Iron absorption by CaCo 2 cells cultivated in serum-free medium as in vitro model of the human intestinal epithelial barrier

A cell culture system consisting of confluent monolayer of human enterocyte-like CaCo 2 cells, cultivated in a serum-free nutritive medium, on microporous synthetic membranes has been used as an in vitro model of the intestinal epithelial barrier. The uptake of ⁵⁵ferric citrate, as well as the transepithelial passage from the apical to the basolateral pole, have been studied. CaCo 2 cells accumulate iron in a time- and concentration-dependent process, largely specific from the apical pole. When ⁵⁵ferric citrate is added at the apical pole, radioiron appears at the basal pole and the clearance rate is ～four times higher than in the opposite direction; the amounts of ⁵⁵Fe increase with the concentration in iron citrate and the duration of incubation. At least two concurrent mechanisms could be involved in iron absorption across monolayers of CaCo 2 cells. A first route would correspond to a paracellular passage of the metal from the apical to the basal pole. The second route would involve a selective intake of iron at the apical pole and could require a reduction of ferric iron, prior to the entry. Iron accumulated by the cells would, for a minor part, be stored within ferritin, whereas the major part would be excreted at the basolateral pole, either as low molecular weight material of undetermined chemical composition but from which iron is easily mobilized by apotransferrin or associated with neosynthesized apotransferrin. Vesicular transport and protein synthesis seem to be required. © 1994 Wiley-Liss, Inc.     

Iron and copper homeostasis and intestinal absorption using the Caco2 cell model

Whole body homeostasis can be viewed as the balance between absorption and excretion, which can be regulated independently. Present evidence suggests that for iron, intestinal absorption is the main site for homeostatic regulation, while for copper it is biliary excretion. There are connections between iron and copper in intestinal absorption and transport. The blue copper plasma protein, ceruloplasmin, and its intracellular homologue, hephaestin, play a role in cellular iron release. The studies reviewed here compare effects of Fe(II) and Cu(II) on their uptake and overall transport by monolayers of polarized Caco2 cells, which model intestinal mucosa. In the physiological range of concentrations, depletion of cellular iron or copper (by half) increased uptake of both metal ions. Depletion of iron or copper also enhanced overall transport of iron from the apical to the basal chamber. Copper depletion enhanced overall copper transport, but iron depletion did not. Pretreatment with excess copper also stimulated copper absorption. Plasma ceruloplasmin (added to the basal chamber) failed to enhance basolateral iron release, and Zn(II) failed to compete with Cu(II) for uptake. Neither copper nor iron deficiency altered expression of IREG1 or DMT1 (-IRE form) at the mRNA level. Thus, in the low-normal range of iron and copper availability, intestinal absorption of both metals appears to be positively related to the need for these elements by the whole organism. The two metal ions also influenced each other's transport; but with copper excess, other mechanisms come into play.     

Effects of C-xylopyranoside derivative on epithelial regeneration in an in vitro 3D oral mucosa model

Identifying substandard tissue-engineered oral mucosa grafts with a poor epithelium before clinical use is critical to ensure quality assurance/control in regenerative medicine, leading to success of grafting. This study investigated the effects of one of the C-xylopyranoside derivatives, β-D-xylopyranoside-n-propane-2-one (XPP), on oral epithelial regeneration. Using a three-dimensional oral mucosa model, we analyzed changes of the epithelial structure, glycosaminoglycan (GAG) synthesis, the expression levels of basement membrane zone markers, and substrates of Akt/mTOR signaling. Compared with the control, 2 mM XPP treatment increased the mean and minimal epithelial thickness, and reduced the variation of epithelial thickness. It also stimulated expressions of decorin and syndecan-1 with change of GAG amount and/or composition, and enhanced the expressions of integrin α6, CD44, and Akt/mTOR signaling substrates. These findings suggest that XPP supplementation contributes to consistent epithelial regeneration. Moreover, upregulation of those markers may play a role in increasing the quality of the oral mucosal epithelium.     

Expression of Rab small GTPases in epithelial Caco-2 cells: Rab21 is an apically located GTP-binding protein in polarised intestinal epithelial cells

Rab proteins belong to a subfamily of small GTP-binding protein genes of the Ras superfamily and play an important role in intracellular vesicular targeting. The presence of members of this protein family was examined in Caco-2 cells by a PCR-based strategy. Twenty-five different partial cDNA sequences were isolated, including 18 Rab protein family members. Seven novel human sequences, representing Rab2B, Rab6A', Rab6B, Rab10, Rab19B, Rab21 and Rab22A, were identified. For one clone, encoding Rab21, full-length cDNA was isolated from a Caco-2 cDNA library. Northern blot analysis showed a ubiquitous expression pattern of Rab21. To study Rab21 protein expression in Caco-2 cells, polyclonal antibodies were raised against GST-Rab21 fusion protein and characterised. The antibodies recognised Rab21 as a protein of approximately 25 kDa. Interestingly, the protein shows a general ER-like staining in nonpolarised Caco-2 cells in contrast to an apically located vesicle-like staining in polarised Caco-2 cells. Furthermore, immunohistochemical staining on human jejunal tissue showed a predominant expression of Rab21 in the epithelial cell layer with high expression levels in the apical region, whereas stem cells in the crypts were negative. We therefore suggest an alternative role for Rab21 in the regulation of vesicular transport in polarised intestinal epithelial cells.     

Rapid in vitro transformation system for liver epithelial cells by iron chelate,Fe-NTA

We have previously found toxic effects of iron chelate, Fe-NTA on cultured normal rat liver epithelial cells (RL34). In the present study, when RL34 cells were exposed to 50 g/ml iron of Fe-NTA for 15 days, besides the expected cytolytic effects in most cells, the appearance of resistant cells was observed. The resistant cells showed drastic morphological transformation, grew in soft agar, and induced hepatocellular carcinomas when transplanted into syngeneic newborn rats in a short period of time. Since DNA instability in the transformed cells was ascertained by differential AO staining, it is suggested that DNA damage by Fe-NTA is of a critical importance for extremely rapid neoplastic transformation of normal epithelial cells.Abbreviations AO acridine orange - DME medium Dulbecco's modified Eagle's medium - FCS fetal calf serum - Fe-HEDTA ferric-N-(2-hydroxyethyl)ethylenediaminetriacetic acid - Fe-NTA ferric-nitrilotriacetate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - PBS Dulbecco's phosphate-buffered saline     

Sensitivity of photosynthesis by spinach chloroplast membranes to osmotic stress in vitro: Rapid inhibition of O2 evolution in presence of magnesium

Thylakoids prepared from spinach (Spinacea oleracea L.) chloroplasts were exposed to osmotic stress in vitro in the presence or absence of different inorganic salts. By an hour after incubation in 1.0 M sorbitol and 10 mM (or more) MgCl₂, the thylakoids lost approximately 80% of their photosystem (PS) II activity, but not PS I. The inhibition occurred only in presence of magnesium as indicated by the combinations of several cations/anions. The PS II activity was relatively insensitive to osmotic stress in the presence of diphenyl carbazide. We therefore conclude that under conditions of water stress in the presence of 10 mM or higher Mg²⁺, the oxygen evolving system in chloroplasts is rapidly inactivated.Abbreviations DCMU 3(3,4-dichlorophenyl)-1,1-dimethylurea - DCPIP 2,6-dichlorophenol indophenol - DPC diphenyl carbazide - MV methyl viologen - PS photosystem Part of this work was included in the thesis submitted by the first author of M.Phil.degree.     

Comparative effects of exopolysaccharides from lactic acid bacteria and fructo-oligosaccharides on infant gut microbiota tested in an in vitro colonic model with immobilized cells

The aim of this study was to compare the effects of purified exopolysaccharides from Lactobacillus rhamnosus RW-9595M with those of a well-known prebiotic (short-chain fructo-oligosaccharides) on infant colonic microbiota using a new three-stage chemostat model with immobilized infant faecal microbiota. Two continuous cultures with different faecal inocula were tested with different compositions of carbohydrate media. During the first fermentation (F1), fructo-oligosaccharides tested at a concentration of 9.8 g L(-1) increased the number of lactobacilli and decreased coliforms both in gel beads and in effluent from all three reactors, in agreement with data from the literature. During the second fermentation (F2), the effect of fructo-oligosaccharides tested at a lower concentration (7.5 g L(-1)) was reduced compared with F1. Fructo-oligosaccharides also increased total organic acid concentration and decreased ammonia production. Results obtained for exopolysaccharide tested at 1.5 g L(-1) indicate that exopolysaccharides from L. rhamnosus RW-9595M was not metabolized by infant microbiota and lacked any prebiotic effect.     

Selective gene silencing of rat ATP-binding cassette G2 transporter in an in vitro blood-brain barrier model by short interfering RNA

The aim of the present study was to specifically silence the rat ATP-binding cassette transporter G2 (rABCG2) gene in brain capillary endothelial cells by transfection of short interfering RNA (siRNA). Four different siRNAs designed to target rABCG2 were each transfected into HEK293 cells with myc-tagged rABCG2 cDNA. Quantitative real-time PCR and western blot analyses revealed that three of the siRNAs were able to reduce exogenous rABCG2 mRNA and protein levels in HEK293 cells. Moreover, rABCG2-mediated mitoxantrone efflux transport was suppressed by the introduction of these three siRNAs into HEK293 cells. In contrast, the other siRNA and non-specific control siRNA did not significantly affect the mRNA expression, the protein level or the transport activity. Endogenous rABCG2 mRNA and protein expression in a conditionally immortalized rat brain capillary endothelial cell line (TR-BBB13) was suppressed by the most potent siRNA among the four siRNAs tested. Furthermore, this siRNA did not affect the mRNA levels of other ABC transporters, such as ABCB1, ABCC1 and ABCG1, and the protein level of ABCB1 in TR-BBB13 cells, suggesting that it can selectively silence rABCG2 at the blood-brain barrier. This should be a useful and novel strategy for clarifying the contribution of rABCG2 to brain-to-blood transport of substrate drugs and endogenous compounds across the blood-brain barrier.     

The changes in the neuronal PC12 and the intestinal epithelial Caco-2 cells during the coculture. The functional analysis using an in vitro coculture system

The interaction between intestinal epithelial cells andperipheral neuronal cells were examined using an invitro coculture system. Two cell lines, Caco-2 and PC12, were usedfor this experiment as an intestinal epithelial and entericneuronal cell model, respectively. By coculturing with fullydifferentiated Caco-2 cells, the neurite outgrowth was inducedin PC12 cells. This neurite outgrowth in PC12 was blocked byanti-nerve growth factor (NGF) polyclonal antibodies,suggesting that the neurite outgrowth in PC12 during thecoculture with Caco-2 cells was due to NGF secreted fromCaco-2 cells. On the other hand, coculturing with fullydifferentiated PC12 cells induced the decrease oftransepithelial electrical resistance in Caco-2 cellmonolayers. The permeability of lucifer yellow alsosignificantly increased, suggesting that the barrier functionand paracellular permeability of Caco-2 monolayers werealtered by coculturing with PC12 cells. The present studysuggests that this in vitro coculture system is a good modelfor the functional analysis of interaction among intestinalepithelial cells with different cell types.     

Cadmium accumulation and interactions with zinc, copper, and manganese, analysed by ICP-MS in a long-term Caco-2 TC7 cell model

The influence of long-term exposure to cadmium (Cd) on essential minerals was investigated using a Caco-2 TC7 cells and a multi-analytical tool: microwave digestion and inductively coupled plasma mass spectrometry. Intracellular levels, effects on cadmium accumulation, distribution, and reference concentration ranges of the following elements were determined: Na, Mg, Ca, Cr, Fe, Mn, Co, Ni, Cu, Zn, Mo, and Cd. Results showed that Caco-2 TC7 cells incubated long-term with cadmium concentrations ranging from 0 to 10 μmol Cd/l for 5 weeks exhibited a significant increase in cadmium accumulation. Furthermore, this accumulation was more marked in cells exposed long-term to cadmium compared with controls, and that this exposure resulted in a significant accumulation of copper and zinc but not of the other elements measured. Interactions of Cd with three elements: zinc, copper, and manganese were particularly studied. Exposed to 30 μmol/l of the element, manganese showed the highest inhibition and copper the lowest on cadmium intracellular accumulation but Zn, Cu, and Mn behave differently in terms of their mutual competition with Cd. Indeed, increasing cadmium in the culture medium resulted in a gradual and significant increase in the accumulation of zinc. There was a significant decrease in manganese from 5 μmol Cd/l exposure, and no variation was observed with copper.     

High-throughput production of recombinant antigens for mouse KIAA proteins in Escherichia coli: computational allocation of possible antigenic regions, and construction of expression plasmids of glutathione-S-transferase-fused antigens by an in vitro recombination-assisted method.

Since the end of 2001, we have conducted a project to isolate and determine entire sequences of mouse cDNA clones which encode the polypeptides corresponding to human KIAA proteins. Towards the ultimate goal of this project to clarify the biological functions of KIAA genes, we have set production of antibodies against mouse KIAA gene products based on their sequence information as the next important stage. As the first step, we developed a high-throughput system utilizing shotgun clones generated during entire sequencing of mouse KIAA cDNAs. The system consists of the following three parts: (1) Shotgun clones encoding regions suitable for production of antigens were selected using a newly developed browser system; (2) the protein-coding sequences of the selected shotgun clones were transferred into an expression vector by in vitro recombination-assisted method in a 96-well format, and expressed as glutathione S-transferase fusion proteins in Escherichia coli; and (3) the solubility of the recombinant antigens were preliminarily assessed in a small-scale culture and then large-scale production and purification was performed using glutathione-affinity beads or retrieval from polyacrylamide gels depending on their solubility. Using these systems, we successfully produced and purified 400 antigens for production of mKIAA antibodies to date.     

Induction of lacI mutations in Big Blue Rat-2 cells treated with 1-(2-hydroxyethyl)-1-nitrosourea: a model system for the analysis of mutagenic potential of the hydroxyethyl adducts produced by 1,3-bis (2-chloroethyl)-1-nitrosourea.

We have investigated the genotoxic effects of 1-(2-hydroxyethyl)-1-nitrosourea (HENU). We have chosen this agent because of its demonstrated ability to produce N7-(2-hydroxyethyl) guanine (N7-HOEtG) and O⁶-(2-hydroxyethyl) 2′-deoxyguanosine (O⁶-HOEtdG); two of the DNA alkylation products produced by 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU). For these studies, we have used the Big Blue Rat-2 cell line that contains a lambda/lacI shuttle vector. Treatment of these cells with HENU produced a dose dependent increase in the levels of N7-HOEtG and O⁶-HOEtdG as quantified by HPLC with electrochemical detection. Treatment of Big Blue Rat-2 cells with either 0, 1 or 5 mM HENU resulted in mutation frequencies of 7.2±2.2×10⁻⁵, 45.2±2.9×10⁻⁵ and 120.3±24.4×10⁻⁵, respectively. Comparison of the mutation frequencies demonstrates that 1 and 5 mM HENU treatments have increased the mutation frequency by 6- and 16-fold, respectively. This increase in mutation frequency was statistically significant (P<0.001). Sequence analysis of HENU-induced mutations have revealed primarily G:C→A:T transitions (52%) and a significant number of A:T→T:A transversions (16%). We propose that the observed G:C→A:T transitions are produced by the DNA alkylation product O⁶-HOEtdG. These results suggest that the formation of O⁶-HOEtdG by BCNU treatment contributes to its observed mutagenic properties.     

Rabies virus glycoprotein expression in Drosophila S2 cells. I: Design of expression/selection vectors, subpopulations selection and influence of sodium butyrate and culture medium on protein expression

The cDNA encoding the rabies virus glycoprotein (RVGP) gene was cloned in expression plasmids under the control of the inductive metallothionein promoter. They were designed in order to bear or not a secretion signal (i) and a cDNA coding for the selection hygromycin. These vectors were transfected into S2 cells, cell populations selected and subpopulations were then obtained by reselection with hygromycin. Cell cultures were examined for kinetics of cell growth, detection of RVGP mRNA and expression of RVGP. All cell populations were shown to express the RVGP mRNA upon induction. S2MtRVGPHy cell population, transfected with one vector that contains RGPV gene and selection gene, was shown to express higher amounts of RVGP as evaluated by flow cytometry (52%) and ELISA (0.64 μg/10⁷ cells at day 7). Subpopulation selection allowed a higher RVGP expression, specially for the S2MtRVGPHy⁺ (5.5 μg/10⁷ cells at day 7). NaBu treatment leading to lower cell growth and higher RVGP expression allowed an even higher RVGP synthesis by S2MtRVGPHy⁺ (8.4 μg/10⁷ cells at day 7). SF900II medium leading to a higher S2MtRVGPHy⁺cell growth allowed a higher final RVGP synthesis in this cell culture. RVGP synthesis may be optimized by the expression/selection vectors design, cell subpopulations selection, chromatin exposure and culture medium employed.     

Synthesis of (+)-(R)- and (−)-(S)-5-hydroxy-2-methyl-2-dipropylaminotetralin: Effects on rat hippocampal output of 5-HT, 5-HIAA,and DOPAC as determined by in vivo microdialysis

Racemic 5-methoxy-2-methyl-2-dipropylaminotetralin ( 3 ) has been prepared by a short synthetic route, in which the N,N-dipropyliminium perchlorate of 5-methoxy-2-tetralone ( 4 ) is a key intermediate. Racemic 3 was resolved by crystallization of the corresponding diastereomeric di-p-toluoyltartrates. The enantiomeric excess (%ee) of the phenolic derivatives of (+)-(R)- and (?)-(S)-3 [(+)-(R)- and (?)-(S)-2] was determined by ¹HNMR spectroscopic analysis of the corresponding diastereomeric (?)-(R)-1,1′-binaphthyl-2,2′-diylphosphoric acid salts utilizing ¹³C satellites. X-ray crystallography established the absolute configuration of (?)-(S)-2 · HCl. The enantiomers of 2 were tested for hippocampal output of 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, and dihydroxyphenylacetic acid in rats by use of in vivo microdialysis. The (?)-(S)-enantiomer appeared to affect 5-HT-turnover, whereas (+)-(R)- 2 was inactive. Results obtained provide support for the previously reported hypothesis that the inactivity of (?)-(S)- 2 at central DA receptors is caused by the steric bulk of the C(2)-methyl group. This makes it possible to define a “DA D2 receptor essential volume.” © 1993 Wiley-Liss, Inc.     

Single-strand breaks in DNA during repair of UV-induced damage in normal human and xeroderma pigmentosum cells as determined by alkaline DNA unwinding and hydroxylapatite chromatography: effects of hydroxyurea, 5-fluorodeoxyuridine and 1-beta-D-arabinofuranosylcytosine on the kinetics of repair.

A simple and sensitive technique for detection of strand breaks in DNA has been further developed. The method has been used to follow UV-induced excision-repair in human fibroblasts. It has been possible to study the kinetics of enzymic reactions in intact cells, in which strand breaks in DNA are produced and sealed again. Hydroxyurea, 5-fluorodeoxyuridine and 1-beta-D-arabinofuranosylcytosine, potent inhibitors of DNA synthesis, drastically increased the number of breaks observed during the repair process. This was probably due to a decreased polymerase activity, which will cause the strand breaks formed by endonuclease to remain open longer. The initial rate of strand-break formation did not seem to be influenced by hydroxyurea or araC, and was about 4000 breaks per minute in a diploid genome, at a dose of 20 J/m2. After 5--30 min, depending on the dose of UV, the number of breaks reached a maximum and started to decrease again. Hydroxyurea decreased the rate of polymerization in the sites under repair. However, there was no concomitant reduction of repair-induced incorporation of [3H]thymidine and no reduction of the excision of pyrimidine dimers. It therefore seems that the action of the polymerase was not a rate-limiting event, but rather an earlier step. It is likely that the endonucleolytic activity determined the rate of repair. As a consequence, the endonuclease and polymerase cannot be bound in a permanent complex. Under certain assumptions, the time for repair of a site, i.e. the time from incision to final ligase sealing, can be estimated as between 3 and 10 min. Essentially no breaks were produced in Xeroderma pigmentosum cells belonging to complementation group A, and there was no enhancement by hydroxyurea. Cells from the variant type of Xeroderma pigmentosum behaved like normal cells in this respect.