and/or has an immunological function.     

Programmed cell death by default in embryonic cells, fibroblasts, and cancer cells.

We recently proposed that most mammalian cells constitutively express all of the proteins required to undergo programmed cell death (PCD) and undergo PCD unless continuously signaled by other cells not to. Although some cells have been shown to work this way, the vast majority of cell types remain to be tested. Here we tested purified fibroblasts isolated from developing or adult rat sciatic nerve, a mixture of cell types isolated from normal or p53-null mouse embryos, an immortalized rat fibroblast cell line, and a number of cancer cell lines. We found the following: 1) All of these cells undergo PCD when cultured at low cell density in the absence of serum and exogenous signaling molecules but can be rescued by serum or specific growth factors, suggesting that they need extracellular signals to avoid PCD. (2) The mixed cell types dissociated from normal mouse embryos can only support one another's survival in culture if they are in aggregates, suggesting that cell survival in embryos may depend on short-range signals. (3) Some cancer cells secrete factors that support their own survival. (4) The survival requirements of a human leukemia cell line change when the cells differentiate. (5) All of the cells studied can undergo PCD in the presence of cycloheximide, suggesting that they constitutively express all of the protein components required to execute the death program.     

Phosphofructokinase D from the epithelial cells of rat small intestine.

Phosphofructokinase from the epithelial cells of rat small intestine was characterized with respect to isoenzyme type in a comparison of its properties with those of the skeletal-muscle, brain and major liver isoenzymes by using five different techniques, namely electrophoresis on cellulose acetate and in polyacrylamide gels, chromatography on DEAE-cellulose, (NH4)2SO4 precipitation and immunotitration. When precautions were taken to inhibit the formation of active proteolytic artifacts by the action of endogenous proteinases, each technique revealed that rat intestinal mucosa contains only a single form of phosphofructokinase. The mucosal isoenzyme was found to be very similar to, although not identical with, the major liver isoenzyme and to be quite distinct from the skeletal-muscle isoenzyme when studied by the techniques of cellulose acetate electrophoresis, chromatography on DEAE-cellulose and immunotitration, whereas the converse was true when studied by the techniques of (NH4)2SO4 precipitation and polyacrylamide-gel electrophoresis. The mucosal isoenzyme was distinct from the brain isoenzyme when studied by each of the five techniques. Tsai & Kemp [(1973) J. Biol. Chem. 248, 785-792] reported that animal tissues contain three principal isoenzymes of phosphofructokinase, type A found as the sole isoenzyme in skeletal muscle, type B found as the major isoenzyme in liver and type C found as a significant isoenzyme in brain. Phosphofructokinase from mucosa is distinct from each of these isoenzymes. Following the nomenclature of Tsai & Kemp (1973), the isoenzyme from the mucosa of rat intestinal epithelial cells is designated phosphofructokinase D. The mucosal and liver isoenzymes behave so similarly with respect to their charge and immunological characteristics, on which the typing of isoenzymes is conventionally based, that it is likely that some tissues reported to contain the liver isoenzyme contain instead the mucosal isoenzyme.     

Programmed cell death of larval tissues induced by juvenile hormone in the bamboo borer, Omphisa fuscidentalis

Programmed cell death (PCD) plays a critical role during animal development through the destruction of unneeded cells and tissues. In some insects, the prothoracic glands (PGs) and anterior silk glands (ASGs) are larval-specific tissues that are normally eliminated by PCD after pupation. Previous studies report that juvenile hormone analog (JHA) terminates the larval diapause of Omphisa fuscidentalis by increasing the hemolymph ecdysteroids that trigger PCD. Because JHA may indirectly induce the PCD of the PGs and ASGs of Omphisa diapausing larvae, the effects of JHA on the induction of PCD were determined. The application of 1μg JHA induced PCD in the PGs and ASGs of larvae identified as stage G0 (prior to pupation). The injection of 1μg 20E triggered the PCD of the ASGs when the larvae expressed a G0-G1 morphology, whereas PCD occurred in the PGs on day 1 post-injection. Histological studies revealed similar patterns of morphological changes during the PG and ASG PCD in the JHA- and 20E-treated larvae. Furthermore, to confirm that PCD was induced by a high ecdysteroid level that increases after JHA application, the expression profiles of EcR-A and EcR-B1 in the PGs and ASGs from the JHA-treated larvae were examined, and the results showed that the expression levels of EcR-A and EcR-B1 mRNA increased during the G0 stage. These results suggest that JHA may be involved in PCD by increasing the ecdysteroid titer, leading to termination of the larval diapause period in Omphisa fuscidentalis.     

Programmed cell death of plant tracheary elements differentiating in vitro

Summary We used various microscopic and labeling techniques to examine events occurring during the programmed cell death (PCD) of plant tracheary elements (TEs) developing in vitro. TEs differentiating in vitro synthesize a secondary cell wall which is complex in composition and pattern at approximately 72 h after hormone manipulation. The timing of PCD events was established relative to this developmental marker. Cytoplasmic streaming continues throughout secondary wall synthesis, which takes 6 h to complete in a typical cell. Vital dye staining and ultrastructural analysis show that the vacuole and plasma membrane are intact during secondary cell wall synthesis, but the cytoplasm becomes less dense in appearance, most likely through the action of confined hydrolysis by small vacuoles which are seen throughout the cell at this time. The final, preeminent step of TE PCD is a rapid collapse of the vacuole occurring after completion of secondary cell wall synthesis. Vacuole collapse is an irreversible commitment to death which results in the immediate cessation of cytoplasmic streaming and leads to the complete degradation of cellular contents, which is probably accomplished by release of hydrolytic enzymes sequestered in the vacuole. This event represents a novel form of PCD. The degradation of nuclear DNA is detectable by TUNEL, an in situ labeling method, and appears to occur near or after vacuole collapse. Our observations indicate that the process of cellular degradation that produces the hollow TE cell corpse is an active and cell-autonomous process which is distinguishable morphologically and kinetically from necrosis. Although TE PCD does not resemble apoptosis morphologically, we describe the production of spherical protoplast fragments by cultured cells that resemble apoptotic bodies but which are not involved in TE PCD. We also present evidence that, unlike the hypersensitive response (HR), TE PCD does not involve an oxidative burst. While this evidence does not exclude a role for reactive oxygen intermediates in TE PCD, it does suggest TE PCD is mechanistically distinct from cell death during the HR.Abbreviations BA 6-benzylamino-purine - DAPI 4,6-diamidino-2-phenylindole diacetate - DCF 2,7-dichlorofluorescein diacetate - DPI diphenyleneiodonium - FDA fluorescein diacetate - HR hypersensitive response - NAA -naphthalene-acetic acid - PCD programmed cell death - ROI reactive oxygen intermediate - TE tracheary element - TUNEL TdT-mediated dUTP nick end labeling     

Programmed cell death by bcl-2-dependent and independent mechanisms in B lymphoma cells.

Programmed cell death (PCD) or apoptosis is a common form of cellular demise during embryogenesis, tumorigenesis and clonal selection in the immune system. The bcl-2 proto-oncogene has been recently implicated as a potential physiological regulator of the PCD pathway. Gene transfer studies have shown that overexpression of bcl-2 blocks apoptosis mediated by several stimuli in cultured cell lines and promotes the survival of B and T lymphocytes in transgenic mice. However, it remains unclear whether under normal conditions bcl-2 is responsible for controlling cell death. We have investigated the role of bcl-2 in the antimembrane IgM (mIgM)-induced apoptotic death of WEHI-231 B cell lymphoma, a model that mimics clonal deletion of immature B cells by antigen. Signalling of mIgM receptors triggered downregulation of both bcl-2 RNA and protein, and induced apoptosis in WEHI-231 B cells. This effect appeared to be specific since (i) the levels of beta 2-microglobulin and beta-actin RNA remain unchanged and (ii) signalling of the apoptosis-resistant B cell lymphoma line BAL-17 with anti-mu was not associated with downregulation of bcl-2 RNA. However, stable expression of bcl-2 by transfection did not rescue WEHI-231 B cells from apoptosis, yet WEHI-231 cells overexpressing bcl-2 were more resistant to programmed cell death induced by heat-shock.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipoproteins of rat small intestine epithelial cells in D-hypovitaminosis

Chemical composition of lipoproteins has been studied in intestinal epitheliocytes of rats in normalcy and under D-hypovitaminosis. It is found that D-hypovitaminosis induces changes in the lipid and protein composition of lipoproteins. It is supposed that disturbances in biosynthesis of the lipoprotein components and their transport may be possible reasons of such changes.     

Catalase particles in the epithelial cells of the guinea-pig small intestine

Synopsis Purified preparations of epithelial cells have been made from the guinea-pig small intestine. Homogenates of these preparations have been analysed by centrifugation in a zonal rotor. The results confirm the presence of lysosomes in these cells and indicate the existence of catalase particles which equilibrate in a sucrose gradient at a density of between 1.21 and 1.23 and which have a different distribution from other subcellular particles except lysosomes. Injection of Triton WR-1339 into fasting animals enables the separation of lysosomes and catalase particles.     

Programmed cell death in the germline

In many organisms, programmed cell death of germ cells is required for normal development. This often occurs through highly conserved events including the transfer of vital cellular material to the growing gametes following death of neighboring cells. Germline cell death also plays a role in such diverse processes as removal of abnormal or superfluous cells at certain checkpoints, establishment of caste differentiation, and individualization of gametes. This review focuses on the cell death events that occur during gametogenesis in both vertebrates and invertebrates. It also examines the signals and machinery that initiate and carry out these germ cell deaths.     

Programmed cell death in neurotumour cells involves the generation of ceramide

Ceramide has been typically thought of as the membrane anchor for the carbohydrate in glycosphingolipids but many studies have suggested that it may cause apoptosis. Apoptosis or programmed cell death (PCD) is thought to be responsible for the death of one-half of neurons surviving the development of the nervous system. The potential involvement of the sphingomyelin-ceramide signaling process as an integral part of PCD was therefore examined in several neurotumour cell lines. We show that synthetic C2-ceramide (N-acetylsphingosine), a soluble ceramide analogue, can rapidly trigger PCD in these cells, characterized by: 1) classic DNA laddering on agarose gels; 2) DNA fragmentation as determined by Hoechst Dye; and 3) cell viability (mitochondrial function and intact nuclei) assays. We report that staurosporine can both activate PCD (by all three criteria above) in neurotumour cells and increase both the formation of ceramide and ceramide mass. Both ceramide formation and the induction of PCD were further enhanced by the co-addition of a ceramidase inhibitor oleoylethanolamine (25 µM). Staurosporine and oleoylethanolamine were similarly effective in inducing ceramide formation and PCD in immortalized hippocampal neurons (HN-2) and immortalized dorsal root ganglion cells (F-11). Our data suggests that formation of ceramide is a key event in the induction of PCD in neuronally derived neurotumour cells.Abbreviations PCD programmed cell death - PKC protein kinase C - HPTLC high-performance thin-layer chromatography - DETAPAC diethylenetriaminepentaacetic acid - DMEM Dubelco's modified Eagle's medium - FCS fetal calf serum - PBS phosphate-buffered saline - DAG diacylglycerol - DDI distilled-deionized - Cer ceramide - SM sphingomyelin Dedicated to Dr Sen-itiroh Hakomori in celebration of his 65th birthday.     

Programmed cell death and the proteasome

A characteristic feature of apoptotic cell death is the activation of a cascade of cytoplasmic proteases that results in the cleavage of a limited number of target proteins. A central role in these proteolytic events has been assigned to members of the capase family. However, the use of low molecular weight proteasomal inhibitors has also demonstrated that protein degradation or processing by the ubiquitin-proteasome system of the cell has a decisive impact on cell survival and death as well, depending on the cell type and/or the proliferative status of the cells studied. Treatment of proliferating cells with proteasome inhibitors leads to cell death, potentially involving an internal signalling conflict between accumulating levels of the cdk inhibitor p27Kip1 and c-myc. In contrast, in terminally differentiated cells the same compounds have the opposite effect of blocking apoptosis, possibly by preventing proteasome-mediated degradation of a capase inhibitor. In this review the role of proteasome-mediated proteolysis in the dying cell is discussed and apparently conflicting results are integrated into a working hypothesis which functionally locates the proteasome upstream of capase3-like enzymes.     

Programmed cell death in the apical ganglion during larval metamorphosis of the marine mollusc Ilyanassa obsoleta

The apical ganglion (AG) of larval caenogastropods, such as Ilyanassa obsoleta, houses a sensory organ, contains five serotonergic neurons, innervates the muscular and ciliary components of the velum, and sends neurites into a neuropil that lies atop the cerebral commissure. During metamorphosis, the AG is lost. This loss had been postulated to occur through some form of programmed cell death (PCD), but it is possible for cells within the AG to be respecified or to migrate into adjacent ganglia. Evidence from histological sections is supported by results from a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, which indicate that cells of the AG degenerate by PCD. PCD occurs after metamorphic induction by serotonin or by inhibition of nitric oxide synthase (NOS) activity. Cellular degeneration and nuclear condensation and loss were observed within 12 h of metamorphic induction by NOS inhibition and occur before loss of the velar lobes, the ciliated tissue used for larval swimming and feeding. Velar disintegration happens more rapidly after metamorphic induction by serotonin than by 7-nitroindazole, a NOS inhibitor. Loss of the AG was complete by 72 h after induction. Spontaneous loss of the AG in older competent larvae may arise from a natural decrease in endogenous NOS activity, giving rise to the tendency of aging larvae to display spontaneous metamorphosis in culture.     

Lactate formation by rat small intestine in vitro.

The formation of lactic acid by mucosal slices, rings and muscle from rat jejunum has been studied for periods of up to 8 min. Lactate output by mucosal slices incubated in the absence of glucose was characterised by two phases: a rapid, initial phase of release lasting about 1 min, followed by a much slower phase extending over the remainder of the incubation period. Glucose addition at 30 s initiated a second rapid phase of lactate release into the medium which was again followed by a slower rate of lactate output up to 8 min. The time course of lactate output suggested that there was a negative Pasteur effect in mucosal slices, which could not be reversed by the addition of ADP or glucose 6-phosphate. By contrast, the rate of lactate formation by rings and muscle from rat jejunum increased steadily over the incubation period, indicating a positive Pasteur effect. When Na+ in the incubating medium were replaced by K+, lactate formation by mucosal slices and rings was considerably reduced. Measurements of tissue lactate content before and during incubation revealed that about three-quarters of the lactate released by mucosal slices during the first 30 s of incubation was present initially in the tissue. After the first 30 s the tissue lactate remained constant both in the presence and absence of glucose so that the lactate released into the incubation medium is equivalent to the lactate formed by the slices. The role of the various tissue components of the small intestine in lactate formation is discussed in relation to sites of glucose entry.     

Induction by activated macrophage-like THP-1 cells of apoptotic and necrotic cell death in intestinal epithelial Caco-2 monolayers via tumor necrosis factor-alpha

Intestinal epithelial cells interact with immune cells located in the intestinal epithelium via soluble factors. An in vitro model system using coculture was constructed to analyze the effect of macrophages on intestinal epithelial cells, and human intestinal epithelial-like Caco-2 monolayers and activated macrophage-like THP-1 cells were used in this study. Coculturing with THP-1 cells resulted in an increase of lactate dehydrogenase release from Caco-2 and a decrease in the transepithelial electrical resistance of the monolayers, showing that coculturing with THP-1 induced cell damage to Caco-2 cells. This disruption was significantly suppressed by adding anti-TNF-alpha antibody and etanercept, strongly suggesting that TNF-alpha secreted from THP-1 had caused cell damage to Caco-2 monolayers. The disrupted Caco-2 monolayers showed both apoptotic and necrotic characteristics by morphological and biochemical analyses. TNFRI and NF-kappaB seem to have been involved in this regulation. It is suggested that this phenomenon is similar in some respects to that observed with IBD and that this in vitro coculture system could be a good model for searching for the drugs or food substances that can be used to treat or prevent IBD.