Regeneration of cultured midgut cells after exposure to sublethal doses of toxin from two strains of Bacillus thuringiensis

Toxin from two strains of Bacillus thuringiensis (Bt), AA 1-9 and HD-73, caused dose-dependent destruction of cultured midgut cells from Heliothis virescens larvae. HD-73 toxin was more effective although, at the doses used, not all cells were killed. After 2 days of exposure to 0.8 pg/μl AA 1-9 or 0.06 pg/μl HD-73, columnar and goblet cell numbers declined to ca 20% of controls. In contrast, stem and differentiating cells increased to 140-200% of controls. The dynamic of depletion and replacement depended on toxin type and concentration. Two days after toxin was washed out, ratios of cell types returned to approximate control levels, suggesting rapid population corrections in vitro. Regulation of the ratio of cell types in each population, and the rate of proliferation and differentiation of stem cells was induced by the cultured midgut cells themselves. Controls and cells treated with toxin from Bt strain AA 1-9 were stained using a polyclonal antibody to Lepidopteran midgut differentiation factor 1 (MDF1). With Bt toxin, 1.5 times more cells stained for MDF1, suggesting increased synthesis of this differentiation factor during increased stem cell differentiation. The response of cultured midgut cells to Bt toxin injury is similar to injured vertebrate tissues dependent on stem cells for replacement and healing.     

Cockroach midgut peptides that regulate cell proliferation, differentiation, and death in vitro

Summary The number of insect midgut cells is maintained homeostatically in vivo and in vitro. However, during starvation, the midgut shrinks and the rate of cell replacement appears to be suppressed. When they undergo metamorphosis, the internal organs of insects are drastically remodeled by cell proliferation, differentiation, and apoptotic processes, and the net number of cells usually increases. An extract of 1650 midguts ofPeriplaneta americana was fractionated by highperformance liquid chromatography (HPLC) to obtain the peptides that regulate these processes. The HPLC fractions were tested for myotropic activity in the foregut and for effects on cell proliferation or loss in primary cultures of larvalHeliothis virescens midgut cells and in a cell line derived from the last-instar larval fat body ofMamestra brassicae. Some fractions stimulated midgut stem cell proliferation and differentiation, while others caused loss of differentiated columnar and goblet cells. Other fractions stimulated cell proliferation in the larval fat body cells. Mention of products in this article does not imply endorsement by the U.S. Department of Agriculture.     

Morphological and functional characterization of isolated stem cells from the midgut of Chilo suppressalis larvae

The proliferation and differentiation of stem cell populations allow the midgut to grow/regenerate in lepidopteran insect. Basic epithelial regenerative functions can be assessed in vitro by purifying these stem and mature cell populations. Therefore, we isolated and purified stem and mature cells from the midgut of C. suppressalis larvae by density gradient centrifugation and observed the morphologies of these cells. A flow cytometry method was used to monitor C. suppressalis stem cell proliferation and differentiation under different cell culture conditions. We observed high proportions of the stem and differentiating cells in third- and fourth-instar larvae, respectively, indicating that, in larvae, stem cells rapidly proliferate early in development and are strongly differentiated at late stages. Incubation in medium supplemented with fat body extract and ecdysone resulted in a significantly increased proportion of stem cells, not of the differentiating cells, indicating that co-culture with fat body extract and ecdysone stimulates the proliferation of C. suppressalis stem cells. Viability bioassays showed that Cry1Ab displayed significant cytotoxic effects on the midgut cell culture of C. suppressalis. The proportion of differentiating cells was significantly increased after a 48-h exposure to sublethal doses of Cry1Ab toxin, and peaked at the Cry1Ab concentration of 0.3 μg/ml, demonstrating that epithelial cells with strong regenerative capacity via the differentiation of stem cells. These results improve our understanding of C. suppressalis stem cell biology and illustrate the potential role of the enhanced midgut regeneration induced by stem cell proliferation or differentiation as a reparation mechanism to Bt toxin.     

TGFβ1 induces growth arrest and apoptosis but not ciliated cell differentiation in rat tracheal epithelial cell cultures

Summary We are studying the regulation of ciliated cell differentiation using an in vitro model of tracheal regeneration. Previously, we reported that removal of growth stimulating compounds such as epidermal growth factor (EGF) and cholera toxin reduced DNA synthesis and cell number while increasing ciliated cell differentiation (Clark et al., 1995). This result suggested that the induction of growth arrest may stimulate terminal differentiation of airway epithelial cells into ciliated cells. Transforming growth factor βs (TGFβs) inhibit epithelial cell proliferation and have also been shown to stimulate epithelial cell differentiation. In this study, the effect of TGFβ₁ on growth and ciliated cell differentiation of rat tracheal epithelial (RTE) cells was examined. TGFβ₁ inhibited [³H]thymidine incorporation by RTE cells in a dose-dependent manner. A 40% inhibition was observed after a 24-h incubation with 10 pM TGFβ₁. Continuous treatment with TGFβ₁ (1–50 pM) also reduced cell number during the time when ciliogenesis occurs. This reduction resulted in part from a loss of cells through exfoliation, in addition to the inhibition of proliferation. The exfoliated cells exhibited several morphological features characteristic of apoptosis, including shrunken cells, condensed and fragmented nuclei, and intact organelles. In addition, electrophoretic analysis of genomic DNA analysis isolated from exfoliated cells demonstrated the presence of a nucleosomal ladder. However, in contrast to the removal of EGF, treatment with TGFβ₁ for 7 d did not increase ciliated cell differentiation. TGFβ1 is, therefore, capable of inhibiting proliferation and increasing apoptosis in RTE cells without stimulating ciliated cell differentiation.     

The role of stem cells in midgut growth and regeneration

Summary TheManduca sexta (L.) [Lepidoptera: Sphingidae] andHeliothis virescens (F.) [Lepidoptera: Noctuidae] midguts consist of a pseudostratified epithelium surrounded by striated muscle and tracheae. This epithelium contains goblet, columnar, and basal stem cells. The stem cells are critically important in that they are capable of massive proliferation and differentiation. This growth results in a fourfold enlargement of the midgut at each larval molt. The stem cells are also responsible for limited cell replacement during repair. While the characteristics of the stem cell population vary over the course of an instar, stem cells collected early in an instar and those collected late can start in vitro cultures. Cultures of larval stem, goblet, and columnar cells survive in vitro for several mo through proliferation and differentiation of the stem cells. One of the two polypeptide differentiation factors which have been identified and characterized from the culture medium has now been shown to be present in midgut in vivo. Thus the ability to examine lepidopteran midgut stem cell growth in vitro and in vivo is proving to be effective in determining the basic features of stem cell action and regulation. Mention of any product in this publication does not imply endorsement by the USDA.     

Implications for the functions of the four known midgut differentiation factors: An immunohistologic study of Heliothis virescens midgut

Antibodies to the peptides that induce differentiation of midgut larval stem cells, the midgut differentiating factors MDF-2, MDF-3, and MDF-4, bind to columnar cells in midgut cultures and in intact midgut of Heliothis virescens, in manners similar to the binding of anti- MDF-1 to those tissues. Antibodies to MDF-2 and MDF-3 also stained droplets in the midgut lumen, suggesting that columnar cells may also release MDF-2- and MDF-3-like cytokines to the lumen. Antibody to MDF-4 exhibited similar staining patterns but also recognized stem and differentiating cells, the presumed targets of peptides that regulate stem cell differentiation. Antibody to MDF-4 also bound to one type of endocrine cell in midgut cultures and in sections of midgut, as well as to the endocrine secretion released both to the midgut lumen and the hemolymph. Antibodies to the MDFs 1, 2, and 3, incubated with cultures of midgut cells, did not appear to prevent differentiation of the stem cells in the cultures but affected viability of mature cells, reflected in increased apoptosis and doubling of the number of differentiating cells compared to controls. Only antibody to MDF-4 induced temporary necrosis and inhibition of population recovery, indicating that MDF4 may be the true differentiation factor. The other MDFs may have additional functions beyond regulation of midgut stem cell differentiation in vivo.     

Rho GTPases mediated integrin α<Subscript>v</Subscript>β<Subscript>3</Subscript> activation in sphingosine-1-phosphate stimulated chemotaxis of endothelial cells

Integrins, a family of transmembrane heterodimeric polypeptides, mediate various biological responses including cell adhesion and migration. In this report, we show that sphingosine-1-phosphate (S1P) activates integrin α_vβ₃ in endothelial cells (ECs) via the sphingosine-1-phosphate receptor subtype 1 (S1P1)-mediated signaling pathway. S1P treatment results in the activation of integrin α_vβ₃ in the lamellipodia region of ECs, suggesting that integrin α_vβ₃ plays a critical role in the S1P-stimulated chemotactic response of ECs. Indeed, S1P treatment induces the association of focal adhesion kinase (FAK) and cytoskeletal proteins with integrin α_vβ₃, the ligation of α_v and β₃ subunits, as well as enhances endothelial migration on vitronectin-coated substrata. Knockdown endothelial S1P1 receptor, treatments with pertussis toxin or dominant-negative-Rho family GTPases abrogates the S1P-induced integrin α_vβ₃ activation in ECs. Consequently, these treatments markedly inhibit the S1P-induced endothelial migratory response on vitronectin-coated substrata. Collectively, these data indicate that the S1P-mediated signaling via the S1P1/G_i/Rho GTPases pathway activates integrin α_vβ₃, which is indispensable for S1P-stimulated chemotactic response of ECs.     

Effects of uniaxial cyclic strain on adipose-derived stem cell morphology,proliferation, and differentiation

Cells and tissues in vivo are subjected to various forms of mechanical forces that are essential to their normal development and functions. The arterial blood vessel wall is continuously exposed to mechanical stresses such as pressure, strain, and shear due to the pulsatile nature of blood flow. Vascular smooth muscle cells (SMCs) populate the media of blood vessels and play important roles in the control of vasoactivity and the remodeling of the vessel wall. It is well documented that the phenotype and functions of vascular SMCs are not only regulated by chemical factors such as transforming growth factor-β₁ (TGF-β₁), but also by mechanical factors such as uniaxial strain. The purpose of our study was to explore the effects of TGF-β₁ alone or in combination with uniaxial cyclic strain on adipose-derived stem cell (ASC) morphology, proliferation, and differentiation. Low passage ASCs were stimulated with 10% strain at 1 Hz for 7 days, with or without TGF-β₁. Cyclic strain inhibited proliferation, and caused alignment of the cells and of the F-actin cytoskeleton perpendicular to the direction of strain. Strain alone resulted in a decrease in the expression of early SMC markers α-SMA and h ₁-calponin. While the response of SMCs and other progenitor cells such as bone marrow stromal cells to mechanical forces has been extensively studied, the roles of these forces on ASCs remain unexplored. This work advances our understanding of the mechanical regulation of ASCs. Presented in part at the third annual meeting of the International Fat Applied Technology Society (IFATS), September 10–13, 2005, Omni Charlottesville Hotel, Charlottesville, VA, USA.     

Histopathology and ultrastructure of midgut of Alabama argillacea (Hübner) (Lepidoptera: Noctuidae) fed Bt-cotton

The interaction of Cry toxins from Bacillus thuringiensis in the midgut of some insect larvae determines their efficacies as insecticides, due to the expression and availability of sites of action of the toxin in the midgut. Researches point out cases of resistance to Cry toxin due to alterations in the binding sites in columnar cell membrane. We analyzed the effects of Cry1Ac toxin expressed by Bt-cotton plants on Alabama argillacea midgut morphophysiology clarifying in levels of morphological and ultrastructural. Larvae in the 4th instar of A. argillacea after 20 min from ingesting Bt-cotton leaves expressing 0.183 ng of Cry1Ac exhibited ultrastructural and morphological modifications in the columnar cells with significant changes in the mitochondrial polymorphism, cytoplasmic vacuolization, microvillus and basal labyrinth. Expressive morphological alterations were also observed in the goblet cells indicating that the columnar cells are not the only target of the Cry1Ac toxin. The regenerative cells did not modify their structures and exhibited decrease in regeneration capacity. In conclusion, the ingestion of 0.183 ± 0.077 ng of Cry1Ac was enough to promote alterations in the columnar and goblet cells, besides reducing significantly the number of regenerative cells, which may have contributed to larval death. Nevertheless, further studies are necessary to determine the true cause of death.     

Differential expression of proliferative, cytoskeletal, and adhesive proteins during postnatal development of the hamster submandibular gland

 Although the submandibular gland (SMG) plays important exocrine and endocrine roles, little is known about the molecular details underlying its development. Previously, we reported that in the postnatally developing hamster SMG, GPT, the protein product of the first N-glycosylation gene, ALG7, was an in vivo marker for salivary cell proliferation. Here we investigated the proliferative, cytoskeletal, and adhesive changes during SMG postnatal development. The cellular localization and abundance of GPT, filamentous actin, and β1 integrin receptor were examined using confocal microscopy and immunoblotting. In neonatal glands, high GPT levels marked extensive cell proliferation throughout the tissue. The apical regions of immature salivary cells displayed intense actin staining, while most of the β1 integrin was diffusely distributed throughout the tissue. As development proceeded, discrete regions of the gland expressed attenuated levels of GPT, an increased organization of actin to the cell cortex, and β1 integrin to the basal lamina. In the adult SMG, differentiated salivary cells displayed low levels of GPT and actin. While the abundance of β1 integrin remained unchanged throughout development, in the adult, it was found exclusively in regions where cells contact the basal lamina. These data indicate that SMG development entails regionalized cell proliferation and polarization, and that these processes are temporally and spatially coordinated with the establishment of stable cell-substratum interactions. Accepted: 26 October 1998     

Two polypeptide factors that promote differentiation of insect midgut stem cells in vitro

Isolated stem cells from the midguts of Manduca sexta and Heliothis virescens can be induced to differentiate in vitro by either of two polypeptide factors. One of the peptides was isolated from culture medium conditioned by differentiating mixed midgut cells; we used high performance liquid chromatographic separation and Edman degradation of the most prominent active peak. It is a polypeptide with 30 amino acid residues (3,244 Da), with the sequence HVGKTPIVGQPSIPGGPVRLCPGRIRYFKI, and is identical to the C-terminal peptide of bovine fetuin. A portion of this molecule (HVGKTPIVGQPSIPGGPVRLCPGRIR) was synthesized and was found to be very active in inducing differentiation of H. virescens midgut stem cells. It was designated Midgut Differentiation Factor 1 (MDF1). Proteolysis of bovine fetuin with chymotrypsin allowed isolation of a pentamer, Midgut Differentiation Factor 2 (MDF2) with the sequence HRAHY corresponding to a portion of the fetuin molecule near MDF1. Synthetic MDF2 was also biologically active in midgut stem cell bioassays. Dose response curves indicate activity in physiological ranges from 10(-14) to 10(-9) M for MDF1 and 10(-15) to 10(-5) M for MDF2.     

Integrin-mediated mechanotransduction in IL-1β stimulated chondrocytes

Mechanical loading and interleukin-1β (IL-1β) influence the release of nitric oxide (^·NO) and prostaglandin E₂ (PGE₂) from articular chondrocytes via distinct signalling mechanisms. The exact nature of the interplay between the respective signalling pathways remains unclear. Recent studies have shown that integrins act as mechanoreceptors and may transduce extracellular stimuli into intracellular signals, thereby influencing cellular response. The current study demonstrates that the application of dynamic compression induced an inhibition of ^·NO and an upregulation of cell proliferation and proteoglycan synthesis in the presence and absence of IL-1β. PGE₂ release was not affected by dynamic compression in the absence of IL-1β but was inhibited in the presence of the cytokine. The integrin binding peptide, GRGDSP, abolished or reversed the compression-induced alterations in all four parameters assessed in the presence and absence of IL-1β. The non-binding control peptide, GRADSP, had no effect. These data clearly demonstrate that the metabolic response of the chondrocytes to dynamic compression in the presence and absence of IL-1β, are integrin mediated.     

Bacillus thuringiensis insecticidal crylab toxin does not affect the membrane integrity of the mammalian intestinal epithelial cells: An in vitro study

Summary The mammalian intestinal epithelium has been found, based on in vivo experiments, to be resistant to insecticidal Cry toxins, which are derived from Bacillus thuringiensis and fatally damage insect midgut cells. Thus, the toxins are commonly used as a genetic resource in insect-resistant transgenic plants for feed. However, Cry toxins bind to the cellular brush border membrane vescle (BBMV) of mammalian intestinal cells. In this study, we investigated the affinity of Cry1Ab toxin, a lepidopteran-specific Cry1-type toxin, to the cellular BBMV of two mammalian intestinal cells as well as the effect of the toxin on the membrane potential of three mammalian intestinal cells compared to its effects on the silkworm midgut cell. We found that Cry1Ab toxin did bind to the bovine and porcine BBMV, but far more weakly than it did to the silkworm midgut BBMV. Furthermore, although the silkworm midgut cells developed severe membrane potential changes within 1 h following the toxin treatment at a final concentration of 2 μg/ml, no such membraneous changes were observed on the bovine, procine, and human intestinal cells. The present in vitro results suggest that, although Cry1Ab toxin may bind weakly or nonspecifically to certain BBMV components in the mammalian intestinal cell, it does not damage the cell’s membrane integrity, thus exerting no subsequent adverse effects on the cell.     

Apoptosis in cultured midgut cells from heliothis virescens larvae exposed to various conditions

We exposed midgut cells from primary cultures of Heliothis virescens larvae to cell-free previously used medium, the Vaughn X and HyQ SFtrade mark media used for serum-free culture of insect cell lines which do not support H. virescens midgut cells, and to toxin from Bacillus thuringiensis. A statistically significant increase in the percent of dying cells was counted in cell populations in Vaughn X medium. Use of the TUNEL method to detect apoptosis indicated a low rate (7.2%) of apoptosis in control cultures grown in Heliothis medium, an increase to approximately 20% in previously used and HyQ SFtrade mark media, and to approximately 45% of cells remaining after exposure to and initial destruction by B. thuringiensis toxin. Apoptotic nuclei were predominant (approximately 6%) in mature columnar cells in control cultures. Approximately 1% of goblet, stem, and differentiating cells were apoptotic. However, apoptosis rose to 12% in stem and differentiating cells exposed to used and unsuitable medium. B. thuringiensis exposure to toxin for 2-3 days resulted in visible membrane damage and necrosis, causing the death of 84% of the cells as measured by both the TUNEL and Annexin methods. Some of the columnar cells and stem and differentiating cells that remained also contained apoptotic nuclei. Stem and differentiating cells normally replace dying mature cells in the midgut. Thus, exposure of cultures of H. virescens midgut cells to adverse environments such as unsuitable or poisonous media appeared to induce down-regulation of the cell populations by apoptosis.     

Functional blockade of α<Subscript>5</Subscript>β<Subscript>1</Subscript> integrin induces scattering and genomic landscape remodeling of hepatic progenitor cells

Background  

Cell scattering is a physiological process executed by stem and progenitor cells during embryonic liver development and postnatal organ regeneration. Here, we investigated the genomic events occurring during this process induced by functional blockade of α₅β₁ integrin in liver progenitor cells.     

Intestinal stem cells in the adult Drosophila midgut

Drosophila has long been an excellent model organism for studying stem cell biology. Notably, studies of Drosophila's germline stem cells have been instrumental in developing the stem cell niche concept. The recent discovery of somatic stem cells in adult Drosophila, particularly the intestinal stem cells (ISCs) of the midgut, has established Drosophila as an exciting model to study stem cell-mediated adult tissue homeostasis and regeneration. Here, we review the major signaling pathways that regulate the self-renewal, proliferation and differentiation of Drosophila ISCs, discussing how this regulation maintains midgut homeostasis and mediates regeneration of the intestinal epithelium after injury.