Identification of telocytes in the lamina propria of rat duodenum: transmission electron microscopy

Recently the new term 'telocytes' has been proposed for cells formerly known as interstitial Cajal-like cells. In fact, telocytes are not really Cajal-like cells, they being different from all other interstitial cells by the presence of telopodes, which are cell-body prolongations, very thin, extremely long with a moniliform aspect. The identification of these cells is based on ultrastructural criteria. The presence of telocytes in others organs was previously documented. We reported for the first time, an ultrastructural study of telocytes in the lamina propria of rat duodenum. Our findings show that typical telocytes are present in the rat duodenum. Telocytes are located in the lamina propria, immediately below mucosal crypts. Telopodes frequently establish close spatial relationships with immune cells, blood vessels and nerve endings. On the basis of their distribution and morphology, we suggest that these cells may be involved in immune response and in our opinion, it may be possible that different locations of telocytes could be associated with different roles.     

The subcellular distribution of zinc in dog prostate studied by X-Ray microanalysis

Summary X-ray microanalysis of zinc in ultrathin sections of dog prostate was performed by electron microscope microanalysis using the potassium pyroantimonate method of preparation. Prostates of both mature and immature dogs were examined and the metal was found to be localised primarily in the nucleolus, nuclear chromatin and secretory granules of epithelial cells. Differences in zinc concentrations were observed between mature and immature tissues, particularly in the nuclear chromatin. The metal was also incorporated into epithelial secretions, lysosomes and fibromuscular stroma. Variable binding of zinc to tissue components was revealed by a combination of histochemical precipitation and subcellular analysis.The authors are grateful to the Tenovus Organisation for general financial support. This work was also supported by the Medical Research Council, Grant No. G974/304B and by a grant of the Austrian Bundesministerium für Wissenschaft und Forschung. One of them (F.S.) was financed by the British Council     

Cellular architecture of the lamina propria of human seminiferous tubules

Summary The lamina propria of human seminiferous tubules is composed of 5 to 7 cellular layers separated by laminae of extracellular connective-tissue components. By means of immunocytochemical methods the different nature of the cellular layers could be defined for the first time. Based on the light-microscopic demonstration of both desmin-like and vimentin-like immunoreactivity in the inner 3 to 4 layers of the lamina propria, these cells can be identified as myofibroblasts. The outermost one or two cellular layers, on the contrary, only show a vimentin-like immunoreactivity indicating the pure fibroblastic nature of these cells. Therefore, the outermost cellular layers are suggested to be derivatives of the interstitium. In cases of disturbed spermatogenesis, the lamina propria is frequently considerably thickened by an increase in the extracellular matrix components between the cellular layers. Whereas the ultrastructural localization of laminin-, collagen type-IV- and fibronectin-like immunoreactivity remains unaffected in the thickened lamina propria, the desmin-like immunoreactive cells of the inner layers strongly decrease in number and staining intensity. Most probably, the myofibro-blasts lose their myoid characteristics to participate in the secretion of increased amounts of extracellular matrix components, which in turn presumably block the mediation of the lamina propria between the interstitium and the germinal epithelium. It is still unclear whether the thickened lamina propria provokes the disturbance of spermatogenesis or vice versa.     

Effect of hypoxia on the binding and subcellular distribution of iron regulatory proteins

Iron regulatory proteins 1 and 2 (IRP1, IRP2) are key determinants of uptake and storage of iron by the liver, and are responsive to oxidative stress and hypoxia potentially at the level of both protein concentration and mRNA-binding activity. We examined the effect of hypoxia (1% O₂) on IRP1 and IRP2 levels (Western blots) and mRNA-binding activity (gel shift assays) in human hepatoma HepG2 cells, and compared them with HEK 293 cells, a renal cell line known to respond to hypoxia. Total IRP binding to an iron responsive element (IRE) mRNA probe was increased several fold by hypoxia in HEK 293 cells, maximally at 4–8 h. An earlier and more modest increase (1.5- to 2-fold, peaking at 2 h and then declining) was seen in HepG2 cells. In both cell lines, IRP1 made a greater contribution to IRE-binding activity than IRP2. IRP1 protein levels were increased slightly by hypoxia in HEK 293 but not in HepG2 cells. IRP1 was distributed between cytosolic and membrane-bound fractions, and in both cells hypoxia increased both the amount and IRE-binding activity of the membrane-associated IRP1 fraction. Further density gradient fractionation of HepG2 membranes revealed that hypoxia caused an increase in total membrane IRP1, with a shift in the membrane-bound fraction from Golgi to an endoplasmic reticulum (ER)-enriched fraction. Translocation of IRP to the ER has previously been shown to stabilize transferrin receptor mRNA, thus increasing iron availability to the cell. Iron depletion with deferoxamine also caused an increase in ER-associated IRP1. Phorbol ester caused serine phosphorylation of IRP1 and increased its association with the ER. The calcium ionophore ionomycin likewise increased ER-associated IRP1, without affecting total IRE-binding activity. We conclude that IRP1 is translocated to the ER by multiple signals in HepG2 cells, including hypoxia, thereby facilitating its role in regulation of hepatic gene expression. Electronic supplementary material The online version of this article () contains supplementary material, which is available to authorized users.     

Expression profiles of iron transport molecules along the duodenum

Duodenal biopsies are considered a suitable source of enterocytes for studies of dietary iron absorption. However, the expression level of molecules involved in iron absorption may vary along the length of duodenum. We aimed to determine whether the expression of molecules involved in the absorption of heme and non‐heme iron differs depending on the location in the duodenum. Analysis was performed with samples of duodenal biopsies from 10 individuals with normal iron metabolism. Samples were collected at the following locations: (a) immediately post‐bulbar, (b) 1–2 cm below the papilla of Vater and (c) in the distal duodenum. The gene expression was analyzed at the mRNA and protein level using real‐time PCR and Western blot analysis. At the mRNA level, significantly different expression of HCP1, DMT1, ferroportin and Zip8 was found at individual positions of duodenum. Position‐dependent expression of other molecules, especially of FLVCR1, HMOX1 and HMOX2 was also detected but with no statistical significances. At the protein level, we observed statistically significantly decreasing expression of transporters HCP1, FLVCR1, DMT1, ferroportin, Zip14 and Zip8 with advancing positions of duodenum. Our results are consistent with a gradient of diminishing iron absorption along the duodenum for both heme and non‐heme iron.     

Mobilisation of recently absorbed Fe in ex vivo perfused rat duodena and the influence of iron status and subsequently absorbed chelators

To investigate the effect of subsequently absorbed metal chelators on recently absorbed ⁵⁹Fe, duodenal segments from iron-deficient and iron-adequate rats were perfused ex vivo until the ⁵⁹Fe tissue load had reached a steady state. Subsequently, the segments were perfused with 3 model chelators and their iron complexes: nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA) and citrate. Of these, NTA and EDTA bind iron much tighter than citrate, and Fe–NTA complexes exchange iron within seconds while Fe-EDTA complexes need 48 h to reach equilibrium.

Duodenal mucosa-to-serosa transport rates were comparable for all 3 chelators and correlated linearly with luminal concentration. Subsequent perfusion with increasing NTA, Fe–NTA(1:2) and EDTA concentrations mobilised increasing amounts of ⁵⁹Fe from the duodenum. Mobilised ⁵⁹Fe moved preferentially back into the luminal perfusate in iron-adequate segments. In iron-deficient segments, ⁵⁹Fe preferentially continued the absorption process across the basolateral membrane. Fe–EDTA(1:1) hardly mobilised any ⁵⁹Fe back into the lumen, though basolateral transfer increased at high concentrations. Citrate and Fe–citrate(1:1) mobilised ⁵⁹Fe only at very high concentrations.

This behaviour is in accordance with the rules of complex chemistry: strong, fast reacting ligands like NTA show most impact. Slowly reacting complexes like Fe–EDTA(1:1) have little mobilising impact in spite of strong affinity between EDTA and iron. The low affinity between iron and citrate can be compensated by large concentration. Moreover, iron-deficient segments show stronger re-uptake of mobilised ⁵⁹Fe from the lumen and a stronger transfer of ⁵⁹Fe from the tissue across the basolateral membrane. Both are compatible with the more marked expression of divalent metal transporter 1 (DMT-1) and IREG-1 at the brushborder and basolateral membrane of iron-deficient enterocytes. The data suggest that iron ions interact with food ligands during their passage from the apical to the basolateral side of duodenal enterocytes.     

Immunocytochemical localization of a calcium-binding protein in the rat duodenum

Summary The cellular localization of the vitamin D-dependent calcium-binding protein (CaBP) in the duodenum of rat was studied using indirect immunofluorescence and immunoperoxidase staining methods. Specific positive reaction product, indicative of the presence of CaBP, was exclusively located within the villous part of the duodenal mucosa. Moreover, CaBP was detected mainly within the supranuclear region of the cytoplasm of absorptive cells and also at the level of their basal laminae. CaBP was not demonstrable either in the nuclei or associated with the brush border membrane of absorptive cells. Also, CaBP was neither detectable in goblet cells nor in sub-epithelial layers. When the specific anti-CaBP antiserum was replaced by nonimmune rabbit serum or when it was preabsorbed on a CaBP-Sepharose conjugate, no positive immunostaining was seen. Together with recent biochemical data our observations agree well with the view that CaBP may act as an intracellular buffer by protecting the cell against too high Ca²⁺ concentrations.     

Cellular and subcellular distribution of laminin in adult rat anterior pituitary

Following the immunodetection of laminin in basal laminae and within some glandular cells of adult rat pituitary (Tougard et al., 1985: In Vitro 21:57), the present work had a double purpose: (a) adjustment of optimal conditions to detect laminin at both the cellular and subcellular level with an immunoperoxidase pre-embedding procedure, and (b) identification of the hormonal specificity of the laminin-containing cells, based on development of a "two-step" method combining the pre- and post-embedding approaches. In addition to extracellular localizations (basal laminae and connective tissue compartment), laminin was detected intracellularly within membrane-bounded compartments in all endocrine cell types and in the folliculo-stellate cells. It was found in rough endoplasmic reticulum cisternae, in a few Golgi saccules, and in vesicles or vacuoles. Labeled secretory granules were observed in gonadotrope, thyrotrope, and corticotrope cells but were very scarce in prolactin cells and absent in somatotrope cells. In addition, exocytotic profiles of labeled granules were frequent, suggesting a granular pathway for laminin export. Labeled vesicles, heterogeneous in size and shape, were observed mostly in prolactin cells. They might represent either a vesicular pathway for laminin export or an endocytic route for membrane-bounded laminin. These results bring complementary data to the concept that epithelial cells elaborate their basement membranes (Pierce and Nakane, 1967: Lab Invest 17:499). Moreover, the subcellular distribution of laminin within organelles involved in the biosynthesis, transport, and even in the storage of secretory products suggests that laminin might be exported by glandular pituitary cells according to different pathways.     

Cellular and subcellular localization of calcium in gravistimulated corn roots

Summary Antimonate staining procedures and energy dispersive X-ray microanalytical techniques were used to determine the patterns of localization of calcium in nonstimulated and gravistimulated corn roots. In horizontally positioned roots within the region of the developing bend there was a change in the staining from that principally localized within cells of the stele to asymmetric staining within the vacuoles of the cortical cells along the upper root surface. There was little staining in the walls. The pattern observed is quite different from that seen in gravistimulated coleoptiles. Staining of mitochondria, plastids and Golgi stacks was seen in most cell types, but no asymmetry of staining was observed. In the rootcap where graviperception is thought to occur, there was little staining of any cellular organelles.     

Cellular and subcellular localization of chromium in the crab Liocarcinus puber (Brachyrhyncha) by transmission electron microscopy,secondary ion mass spectrometry and electron microprobe analysis

The swimming crab Liocarcinus puber (Crustacea Brachyrhyncha) was exposed for 2 weeks to CrCl₃ (chromium occurs principally in the trivalent state in the natural environment). The gills, digestive gland and muscle were examined by several analytical techniques for cellular and subcellular localization of chromium. The techniques applied were secondary ion mass spectrometry (ion microscopy and ion microprobe analysis) associated with photon microscopy and X-ray spectrometry (electron microprobe analysis) together with transmission electron microscopy. The digestive gland was found to be free of chromium, whereas chromium was adsorbed onto the gill exoskeleton. The muscle was the only tissue with intracellular electron-dense precipitates with no surrounding membrane. The metal was detected in the heterophagic vacuoles of amoebocytes where it was associated with phosphorus and trapped in an unsoluble form. Mechanisms of chromium cellular and subcellular metabolism were compared between crabs and other aquatic organisms. L. puber does not appear to be a suitable bioindicator of chromium pollution because of molting and its low chromium bioaccumulation capability.     

Uptake of iron nanoparticles by Aphanorrhegma patens (Hedw.) Lindb.

Iron nanoparticles were produced in a radiofrequency levitation furnace and their dimensions and purity determined by scanning electron microscopy and x-ray microanalysis. Confocal microscopy revealed that these particles penetrated the leaves of Aphanorrhegma when applied to the plants as mineral water suspensions. This, the first demonstration of nanoparticle uptake by a bryophyte, opens the way to nanotoxicological studies paralleling those in higher plants. The actual penetration mechanism in both groups remains unknown.     

Localization of thrombomodulin in pericryptal fibroblasts of the rat duodenum

Thrombomodulin (TM) contributes to the inactivation of thrombin which activates protein C, a major anticoagulant and anti-inflammatory protein. This report describes the distribution and localization of TM in the rat duodenum by light and electron microscopic immunohistochemistry. In addition to the endothelium of the entire vasculature, TM was found on the non-vascular structures, cellular networks of pericryptal fibroblasts surrounding the bases of the glandular crypts. TM was localized on the plasma membrane of the pericryptal fibroblasts which existed in the pericryptal spaces between the basal lamina of the epithelium and the collagenous sheets around the base of the crypt. TM-immunoreactive pericryptal fibroblasts were stellate-shaped cells and contacted with each other by their cytoplasmic processes forming an anastomosing syncytium around the base of the crypt. In contrast to pericryptal fibroblasts, TM was not localized in subepithelial fibroblasts in the villi or smooth muscle cells in the lamina muscularis mucosae and the muscularis. The functional significance of TM in pericryptal fibroblasts is discussed.     

Current knowledge of iron metabolism

Iron plays many roles in human physiology. In this article, we summarize the basic and current knowledge of this essential micronutrient on human metabolism.     

Excessive iron accumulation in the pea mutants dgl and brz: subcellular localization of iron and ferritin

The pea (Pisum sativum L.) mutants dgl and brz are defective in the regulation of iron uptake. Enhanced proton extrusion and constitutively high Fe(III) reductase activities in the roots lead to an accumulation of iron and other divalent cations in different organs of the mutants. Ultrastructural investigations of the basal leaflets of the mutants revealed in the cytoplasm, in mitochondria and especially in, or close to the endoplasmic reticulum numerous electron-dense particles which were absent in the corresponding wild type plants DGV and Sparkle. By means of electron-spectroscopic imaging and electron-energy-loss spectroscopy it could be shown that these electron-dense particles consist mainly of iron. Some of the iron deposits were immunocytochemically identified as the iron-storage protein ferritin. It is suggested that the precipitation of excessive iron in the dgl and brz mutant leaves in the form of electron-dense iron particles combined with the accumulation of ferritin is part of the plant defense mechanism against Fe-mediated oxidative stress. Received: 17 February 1998 / Accepted: 4 July 1998     

Rapid reduction of iron in horse spleen ferritin by thioglycolic acid measured by dispersive X-ray absorption spectroscopy

Summary The release of iron from ferritin is important in the formation of iron proteins and for the management of diseases in both animals and plants associated with abnormal accumulations of ferritin iron. Much more iron can be released experimentally by reduction of the ferric hydrous oxide core than by chelation of Fe³⁺ which has led to the notion that reduction is also the major aspect of iron release in vivo. Variations in the kinetics of reduction of the mineral core of ferritin have been attributed to the redox potential of the reductant, redox properties of the iron core, the structure of the protein coat, the analytical method used to detect Fe²⁺ and reactions at the surface of the mineral. Direct measurements of the oxidation state of the iron during reduction has never been used to analyze the kinetics of reduction, although Mössbauer spectroscopy has been used to confirm the extent of reduction after electrochemical reduction using dispersive X-ray absorption spectroscopy (DXAS). We show that the near edge of X-ray absorption spectra (XANES) can be used to quantify the relative amounts of Fe²⁺ and Fe³⁺ in mixtures of the hydrated ions. Since the nearest neighbors of iron in the ferritin iron core do not change during reduction, XANES can be used to monitor directly the reduction of the ferritin iron core. Previous studies of iron core reduction which measured by Fe²⁺ · bipyridyl formation, or coulometric reduction with different mediators, suggested that rates depended mainly on the redox potential of the electron donor. When DXAS was used to measure the rate of reduction directly, the initial rate was faster than previously measured. Thus, previously measured differences in reduction rates appear to be influenced by the accessibility of Fe²⁺ to the complexing reagent or by the electrochemical mediator. In the later stages of ferritin iron core dissolution, reduction rates drop dramatically whether measured by DXAS or formation of Fe²⁺ complexes. Such results emphasize the heterogeneity of ferritin core structure.     

On structural patterns of the lamina propria of human seminiferous tubules

Summary The ultrastructure of the lamina propria of human seminiferous tubules was analyzed in normal specimens and compared to biopsies showing great thickenning of this area in light microscopy.The contractile cells are stellate in shape, the intercellular gaps between their branchings being less than 150 Å. The cytoplasmic features of these cells are similar to those described by Ross and Long (1966) and do not differ significantly in the pathological cases examined.The intercellular components, namely collagen fibers, microfibrils and an incomplete basement membrane-like coating of the contractile cells, are strikingly increased in the thickenned lamina propria, although the number of layers making up this structure needs not be increased. Occasionally, the intercellular space is occupied by only one of these materials.The distribution of collagen permits identification of two main patterns in the thickenned lamina propria: a) one where the basement membrane of the seminiferous epithelium is separated from the first layer of contractile cells by a wide collagen zone, and b) another case where the layer displaying greater thickness because of increased collagen deposition is located further away from the germinal epithelium.The functional activity of the contractile cells, the physiological implication of structural alterations of the lamina propria and the necessity to correlate these observations to andrological findings, are discussed.Presented in part at the 68. Meeting of the Anatomische Gesellschaft, Lausanne, April 1973.Fellow of the Alexander von Humboldt Foundation, on leave of absence from Depto. de Biología y Genética, Sede Norte, Universidad de Chile, Santiago.Supported by Grants from the Deutsche Forschungsgemeinschaft.     

Effects of acid on the basal lamina of the rat stomach and duodenum

Rapid restitution of the gastric and intestinal epithelium after acute injury involves emigration of cells from the gastric glands and basal half of the intestinal villi. An intact basal lamina is prerequisite to the restitution process. The present study was performed to determine the effects of acid on the rat gastric and duodenal basal lamina. The basal lamina was denuded in vitro by ultrasonic vibration. The tissue was then immersed in 0.2 M mannitol (control) or in HCl (5-50 mM) for 10 min. Samples of the tissues were examined by transmission and scanning electron microscopy. Some samples were stained with ruthenium red to demonstrate glycosaminoglycans. The lower concentrations of acid (5 and 10 mM) had little or no effect on the structure of the basal lamina. However, exposure to 20 and 50 mM HCl caused extensive damage to the basal lamina and exposed the underlying connective tissue matrix of the lamina propria. Ruthenium red staining demonstrated differences in size and location of glycosaminoglycans within the basal laminae of stomach and intestine. Exposure to acid at concentrations of 20 or 50 mM caused total loss of ruthenium red staining in both intestinal and gastric basal laminae. Exposure to 10 mM acid resulted in loss of the outermost (luminal) layer of anionic sites from the gastric basal lamina. These studies demonstrate that brief exposure to acid, in concentrations which are necessary for the formation of hemorrhagic erosions in the stomach, caused damage to the basal lamina. This damage may impair epithelial restitution and thus account, in part, for the role of acid in ulcerogenesis.