Ultrastructural changes in colonic epithelial cells in a rat model of inflammatory bowel disease

Inflammatory bowel disease (IBD) is a global, chronic intractable disease. The functions of drugs and food components have been evaluated in models of IBD induced by 2,4,6‐trinitrobenzene sulfonic acid (TNBS). Here, we used transmission (TEM) and osmium‐maceration scanning (SEM) electron microscopy to evaluate the ultrastructure of colonic epithelial cells in rat models of IBD induced by TNBS. Histological evaluation revealed that the intestinal crypts in the most regions of the IBD‐model colons were deformed and we classified them as having high cell migration rates (HMIG). The remaining regions in the intestinal crypts retained a relatively normal structure and we classified them as having low cell migration rates (LMIG). Osmium‐maceration SEM revealed the mucosal fluid flowing in spaces without secretory granules in crypt goblet cells of both HMIG and LMIG regions, indicating the depletion of goblet cell mucin that is found in patients with IBD. The Golgi apparatus in absorptive cells was stacked and curled in both regions. Osmium‐maceration SEM showed membrane network structures resembling endoplasmic reticulum that were large and expanded in absorptive cells with HMIG rather than with LMIG regions in IBD‐model colons. These findings indicated that endoplasmic reticulum stress is associated with susceptibility to IBD and that the effects of various agents can be evaluated according to endoplasmic reticulum stress revealed by using electron microscopy in models of IBD induced by TNBS.     

Renal cell culture

Methods for the establishment and growth of renal cell types in culture are reviewed, with emphasis on current trends. General techniques available for the isolation and culture of glomerular cells have progressed from explant to enzyme dissociation and cloning techniques. The growth characteristics and properties of cultured glomerular endothelial, epithelial, mesangial, and bone-marrow-derived cells are discussed. Studies are described in which cultures of contractile mesangial cells have led to an elucidation of their role both in normally functioning glomeruli and in disease states. Renal tubule culture techniques also have progressed from mixed tissue explants and cell isolates to fractionation of enriched tubule populations and growth of specific, individually microdissected proximal convoluted, proximal straight, thick ascending limb of Henle's loop, and collecting tubules. The differentiated tubule epithelial-specific properties of such primary cultures are discussed in relation to those of permanently growing cell lines such as MDCK and LLC-PK1. Renal tubule cultures will be invaluable for the study of the role of hormones and extracellular matrix in epithelial growth and polarity of normal structure and function. In addition, in vitro models of cultured renal tubules have been established to study the effects of age, nephrotoxins, and anoxic injury.     

Localization of fatty acids with selective chain length by imaging time-of-flight secondary ion mass spectrometry

Localization of fatty acids in biological tissues was made by using TOF-SIMS (time-of-flight secondary ion mass spectrometry). Two cell-types with a specific fatty acid distribution are shown. In rat cerebellum, different distribution patterns of stearic acid (C18:0), palmitic acid (C16:0), and oleic acid (C18:1) were found. Stearic acid signals were observed accumulated in Purkinje cells with high intensities inside the cell, but not in the nucleus region. The signals colocalized with high intensity signals of the phosphocholine head group, indicating origin from phosphatidylcholine or sphingomyelin. In mouse intestine, high palmitic acid signals were found in the secretory crypt cells together with high levels of phosphorylinositol colocalized in the crypt region. Palmitic acid was also seen in the intestinal lumen that contains high amounts of mucine, which is known to be produced in the crypt cells. Linoleic acid signals (C18:2) were low in the crypt region and high in the villus region. Oleic acid signals were seen in the villi and stearic acid signals were ubiquitous with no specific localization in the intestine. We conclude that the results obtained by using imaging TOF-SIMS are consistent with known brain and intestine biochemistry and that the localization of fatty acids is specific in differentiated cells.     

Adhesion complexes implicated in intestinal epithelial cell-matrix interactions

This article review summarizes data on cell-substratum adhesion complexes involved in the regulation of cellular functions in the intestine. We first focus on the molecular composition of the two main adhesion structures-the beta1 integrin-adhesion complex and the hemidesmosome-found in vivo and in two human intestinal cell lines. We also report the key findings on the cellular behavior and response to the extracellular matrix that involve integrins, the main transmembrane anchors of these complexes. How the dynamics of cell/extracellular matrix interactions contribute to cell migration, proliferation, differentiation, and tumorigenicity is discussed in the light of the data provided by the human intestinal cells.     

Integrins as mediators of epithelial cell-matrix interactions in the human small intestinal mucosa

The intestinal epithelium is a highly dynamic tissue, which depends on a variety of factors for the regulation of its rapid renewal and expression of digestive functions. Over the last 10 years, it has become evident that among these factors are cell interactions with the extracellular matrix, more specifically with the underlying basement membrane, through a series of specific cell membrane receptors, many of which are integrins. Integrins regulate the assembly of adhesive junctions as well as the activation of various signaling pathways, leading to the modulation of gene expression. The analysis of the integrin repertoire along the crypt-villus axis in the human small intestinal epithelium identifies a number of beta1 and beta4 integrins, showing differential patterns of expression relative to its two functional compartments. Among them are the integrins alpha3beta1, alpha7Bbeta1 and the functional form of alpha6beta4 that appear to be related, in concert with the distribution of their ligands, to the process of intestinal cell differentiation, and the integrins alpha2beta1, alpha1beta1, alpha5beta1, and the non-functional form of alpha6beta4 that seem to be coupled with the undifferentiated/proliferative status of crypt cells. These observations delineate the potential complexity of the organization of epithelial cell-matrix interactions involved in the maintenance of the human intestinal crypt-villus axis.     

Cytoskeleton as a target for injury in damaged intestinal epithelium

This report summarizes the findings of a series of studies undertaken to discern the role of the cytoskeleton in intestinal injury and defense. Two established cell lines were used for these studies. IEC-6 cells (a rat intestinal cell line) were incubated in Eagle's minimal essential medium with and without 16, 16 dimethyl prostaglandin E(2) (dmPGE(2); 2.6 microM) for 15 minutes and subsequently incubated in medium containing 10% ethanol (EtOH). The effects on cell viability and the actin cytoskeleton were then determined. Using a similar protocol, Caco-2 cells (a human colonic cell line) were employed to assess the microtubule cytoskeleton under these conditions. In both cell lines, EtOH extensively disrupted the cytoskeletal component being evaluated coincident with adversely affecting cell viability. Pretreatment with dmPGE(2) increased cell viability and abolished the disruptive effects on both the actin and microtubule cytoskeleton in cells exposed to EtOH. Prior incubation with cytochalasin D, an actin disruptive agent, prevented the protective capabilities of dmPGE(2) in IEC-6 cells challenged with EtOH. Phalloidin, an actin stabilizing agent, demonstrated similar effects to that of dmPGE(2) by stabilizing the actin cytoskeleton and preserving cellular viability in IEC-6 cells in response to EtOH. In Caco-2 cells, taxol, a microtubule stabilizing agent, mimicked the effects of dmPGE(2) by increasing cell viability in cells exposed to EtOH and enhancing microtubular integrity. In contrast, pretreatment with colchicine, an inhibitor of microtubule integrity, prevented the protective effects of dmPGE(2). These findings support the hypothesis that the cytoskeleton may be a major target for injury in damaged intestinal epithelium, and that the protective action of dmPGE(2) is orchestrated through preservation of this target.     

Atomic force microscopy imaging of living cells: a preliminary study of the disruptive effect of the cantilever tip on cell morphology

Recent studies have demonstrated that atomic force microscopy (AFM) is a potential tool for studying important dynamic cellular processes in real time. However, the interactions between the cantilever tip and the cell surface are not well understood, and the disruptive effect of the cantilever tip on cell morphology has not been well characterized. In this study, the disruptive effect of the scanning cantilever tip on cell morphology, in the AFM contact mode, has been investigated. The aims of this study are to identify what kinds of cell morphological changes generally occurred under normal AFM imaging conditions and to find out how long cells remain viable during scanning. Two cell lines, SK-N-SH (human neuroblastoma cells) and AV12 (Syrian hamster cells) were studied in the experiment because these are widely used in biomedical research as an expression system for studying cellular functions of neuronal receptors. The experimental results suggest that the sensitivity of cells to the cantilever disruptive effect is dependent on cell type and that there are patterns observed in the changes of cell morphology induced by the cantilever force in these two cell lines.     

Use of immunoelectron microscopy and intestinal models to explore the elaboration of apolipoproteins required for intraenterocyte lipid transport

The intestine is the organ that contributes the majority of circulating alimentary lipoproteins. Intestinal epithelial cells have the unique ability to elaborate chylomicrons, the largest triglyceride-rich lipoproteins and the main vehicle for the transport of dietary lipids. The final intracellular assembly and exocytosis of chylomicrons require enterocyte-derived apolipoproteins (apo). As research on lipoprotein metabolism evolved, it has become increasingly evident that apo B is a crucial protein for the normal packaging of triglyceride-rich lipoproteins. Immunocytochemical techniques have successfully been used to demonstrate the presence of two types of apo B, the B-100 and the B-48, in different subcellular compartments of the human enterocyte. Confirmation was obtained by biochemically analyzing human lymph and intestine from pediatric patients. In addition, the immunoelectron microscopic approach revealed the location of apo A-I in the rough endoplasmic reticulum (ER) and predominantly in the Golgi apparatus and the basolateral membrane, which confirms the rapid transport of apo A-I documented by other studies. Proven utility and experimental conditions were defined to demonstrate the ability of Caco-2 cells, a colon carcinoma cell line, to esterify lipids, synthesize apo, and assemble lipoproteins. Thus, immunocytochemical and biochemical techniques can be combined with in vivo and in vitro intestinal models for the study of the intestinal lipid transport.     

Cell surface distribution of fibronectin in cultures of fibroblasts and bladder derived epithelium: SEM-immunogold localization compared to immunoperoxidase and immunofluorescence

Expression of cell surface fibronectin in cultures of untransformed fibroblasts is well documented, but little is known of its presence and distribution in cultured epithelial cells. Using species monospecific anti-fibronectin antibodies, the distribution of fibronectin in untransformed fibroblasts and in normal and neoplastic bladder epithelial cells was characterized by indirect labelling experiments using immunogold scanning electron microscopy (SEM). The surface matrix of fibronectin expressed in rodent and human fibroblast cell lines was demonstrated with ease by SEM of gold-tagged second antibodies. However, no fibronectin could be detected on any of the mouse and human bladder epithelium-derived cells studied in single or in mixed epithelial-fibroblast cultures. These SEM-immunogold observations were compared to and confirmed by immunofluorescence and immunoperoxidase microscopy. Immunofluorescence and SEM localization of the fibronectin in the extracellular matrix presented similar distribution patterns but the higher resolution of the SEM provided a more detailed analysis.     

Regulation of intestinal regeneration: new insights

Intestinal regeneration is the process by which intestinal injury penetrating deep to the lamina propria heals. The regenerative process involves epithelial cell migration and proliferation, changes in cellular function, adaptation of subepithelial tissues, and contraction of the injured area. This requires interaction of multiple cell types. While many observations have been made about the process of regeneration, its regulation is not well understood. Previous studies, performed primarily in a serosal patch model, have identified many potential regulatory factors. These include location and size of the injury, other associated injury, e.g., resection, and a variety of agents that influence one or more of the primary processes involved. Epidermal growth factor (EGF), in particular, appears to play a role in many aspects of regeneration. Recent advances in the understanding of intestinal growth regulation have provided new insights into the regulation of intestinal regeneration. Developmental studies in genetically manipulated mice suggest a role for gene products not previously implicated in regeneration. The importance of apoptosis in growth regulation has recently been emphasized. Mesenchymal-epithelial interactions have gained greater appreciation. Finally, it has become clear that immune cells and cytokines are important factors in this process. Transforming growth factor-beta (TGFbeta) has been implicated as another important regulator of several of the processes involved in intestinal regulation. Improved understanding of the regulation of intestinal regeneration will lead to new therapeutic approaches to stimulate intestinal healing in the clinical setting.     

Differential expression of CD95, Bcl-2, and Bax in rat gastric chief and parietal cells

Apoptotic cell death is common in the inflamed gastric mucosa, but its role in the regulation of cell homeostasis in normal gastric mucosa is unknown. We investigated the expression of CD95, Bcl-2, and Bax and their roles in the regulation of apoptosis in normal rat gastric mucosa and in cultures of highly enriched rat chief and parietal cells by immunostaining, Western blotting, and FACS. In intact tissue CD95, Bcl-2, and Bax were localized predominantly in the glandular base region in chief cells. In freshly isolated cells, expression of CD95, Bcl-2, and Bax was much more pronounced in chief cells than in parietal cells. A lower intracellular Bcl-2/Bax ratio suggesting a higher susceptibility to apoptosis was noticed in chief rather than in parietal cells. In extended cultures of parietal and chief cells, Bax expression was upregulated and Bcl-2 expression was downregulated. These regulatory changes, presumably caused by in vitro effects, were not associated with an increase in spontaneous apoptosis. Treatment of chief and parietal cells with Fas-ligand induced apoptosis of all CD95 expressing cells. Expression of CD95, Bcl-2, and Bax predominantly in chief cells suggests that in this cell type regulation of apoptosis may differ from that in parietal cells. Binding of FasL with functionally active CD95 receptors on chief and parietal cells may be relevant for induction of apoptosis in inflamed gastric mucosa.     

体外培养的不同亚型肺癌细胞株差异蛋白的初步分析

分析体外培养的不同亚型肺癌细胞株蛋白质表达差异,筛选肺癌细胞的标志蛋白并与肺癌病人血清中的标志蛋白进行对比分析。采用SELDI(Surface Enhanced LaserDesorption/Ionization)蛋白质芯片技术检测了三种肺癌细胞株A549(肺癌)、Calu-6(腺癌)和PG(大细胞癌)以及人胚肺二倍体成纤维细胞(2BS)的蛋白质谱。结果显示与2BS细胞比较,肺癌细胞有24个蛋白质表达发生明显改变。     

Expression and regulatory role of receptors for vasoactive intestinal peptide in bone cells

An intense network of nerve fibers can be demonstrated in skeletal tissues, not only in the periosteum but also within cortical bone, growth plate, and bone marrow. This neuro-osteogenic network expresses a restricted number of signalling molecules, including neuropeptides, neurotransmitters, and neurotrophins. Several lines of evidence indicate that receptors for these molecules are present on bone cells and that activation of these receptors leads to changes in bone cell activities. In addition, deletion of signalling molecules has been shown to alter bone metabolism. In the present review, these studies are summarized with a focus on distribution and effects of vasoactive intestinal peptide.     

Cell cycle behavior and MyoD expression in response to T3 differ in normal and mdx dystrophic primary muscle cell cultures

Since mdx limb muscle regeneration in vivo is accompanied by rapid myoblast proliferation and differentiation compared to normal, we tested the possibility that proliferation and differentiation were differentially regulated in normal and mdx dystrophic muscle cells. Cell cycle behavior, MyoD expression, and the effects of thyroid hormone (T3) treatment were examined in primary cultures. Using a 4-hour pulse time for bromodeoxyuridine (BrdU) incorporation during S-phase, the phases of the cell cycle (early S, late S, G(2)/M, and G(0)/G(1)) were separated by 2-colour fluorescence (BrdU/PI) analysis using flow cytometry. The G(0)/G(1)-early S and the late S-G(2)/M transitions were examined under the influence of T3 in cycling normal and mdx muscle cell cultures over a 20-hour time period. Myogenesis and differentiation were assessed morphologically and by immunostaining for MyoD protein. Mdx cultures had fewer cells in G(0)/G(1) at 20 hours and more cells in early and late S-phase compared to normal cultures. T3 significantly increased the proportion of normal cells in early S-phase by 20 hours, and reduced the proportions in G(2)/M phase. Over the same time interval in parallel cultures, the proportion of MyoD+ normal cells decreased significantly. In the absence of T3, mdx cell cultures showed greater proportions of cells in S-phase than normal cultures, and similar increases in S-phase and loss of MyoD expression over time. However, mdx cultures had no change in the proportion that were MyoD+ during T3 treatment. The results confirm that T3 in primary cultures increased proliferation and prevented the de-differentiation of mdx cells to a greater degree than was typical of normal cells. The different susceptibilities to T3-related shifts between proliferation and differentiation observed in vitro support the idea that committed mdx myoblasts may be more activated and proliferative than normal myoblasts during regeneration in vivo.     

QuantitativeProfilingofS-NitrosylatedProteinsinParkinson’sDiseaseParadigmswiththeEffectsofBotanicalPhenolics

A convergent feature for most aging-related neurological diseases, such as Parkinson’s Disease (PD), is excessive generation of free radicals – reactive nitrogen and oxygen species (RNS/ROS), which can contribute to neuronal cell death and link to the disease pathogenesis. Free radical nitric oxide (NO) is a signaling molecule involving in the regulation of a wide range of cellular functions from development to disease. Emerging evidence suggests that nitrosative stress due to NO over-production induces post-translational modifications of protein cysteine and modulates protein enzymatic activity in cells. S-Nitrosylation, the covalent adduction of NO to specific protein cysteine thiol, is considered as a predominant, redox-based prototypical mechanism for cell signaling. Previously, endogenous protein S-nitrosylation was detected by the biotin switch assay. Taking the advantages of both biotin switch assay and differential in-gel electrophoresis (DIGE), we developed a gel-based proteomics method, named as NitroDIGE, to globally and quantitatively investigate protein S-nitrosylation. Using this method, we identified a subset of S-nitrosylated proteins from both in vitro and in vivo models of Parkinsonism including pesticide rotenone-induced PD-relevant insults in SH-SY5Y cells. Moreover, we determined whether protein S-nitrosylation in cellular PD models could be modulated by different botanical phenolic compounds, including epigallocatechin gallate (EGCG) from green tea, and apocynin from Picrorhiza kurrooa, a herbal plant grown in the Himalayan. The NitroDIGE results demonstrated that the treatment of botanical compounds could reduce excessive S-nitrosylated proteins in SH-SY5Y cells exposed to rotenone, indicating that these botanical phenolics could serve as effective scavengers to attenuate nitrosative stress and PD-relevant insults.     

Calculation of surface area and volume of human erythrocytes from scanning electron micrographs

This article demonstrates that surface and volume measurements of individual human erythrocytes with varying shapes can be obtained from scanning electron micrographic stereopairs using an approach based on established principles of photogrammetry. Instead of calculating the coordinates of several hundred points plotted for each cell, a procedure that proves tedious and time-consuming, we show that a reasonable approximation of cell surface and volume can be achieved from simple geometrical models constructed with a small number of carefully measured points and angles, using a stereocomparator. The values obtained for two normal erythrocytes and for two distorted red cells from a patient with congenital pyruvate kinase deficiency haemolytic anaemia are consistent with available information on the geometry of these cells. Because scanning electron microscopy requires extensive manipulation of the cells, the values obtained cannot be applied to fresh living material, but appear fairly accurate for the purpose of comparison between cells prepared in the same manner.     

Cell-substrate interactions in cnidaria

Studies on morphogenesis and regeneration in cnidarians have a long history, and the importance of cell-ECM (extracellular matrix) interactions for these processes has been well recognized and studied since the middle of the 20th century. Cnidarians have a life cycle with a larva, a polyp, and often a medusa generation. In the medusa, the ECM (mesoglea) is very prominent and essentially shapes the animal. In the larva and the polyp, the ECM is a thin layer. Some of the ECM components known from vertebrates have been identified in cnidarians by immunohistochemistry, electron microscopy, rotary shadowing, biochemistry, and molecular cloning. In vivo and in vitro experiments suggest that the cnidarian ECM plays a role in cell migration and morphogenesis comparable to that known from other developmental systems. In the fresh water polyp Hydra, regeneration of body patterns and migration of nematocytes seems to require the presence of ECM ligands and the corresponding cell receptors. In hydrozoan medusae, DNA replication and the stability of the differentiated state of isolated tissue can be influenced by altering the properties of the ECM substrate. When cultured, most cnidarian cells survive only when attached to ECM substrates, they rarely divide and die within short times.     

In vitro and in situ experimental model for X-ray microanalysis of intestinal epithelium

Intestinal chloride (Cl) transport is disturbed in a number of diseases. X-ray microanalysis can be used to study the distribution of Cl and other ions in intestinal epithelial cells. In this study it was attempted to establish an experimental system that retains the in vivo elemental composition of intestinal epithelial cells. An in vitro system was set up in which a segment of rat intestine was mounted in a bath and perfused with different fluids. The chloride in the bath or in the perfusion fluid could be exchanged for gluconate or bromide to determine the direction of chloride fluxes. An in situ system was set up in which the animal was anesthetized and a segment of the intestine was perfused with different solutions. In the in vitro experiments the concentration of Na and Cl in the epithelial cells increased and that of K decreased. These changes occurred within the first 30 minutes of incubation. Uptake of chloride occurred mainly from the bath, as seen in experiments where bromide was used as a chloride analog. The concentration gradient between bath and tissue determined the extent of chloride uptake. Addition of glucose to the perfusion fluid and bath did not improve the results. In the in situ system, preservation of the intracellular ion composition was better. Acceptable results were obtained with perfusion with Krebs-Ringer's buffer without glucose for 30 minutes. In this case, the elemental content of the cell did not change appreciably during incubation. If glucose was added, the Na concentration increased in comparison to the control, both in crypt and villus cells. It is concluded that the intestinal epithelium is a sensitive system, very prone to disturbance of its homeostasis. However, the in situ system can be used in studies of agonist-induced ion transport.     

Detection of mitochondrial DNA in living animal cells with fluorescence microscopy

The detection of mitochondrial DNA (mtDNA) in living human cells could be useful for understanding mitochondrial behaviour during cellular processes and pathological mtDNA depletions. However, until now, human mtDNA has not been visualized in living cells with fluorescence microscopy, although it has been easily detected in organisms with larger mtDNA. Previous reports have stated that mtDNA staining results in homogeneous fluorescence of mitochondria or that animal mitochondria are refractory to DAPI staining. This paper shows that mtDNA of cultured green monkey kidney CV-1 can be stained using a very low concentration of DAPI, then detected by a cooled Photometrics CCD camera with 14-bit resolution detection. Indeed, under these conditions CV-1 cells have small fluorescent spots in the cytoplasm that colocalize with mitochondria, even after mitochondrial movements, uncoupling by carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone and swelling. These observations have been reproduced for the human fibroblast foreskin cell line HS68. These results and known properties of DAPI as a specific DNA stain strongly suggest that mtDNA can be detected and visualized by fluorescence microscopy in human living cells, with potential developments in the study of mtDNA in normal and pathological situations.