Environmental fate of 2,4-dinitroanisole (DNAN) and its reduced products.

Several defense departments intend to replace 2,4,6-trinitrotoluene (TNT) in munitions formulations by the less sensitive 2,4-dinitroanisole (DNAN). To help understand environmental behavior and ecological risk associated with DNAN we investigated its key initial abiotic and biotic reaction routes and determined relevant physicochemical parameters (pKa, logKow, aqueous solubility (Sw), partition coefficient (Kd)) for the chemical and its products. Reduction of DNAN with either zero valent iron or bacteria regioselectively produced 2-amino-4-nitroanisole (2-ANAN) which, under strict anaerobic conditions, gave 2,4-diaminoanisole (DAAN). Hydrolysis under environmental conditions was insignificant whereas photolysis gave photodegradable intermediates 2-hydroxy-4-nitroanisole and 2,4-dinitrophenol. Physicochemical properties of DNAN and its amino products drastically depended on the type and position of substituent(s) on the aromatic ring. Sw followed the order (TNT

Microaerophilic degradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by three Rhodococcus strains.

AIM: The goal of this study was to compare the degradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by three Rhodococcus strains under anaerobic, microaerophilic (<0.04 mg l(-1) dissolved oxygen) and aerobic (dissolved oxygen (DO) maintained at 8 mg l(-1)) conditions. METHODS AND RESULTS: Three Rhodococcus strains were incubated with no, low and ambient concentrations of oxygen in minimal media with succinate as the carbon source and RDX as the sole nitrogen source. RDX and RDX metabolite concentrations were measured over time. Under microaerophilic conditions, the bacteria degraded RDX, albeit about 60-fold slower than under fully aerobic conditions. Only the breakdown product, 4-nitro-2,4-diazabutanal (NDAB) accumulated to measurable concentrations under microaerophilic conditions. RDX degraded quickly under both aerated and static aerobic conditions (DO allowed to drop below 1 mg l(-1)) with the accumulation of both NDAB and methylenedinitramine (MEDINA). No RDX degradation was observed under strict anaerobic conditions. CONCLUSIONS: The Rhodococcus strains did not degrade RDX under strict anaerobic conditions, while slow degradation was observed under microaerophilic conditions. The RDX metabolite NDAB was detected under both microaerophilic and aerobic conditions, while MEDINA was detected only under aerobic conditions. IMPACT AND SIGNIFICANCE OF THE STUDY: This work confirmed the production of MEDINA under aerobic conditions, which has not been previously associated with aerobic RDX degradation by these organisms. More importantly, it demonstrated that aerobic rhodococci are able to degrade RDX under a broader range of oxygen concentrations than previously reported.

Fluorescence microscopy as a tool for in situ life detection.

The identification of extant and, in some cases, extinct bacterial life is most convincingly and efficiently performed with modern high-resolution microscopy. Epifluorescence microscopy of microbial autofluorescence or in conjunction with fluorescent dyes is among the most useful of these techniques. We explored fluorescent labeling and imaging of bacteria in rock and soil in the context of in situ life detection for planetary exploration. The goals were two-fold: to target non-Earth-centric biosignatures with the greatest possible sensitivity and to develop labeling procedures amenable to robotic implementation with technologies that are currently space qualified. A wide panel of commercially available dyes that target specific biosignature molecules was screened, and those with desirable properties (i.e., minimal binding to minerals, strong autofluorescence contrast, no need for wash steps) were identified. We also explored the potential of semiconductor quantum dots (QDs) as bacterial and space probes. A specific instrument for space implementation is suggested and discussed.

Foxl1 controls the Wnt/beta-catenin pathway by modulating the expression of proteoglycans in the gut.

Foxl1 is a winged helix transcription factor expressed in the mesenchyme of the gastrointestinal tract. Foxl1 null mice display severe structural defects in the epithelia of the stomach, duodenum, and jejunum. Here we addressed the molecular mechanisms by which Foxl1 controls gastrointestinal differentiation. First we showed that the abnormalities found in the epithelia of the null mice are the result of an increase in the number of proliferating cells and not a change in the rate of cell migration. Next we investigated the regulatory circuits affected by Foxl1. We focused on the Wnt/beta-catenin signaling pathway as a possible target of Foxl1 as it has been shown to play a central role in gastrointestinal proliferation. We demonstrated that Foxl1 activates the Wnt/beta-catenin pathway by increasing extracellular proteoglycans, which act as co-receptors for Wnt. Thus we establish that Foxl1 is involved in the regulation of the Wnt/beta-catenin pathway, providing a novel link in mesenchymal/epithelial cross-talk in the gut. Moreover, we provide the first example implicating proteoglycans in the regulation of cellular proliferation in the gastrointestinal tract.

Oxygen tension beneath piggyback contact lenses and clinical outcomes of users.

PURPOSE: Patients with keratoconus sometimes wear a rigid contact lens as a piggyback over a countersunk soft one. This geometry restricts movement of the rigid lens over the central cornea and may also reduce corneal oxygenation. This aim of this study was to estimate the equivalent oxygen percentage beneath such lenses and to review the clinical records of users to determine their success rate. METHODS: The right eyes of four normal subjects were covered for 5 minutes with a combination of rigid gas permeable (Boston ES or 7 [Polymer Technology Corp., Rochester, NY]) and soft (Acuvue [Johnson & Johnson, Jacksonville, FL], Keraflex [KF] 3 or 5 [Blanchard, Sherbrooke, QC, Canada]) contact lenses. After removal of the lenses, oxygen tension was measured with a Clark electrode. In order to determine whether measurements of oxygen tension were supported by clinical data, the clinical files of 11 patients fitted with KF 5-Boston ES piggyback lenses were reviewed for the presence of corneal edema. RESULTS: Mean relative equivalent oxygen percentage ranged from 6.9% to 11.2% beneath the following lens combinations: KF 3-Boston ES; KF 3-Boston 7; KF 5-Boston ES; KF 5-Boston 7; Acuvue-Boston ES; and Acuvue-Boston 7. Of the ten patients followed after dispensing, none had any sign of corneal edema. Nine had worn their contact lenses for periods up to 6 years. CONCLUSIONS: Based on the measured equivalent oxygen percentage and on the absence of corneal edema in patients, the combination of countersunk KF 5 with Boston ES lenses provides enough oxygen to fulfill corneal needs in oxygen during daily wear.

Human cell models to study small intestinal functions: recapitulation of the crypt-villus axis.

The intestinal epithelium is continuously and rapidly renewed by a process involving cell generation, migration, and differentiation, from the stem cell population located at the bottom of the crypt to the extrusion of the terminally differentiated cells at the tip of the villus. Because of the lack of normal human intestinal cell models, most of our knowledge about the regulation of human intestinal cell functions has been derived from studies conducted on cell cultures generated from experimental animals and human colon cancers. However, important advances have been achieved over recent years in the generation of normal human intestinal cell models. These models include (a) intestinal cell lines with typical crypt cell proliferative noncommitted characteristics, (b) conditionally immortalized intestinal cell lines that can be induced to differentiate, and (c) primary cultures of differentiated villuslike cells that can be maintained in culture for up to 10 days. Each of these models should help in the investigation of the specific aspects of human intestinal function and regulation. Furthermore, taken together, these models provide an integrated system that allows an in vitro recapitulation of the entire crypt-villus axis of the normal human small intestine.

A new primary culture system representative of the human gastric epithelium.

The gastric pit-gland unit is a highly dynamic and compartimentalized structure which assumes important key functions such as acid secretion, digestion of dietary proteins and triglycerides, protection, and epithelial restitution following injury. However, in vitro models representative of the intact gastric epithelium are still lacking. The current study was undertaken to investigate the possibility of generating such primary cultures from human fetal stomach. The use of Matrisperse, a nonenzymatic solution, allowed complete dissociation of the epithelial layer and the maintenance for at least 7 days of all gastric epithelial cell types in primary culture on plastic. Indirect immunofluorescence and Western blot analyses confirmed the purity of epithelial cultures, composed of 60% mucus-secreting cells, 25% zymogenic chief cells, 5% parietal cells, and a small proportion of mitotic precursors. Their functionality was demonstrated by the presence of zonulae occludens and adherens at cell to cell contacts, [(3)H]thymidine incorporation, Periodic acid Schiff staining, and expression of growth factor receptors (EGF/TGFalpha, IGF1, HGF, KGF), gastric H(+)/K(+)-ATPase, pepsinogen (Pg5), and human gastric lipase (HGL). Chief cells were able to produce and secrete both Pg5 and HGL and to respond to EGF treatment. In conclusion, we developed a new primary culture system of human gastric epithelium characterized for the first time by the absence of added matrix and the maintenance of functional chief cells. It represents an experimental breakthrough that will serve applications in investigating the actions of hormones, mesenchymal growth factors, and basement membrane proteins on human gastric functions in vitro.

Expression of functionally distinct variants of the beta(4)A integrin subunit in relation to the differentiation state in human intestinal cells.

Integrins are important mediators of cell-laminin interactions. In the small intestinal epithelium, which consists of spatially separated proliferative and differentiated cell populations located, respectively, in the crypt and on the villus, laminins and laminin-binding integrins are differentially expressed along the crypt-villus axis. One exception to this is the integrin alpha(6)beta(4), which is thought to be ubiquitously expressed by intestinal cells. However, in this study, a re-evaluation of the beta(4) subunit expression with different antibodies revealed that two forms of beta(4) exist in the human intestinal epithelium. Furthermore, we show that differentiated enterocytes express a full-length 205-kDa beta(4)A subunit, whereas undifferentiated crypt cells express a novel beta(4)A subunit that does not contain the COOH-terminal segment of the cytoplasmic domain (beta(4)A(ctd-)). This new form was not found to arise from alternative beta(4) mRNA splicing. Moreover, we found that these two beta(4)A forms can associate into alpha(6)beta(4)A complexes; however, the beta(4)A(ctd-) integrin expressed by the undifferentiated crypt cells is not functional for adhesion to laminin-5. Hence, these studies identify a novel alpha(6)beta(4)A(ctd-) integrin expressed in undifferentiated intestinal crypt cells that is functionally distinct.

Primary cultures of fully differentiated and pure human intestinal epithelial cells.

The epithelium of the small intestinal mucosa is a highly dynamic system particularly well suited for analyzing key biological phenomena such as cell differentiation and migration, cell-matrix interactions, and apoptosis. However, in vitro models of fully differentiated normal human enterocytes are still lacking. The objective of the present study was to investigate the possibility of generating such differentiated intestinal cell cultures from the fetal small intestine. For this purpose, various dissociation methods were tested in order to obtain pure, viable, and functional enterocytes. One of these methods, based on the procedure to recover epithelial cells grown on Matrigel and involving the use of Matrisperse, a nonenzymatic dissociating solution, was found to allow the isolation of the integral epithelial lining from the mesenchyme. In culture, these epithelial fractions plated on collagen I spread rapidly and reached confluence after 3-4 days. When tested after 5-7 days, these primary cultures of differentiated enterocytes (PCDE) remained well preserved. Both goblet and absorptive cells exhibit all the main characteristics of intact villus intestinal cells as assessed by electron microscopy. Indirect immunofluorescence and Western blot analyses confirmed the purity of PCDE. The functional status of these cells was demonstrated by the presence of uniformly distributed tight junction, zonula adherens, and desmosomal components at the region of cell to cell contact as well as expression of various brush border enzymes, namely sucrase-isomaltase and lactase, and goblet cell mucins. As expected, cell proliferation was found to be negligible as assessed by DNA synthesis. Taken together, these data show that primary cultures of pure and viable differentiated enterocytes can be generated from the human fetal small intestine.

Regulated expression of the integrin alpha9beta1 in the epithelium of the developing human gut and in intestinal cell lines: relation with cell proliferation.

The integrin alpha9beta1 is one of the recently identified integrins whose expression is restricted to specialized tissues. Its exact function is still unknown. In the present study, we have analyzed the expression of the alpha9 subunit in human fetal and adult small intestinal and colonic epithelia as well as in intestinal cell lines by indirect immunofluorescence, immunoprecipitation, Western blot, and Northern blot. In intact tissues, the antigen was restricted to the basolateral domain of epithelial cells in intestinal crypts at the fetal stage and was absent in the adult. The alpha9beta1 integrin was also detected in the intestinal cell lines HIEC-6 and Caco-2/15. The presence of alpha9beta1 in HIEC-6 was found to be consistent with their proliferative crypt-like status. In Caco-2/15 cells, the integrin was present at high levels in proliferating cells but was downregulated when cells cease to grow and undertake their differentiation. EGF treatment, which is known to maintain Caco-2/15 cells in a proliferative state, resulted in higher levels of alpha9 as compared to control cells. Taken together, these observations suggest a relation between integrin alpha9beta1 expression and proliferation in human intestinal cells.

Identification, distribution, and tissular origin of the alpha5(IV) and alpha6(IV) collagen chains in the developing human intestine.

The basement membrane type IV collagen is a family composed of six genetically distinct but structurally similar polypeptide chains, alpha1-alpha6. The alpha1(IV) and alpha2(IV) chains are ubiquitous components of all BMs whereas the other four have a restricted tissue distribution. In the present study, we have analyzed the expression, distribution, and cellular origin of the alpha5(IV) and alpha6(IV) chains in the developing and adult human small intestine and in well-characterized in vitro models by indirect immunofluorescence, Western blot, and RT-PCR. We have found that in the fetal small intestine, alpha(IV) and alpha6(IV) are present in the epithelial BM and, in contrast to alpha1(IV) and alpha2(IV), are produced by both epithelial and mesenchymal cells. A distinct tissular origin for the alpha1/alpha2(IV) and alpha5/alpha6(IV) chains suggests that alpha5(IV) and alpha6(IV) associate as a heterotrimer in this organ. We have also found that a particular situation of alpha5(IV)/alpha6(IV) chain expression occurs in the adult intestine. Indeed, as compared with the fetal intestine, alpha6(IV) chain production is maintained while the expression of the alpha5(IV) chain is substantially reduced. Altered expression of the alpha5(IV) chain was also observed in the differentiating enterocytic-like Caco-2/15 cells, suggesting that in the intestinal model, the alpha5(IV) chain is subject to a regulated expression. Taken together, these observations indicate that the human intestinal epithelial BM contains up to four type IV collagen chains: the classical alpha1(IV)/alpha2(IV) chains, which originate from mesenchymal cells, and the alpha5(IV)/alpha6(IV) chains, which are of both epithelial and mesenchymal origin and have their expression regulated throughout development.

Epithelial vs mesenchymal contribution to the extracellular matrix in the human intestine.

The basement membrane (BM) underlying the epithelium of the intestine is generally believed to be of both epithelial and mesenchymal origin but the exact contribution of each tissue has not been directly examined in the human. In this study, we have used a newly described procedure to dissociate the human intestine into pure epithelial and corresponding mesenchymal fractions. Northern blot and RT-PCR analyses of the fractions for the presence of transcripts encoding extracellular matrix molecules revealed that the epithelium produces the formal BM molecules such as the alpha 1, alpha 2, and beta 1 chains of laminin-1 and laminin-2 and the alpha 5(IV) and alpha 6(IV) chains of collagen as well as fibronectin, a BM-associated molecule. Interestingly, the alpha 1(IV) chain of collagen, which associates with the alpha 2(IV) chain to form the main BM collagen network, as well as tenascin-C and decorin, two BM-associated molecules, was found to be exclusively of mesenchymal origin. Taken together, these data support the concept that in the human, as in experimental animals, the intestinal BM is composed of components produced from both the epithelium and the mesenchyme.

Relation between integrin alpha7Bbeta1 expression in human intestinal cells and enterocytic differentiation.

BACKGROUND & AIMS: Cell-laminin interactions are principally mediated by specific membrane receptors of the integrin family. The integrin alpha7beta1 is one of them. Its expression in the intestine has not yet been investigated although it appears to be a key element in muscle cell differentiation. In this study, the expression of its three known isoforms has been analyzed in developing and adult small intestine and in intestinal cell lines. METHODS: The expression of the integrin alpha7beta1 was analyzed by indirect immunofluorescence, Western blotting, immunoprecipitation, and reverse-transcription polymerase chain reaction. RESULTS: The alpha7B isoform, but not the alpha7A and C isoforms, was detected in intestinal epithelial cells. In vivo, the presence of the alpha7B subunit was closely paralleled with (1) acquisition of differentiation characteristics during development and along the crypt-villus axis in the adult small intestine and (2) loss of enterocytic functions in the re-differentiated colonic epithelium. In vitro, the expression of alpha7B was also shown to correlate with the acquisition of enterocytic functions. In Caco-2 cells, the alpha7Bbeta1 integrin was found transiently up-regulated at the onset of sucrase-isomaltase expression. CONCLUSIONS: Taken together, these results suggest that alpha7Bbeta1 expression is correlated with human intestinal cell differentiation.

Use of the dissociating enzyme thermolysin to generate viable human normal intestinal epithelial cell cultures.

The regulation of intestinal cell proliferation, migration, and differentiation has been the subject of numerous studies. However, in human, progress in this field has been traditionally hampered by the lack of normal epithelial cell models. The aim of the present study was to define conditions in order to isolate, and more importantly to grow in a continuous manner, human small intestinal epithelial cells. A number of mechanical and/or enzymatic dissociation methods have been tested to isolate viable epithelial cells from the fetal small intestine. Cultured cells were characterized by indirect immunofluorescence and Western blot analysis. It was found that the use of thermolysin (50 microgram/ml, 2-3 h at 37 degrees C) can be advantageously applied to the isolation of viable epithelial cells free from contaminating fibroblasts when obtained from the 17- to 19-week fetal ileum. Furthermore, this procedure allowed the generation of continuously growing human intestinal epithelial cell cultures, which retain the ability to express specific cytokeratins as well as intestinal cell markers over a number of passages. This study shows that normal epithelial cell cultures can be relatively easily and reproducibly generated from the human fetal small intestine.

Uncoordinated, transient mosaic patterns of intestinal hydrolase expression in differentiating human enterocytes.

The heterogenous expression of brush border membrane hydrolases by the human enterocyte-like Caco-2 cell line during morphological and functional differentiation in vitro was investigated at the cellular level. Indirect immunofluorescence revealed that the heterogeneous ("mosaic") expression of sucrase-isomaltase, lactase, aminopeptidase N, and alkaline phosphatase was, in fact, transient in nature. The labeling indexes for each hydrolase gradually increased during culture at postconfluence in order to reach a maximum (> or = 90%) after 30 days, concomitant with an upregulation of their respective protein expression levels. In contrast, dipeptidylpeptidase IV labeling remained relatively constant. Backscattered electron imaging analysis in midstage (12 days postconfluence) monolayers demonstrated a lack of correlation between brush border membrane development and expression of each enzyme studied. Moreover, double immunostaining revealed that none of the other four hydrolases correlated directly with sucrase-isomaltase expression. Finally, immunodetection for the proliferation-associated antigen KI-67 revealed a transient mosaic pattern of proliferation which was inversely related to Caco-2 cell differentiation. These data indicate that enterocytic differentiation-related (as well as proliferation-related) gene expression in Caco-2 cells is regulated but uncoordinated at the cellular level, suggesting that an overall control mechanism is lacking.

Appearance and distribution of laminin A chain isoforms and integrin alpha 2, alpha 3, alpha 6, beta 1, and beta 4 subunits in the developing human small intestinal mucosa.

BACKGROUND: Laminin, a major component of basement membranes, is well known in its classical heterotrimeric form (B1-A-B2) to regulate diverse biological functions, including cell polarization and differentiation. However, the role of merosin, a laminin-like molecule in which an M chain is substituted for its homologous A chain, remains largely unknown. METHODS: In the present study, we analyzed by indirect immunofluorescence the expression and distribution of these four laminin chains as well as the integrins alpha 2 beta 1, alpha 3 beta 1, alpha 6 beta 1, and alpha 6 beta 4, four potential receptors, at the epithelial-mesenchymal interface of the developing human small intestine, with a panel of specific monoclonal antibodies. RESULTS: Beginning at 7 weeks of gestation and throughout mucosal organogenesis, the B1 and B2 chains were uniformly detected at the epithelial basement membrane. The A chain also was detected beginning at 7 weeks, and its distribution at the basement membrane remained uniform throughout villus (9+ weeks) and crypt (16+ weeks) formation. In contrast, M chain expression was not observed until 16 weeks; between 16 and 20 weeks, it was exclusively associated with the base of epithelial cells that comprised the forming crypts. Integrins alpha 6 beta 1 and alpha 6 beta 4, as determined by their subunit immunolocalization, appeared to be expressed by all enterocytes from 7 to 20 weeks. In contrast, the expression of the alpha 2 beta 1 and alpha 3 beta 1 integrins was found time- and site-restricted. The alpha 2 subunit was predominantly detected in the epithelial cells of the intervillous area and its derivative, the crypt, whereas the alpha 3 subunit was strongly expressed by all epithelial cells except those located at the bottom of 19-20-week-old crypts. CONCLUSIONS: Taken together, these observations demonstrate that both compositional changes in the basement membrane and differential expression of receptors occur during human intestinal organogenesis, suggesting that epithelial cell-matrix interactions play a role during development.

Expression of the alpha-5(IV) collagen chain in the fetal human small intestine.

BACKGROUND/AIMS: The basement membrane type IV collagen is a family composed of at least five genetically distinct but structurally similar polypeptide chains, alpha 1-alpha 5. The alpha 1(IV) and alpha 2(IV) chains are ubiquitous components of basement membranes, whereas the alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains have a restricted tissue distribution. The aim of this study was to analyze the presence of these minor type IV collagen chains in the small intestinal mucosa. METHODS: The expression of type IV collagen chains in the developing and adult human small intestine was determined by indirect immunofluorescence with monoclonal and polyclonal antibodies. Western blotting and Northern hybridization analysis were also used to additionally investigate the expression of the alpha 1(IV) and alpha 5(IV) chains. RESULTS: The alpha 3-alpha 5(IV) chains were absent from the adult epithelium, but, surprisingly, the alpha 5(IV) chain was consistently detected in the fetal mucosa. Its expression was confirmed by Western blotting, complementary DNA polymerase chain-reaction amplification, and Northern hybridization analysis. CONCLUSIONS: The alpha 5(IV) chain of collagen is expressed in the fetal but not adult human intestinal epithelium. Its position at the basolateral domain of epithelial cells suggests a potential role for this molecule during development.

Relationship between tenascin and alpha-smooth muscle actin expression in the developing human small intestinal mucosa.

The expression of tenascin (Tn) and alpha-smooth muscle actin (alpha-SMA) was analyzed in the developing and adult human small intestine by means of double immunofluorescent staining with specific antibodies. By 7 weeks of gestation, the gut anlage has a simple tubular shape and is formed of a stratified undifferentiated epithelium surrounded by a poorly organized mesenchyme. Both Tn and alpha-SMA were found exclusively at the periphery of the tissue, corresponding to the presumptive muscularis propria. By 9 weeks, villus rudiments had formed but Tn and alpha-SMA remained restricted to the muscularis propria. Tn was first detected in the mesenchyme at 11 weeks. By 13 weeks, a preferential distribution of Tn in the subepithelial region of the mesenchyme was readily observed while alpha-SMA was still absent. From this stage to 20 weeks, Tn gradually concentrated in this region that, as determined by alpha-SMA detection, corresponded to the future muscularis mucosa area. As shown by double staining of Tn and alpha-SMA, deposition of Tn also preceded the appearance of the other alpha-SMA-expressing cells in the mucosa. These observations suggest that Tn could have a role in the differentiation of intestinal contractile cells.

Expression of tenascin in the developing human small intestine.

The expression of tenascin (Tn), an extracellular matrix glycoprotein, was analyzed in the human small intestine. Three major oligomeric forms of intestinal Tn (320 kDa, 220 kDa and 200 kDa) were identified in both the fetus and the adult. The intestinal 200 kDa form was not found expressed by intestinal mesenchymal cells in culture. During development, the amounts of the 320 and 220 kDa forms remained relatively constant between 9-20 weeks of gestation as compared to sucrase-isomaltase and a-smooth muscle actin while the 200 kDa immunoreactive component increased substantially during this period of active intestinal morphogenesis. Further experiments by microdissection revealed that the 200 kDa component was the major form of Tn in the intestinal villus. These results suggest that this newly identified 200 kDa form of Tn in the small intestine is predominantly expressed in the villus under a developmentally regulated pattern.