Entire Villus Research Articles

P2.29: IGL-1 solution for the preservation of human intestine: the first report in the literature

Introduction: IGL-1 is a newer preservation solution largely resembling University Wisconsin (UW) solution in terms of ingredients but where the viscous starch has been replaced by polyethylene glycol resulting in lower viscosity. In addition, IGL-1 has low potassium and high sodium concentrations (extracellular-type solution). The solution has been used in several European countries for the preservation of kidneys, pancreases and livers with efficacy and safety comparable to those of UW or Custodiol. However, a systematic appraisal of the intestinal preservation injury using ILG-1 is missing. Methods: Following aortic retrograde perfusion with 4 liters IGL-1, the ileum of fifteen deceased, brain dead multiorgan donors was retrieved and stored at 4C. Samples were obtained after 8h, 14h and 24 hours of cold storage. Histology (Chiu/Park scale), Goblet cells (GC) count, apoptosis (active caspase 3) and tight junction proteins claudin 3 and Zonula occludens (ZO)-1 were studied. Results: Eight hours of cold storage resulted in a moderate epithelial detachment form lamina propria (median Chiu grade 2 (range 1–3) and minimal GC depletion. Preservation injury progressed to a median grade of 3 (range 2–5) after 14 hours and remained at similar levels at 24 hours. Active Caspase-3 was very low or absent at all time-points. After 8h ZO-1 expression was well preserved along the entire villus but became discontinuous after 14 hours of cold storage. Claudin 3 staining pattern remained well preserved throughout the entire cold storage. Conclusions: The development of the preservation injury in the human intestine following perfusion and storage in IGL-1 appears similar with the other solutions. Interestingly, the structural preservation injury did not seem to continue to worsen after 14 hours while the injury at subcellular level advanced further.

MT1-MMP and its potential role in the vertebrate intestinal morphogenesis

Membrane type 1-matrix metalloproteinase (MT1-MMP) is involved in numerous biological processes, including morphogenesis. However, the role of MT1-MMP in the development of the vertebrate intestine is poorly understood. This study aimed to evaluate the expression of MT1-MMP in the intestine of rats and chickens along the embryonic and postnatal periods using immunohistochemistry. Results revealed a remarkable spatiotemporal correlation between MT1-MMP expression and intestinal villi morphogenesis in both vertebrates. However, the villi morphogenesis process was found to be different in chickens to that of rats. Moreover, extensive MT1-MMP labeling was observed in the entire villus epithelium from birth until the complete maturation of the small intestinal mucosa in both vertebrates. From these results, we suggest that MT1-MMP contributes to intestinal development, particularly to villi morphogenesis, in both vertebrates. However, further studies are necessary to confirm the role of MT1-MMP in this cellular process. In addition, we performed validation of the primary antibody against human MT1-MMP for adult chickens.

High-resolution 3D analysis of mouse small-intestinal stroma.

Here we detail a protocol for whole-mount immunostaining of mouse small-intestinal villi that can be used to generate high-resolution 3D images of all gut cell types, including blood and lymphatic vessel cells, neurons, smooth muscle cells, fibroblasts and immune cells. The procedure describes perfusion, fixation, dissection, immunostaining, mounting, clearing, confocal imaging and quantification, using intestinal vasculature as an example. As intestinal epithelial cells prevent visualization with some antibodies, we also provide an optional protocol to remove these cells before fixation. In contrast to alternative current techniques, our protocol enables the entire villus to be visualized with increased spatial resolution of cell location, morphology and cell-cell interactions, thus allowing for easy quantification of phenotypes. The technique, which takes 7 d from mouse dissection to microscopic examination, will be useful for researchers who are interested in most aspects of intestinal biology, including mucosal immunology, infection, nutrition, cancer biology and intestinal microbiota.

Molecular support for high paracellular absorption in the intestine of flying mammals (1109.18)

Flying mammals absorb water‐soluble nutrients (e.g. glucose) at the intestine mainly by the paracellular route [across tight junctions (TJ) between adjacent enterocytes], while non‐flying mammals mainly rely on the transcellular route (across cell membrane using transporter proteins). We predicted that insectivorous bats Tadarida brasiliensis, compared to Mus musculus, would show enhanced expression of CLDN‐2 (CLDN=claudin) and OCLN (occludin) TJ proteins, which have been described as responsible for the permeability of polar water‐soluble solutes. We assessed the level and spatial distribution of CLDN‐2, ‐3, ‐5, ZO‐1 and OCLN by immunohistochemistry along the villus axis in the proximal intestine. Immuno‐specificity of the antibodies was checked by western blot. No differences were found for CLDN‐3 and ZO‐1 between species. We confirmed low expression of CLDN‐2 in mice (Holmes et al. 2006, Gene Expr Patterns 6: 581‐8; no signal detected in our preparations), but in bats the villus tips exhibited CLDN‐2. OCLN was expressed along the entire villus axis but with a stronger signal for the bat than for mice, giving additional support to our prediction. A third difference was that CLDN‐5 was strongly expressed along the villus in bats, whereas in mice its presence was not detected. Findings suggest potential molecular mechanism(s) underlying the observed higher paracellular absorption in bats compared to mice.Grant Funding Source: NSF IOS 1025886

Developmental Profile of Claudin‐3, ‐5, and ‐16 Proteins in the Epithelium of Chick Intestine

Proteins in the claudin family are a main component of tight junctions and form a seal that modulates paracellular transport in intestinal epithelium. This research tests the hypothesis that claudins 3, 5, and 16 will appear in the epithelium of embryonic intestine during functional differentiation. Immunohistochemistry is utilized to explore the developmental patterns of claudin-3, -5, and -16 proteins in the epithelium of embryonic chick intestine from 9 days prior to hatching through the early post-hatch period. These claudin proteins either changed their cellular localization or first appeared around the time of hatching. After hatching, claudin-3 expression was prominent in basal-lateral regions of the epithelium along the entire villus, but was absent from crypts. Claudin-5 was expressed most strongly in the crypt and lower villus epithelium within junctional complexes, whereas immunostaining of claudin-16 was localized within goblet cells of the upper villus region. The relative mRNA levels of claudin-3, -5, and -16 showed similar patterns; transcript levels rose between 18 and 20 days of development, then dropped by 2 days post-hatch. Results of this work indicate that the claudin proteins assume their final locations within the epithelium around the time of hatching, suggesting that in addition to their known barrier and fence functions within tight junctions, these claudins may have additional roles in the differentiation and/or physiological function of chick intestine. The localization of claudin-16 to goblet cells and its distribution in the more mature cells of the upper villus region suggest an unexpected role in goblet cell maturation and mucus secretion.

Tissue-Specific Alterations in Zinc Transporter Expression in Intestine and Liver Reflect a Threshold for Homeostatic Compensation during Dietary Zinc Deficiency in Weanling Rats

Intestinal zinc (Zn) absorption and liver Zn mobilization are presumed to regulate Zn homeostasis. Several Zn transporters have been identified; however, their contribution to Zn homeostasis is poorly understood. Moreover, their regulation during periods of growth is unknown. To characterize the mechanisms that maintain Zn status, weanling rats were fed control (25 mg/kg), marginally low (MLZ; 15 mg/kg), low (LZ; 7 mg/kg), or very low (VLZ; <1 mg/kg) Zn diets for 3 wk and effects on jejunum Zip4 and ZnT1 and hepatic Zip1 and ZnT1 were assessed. Another control group was pair-fed (PF) to VLZ. The MLZ rats had lower jejunum ZnT1 protein abundance than the control. In the LZ group, we detected increased jejunum Zip4 mRNA expression and hepatic ZnT1 protein abundance and reduced jejunum Zip4 and ZnT1 and hepatic Zip1 protein abundance. VLZ had lower jejunum ZnT1 mRNA and protein abundance and hepatic Zip1 and ZnT1 protein abundance compared with the PF group. Zip4 protein was present at the intestinal villus tip in controls but was detected on the apical membrane throughout the entire villus in LZ rats. ZnT5 protein in jejunum was always detected at the apical membrane and also at the basolateral membrane of VLZ rats. In contrast, ZnT7 was found intracellularly in jejunum. Our data suggest that effects of Zn deficiency on Zn homeostasis occurs biphasically during marginal Zn deficiency through increased intestinal Zn uptake capacity and reduced intestinal Zn efflux, then during more pronounced degrees of Zn deficiency through decreased liver Zn accretion and increased hepatic Zn efflux back into circulation. These results assist in our understanding of how mammals regulate Zn homeostasis.

Noncytotoxic Clostridium perfringens Enterotoxin (CPE) Variants Localize CPE Intestinal Binding and Demonstrate a Relationship between CPE-Induced Cytotoxicity and Enterotoxicity

Clostridium perfringens enterotoxin (CPE) causes the symptoms of a very common food poisoning. To assess whether CPE-induced cytotoxicity is necessary for enterotoxicity, a rabbit ileal loop model was used to compare the in vivo effects of native CPE or recombinant CPE (rCPE), both of which are cytotoxic, with those of the noncytotoxic rCPE variants rCPE D48A and rCPE(168-319). Both CPE and rCPE elicited significant fluid accumulation in rabbit ileal loops, along with severe mucosal damage that starts at villus tips and then progressively affects the entire villus, including necrosis of epithelium and lamina propria, villus blunting and fusion, and transmural edema and hemorrhage. Similar treatment of ileal loops with either of the noncytotoxic rCPE variants produced no visible histologic damage or fluid transport changes. Immunohistochemistry revealed strong CPE or rCPE(168-319) binding to villus tips, which correlated with the abundant presence of claudin-4, a known CPE receptor, in this villus region. These results support (i) cytotoxicity being necessary for CPE-induced enterotoxicity, (ii) the CPE sensitivity of villus tips being at least partially attributable to the abundant presence of receptors in this villus region, and (iii) claudin-4 being an important intestinal receptor for CPE. Finally, rCPE(168-319) was able to partially inhibit CPE-induced histologic damage, suggesting that noncytotoxic rCPE variants might be useful for protecting against some intestinal effects of CPE.

Arginine Activates Intestinal p70S6k and Protein Synthesis in Piglet Rotavirus Enteritis

We previously showed that phosphorylation of p70 S6 kinase (p70S6k) in the intestine is increased during viral enteritis. In this study, we hypothesized that during rotavirus infection, oral Arg, which stimulates p70S6k activation, will further stimulate intestinal protein synthesis and mucosal recovery, whereas the p70S6k inhibitor rapamycin (Rapa) will inhibit mucosal recovery. Newborn piglets were fed a standard milk replacer diet supplemented with Arg (0.4 g · kg−1 · d−1, twice daily by gavage), Rapa (2 mg · m−2 · d−1), Arg + Rapa, or saline (controls). They were infected on d 6 of life with porcine rotavirus. Three days postinoculation, we measured the piglets’ body weight, fecal rotavirus excretion, villus-crypt morphology, epithelial electrical resistance in Ussing chambers, and p70S6k activation by Western blotting and immunohistochemistry. We previously showed a 2-fold increase in jejunal protein synthesis during rotavirus diarrhea. In this experiment, Arg stimulated jejunal protein synthesis 1.3-fold above standard medium, and the Arg stimulation was partially inhibited by Rapa. Small bowel stimulation of p70S6k phosphorylation and p70S6k levels were inhibited >80% by Rapa. Immunohistochemistry revealed a major increase of p70S6k and ribosomal protein S6 phosphorylation in the crypt and lower villus of the infected piglets. However, in Arg-treated piglets, p70S6k activation occurred over the entire villus. Jejunal villi of the Rapa-treated group showed inactivation of p70S6k and a decrease in mucosal resistance (reflecting increased permeability), the latter of which was reversed by Arg. We conclude that, early in rotavirus enteritis, Arg has no impact on diarrhea but augments intestinal protein synthesis in part by p70S6k stimulation, while improving intestinal permeability via a mammalian target of rapamycin/p70S6k-independent mechanism.

MTOR SIGNALING IS A COMPONENT OF INTESTINAL REPAIR IN PIGLET ROTAVIRUS ENTERITIS

Recent identification of the mTOR pathway as a key regulator of amino acid sensing, mRNA translation, and protein synthesis led us to investigate its role in viral diarrhea. We hypothesized that during rotavirus enteritis, malnutrition would reduce jejunal protein synthetic rate and mTOR signaling via its immediate downstream target, ribosomal p70 s6 kinase (p70s6k). Methods: Newborn Yorkshire piglets were artificially fed from birth and half were infected with porcine rotavirus on d5 of life. Study groups included controls, infected, infected/50% protein-calorie malnourished, and infected and treated with arginine (ARG, 0.3 g/kg/d) and/or rapamycin (1 mg/m2). Results: At the peak of diarrhea (on days 3 and 5 post-inoculation), western blotting revealed four-fold induction of p70s6k phosphorylation and p70s6k level (p < 0.05) compared with uninfected tissue. In vitro jejunal protein synthetic rate increased 2-fold (p < 0.05) during infection. Malnutrition did not alter the level of p70s6k activation or the increase in gut permeability caused by infection. Immunolocalization revealed that infection produced a major induction of cytoplasmic p70s6k and of nuclear phospho-p70s6k in the crypt and lower villus. In vitro jejunal protein synthesis and p70s6k phosphorylation increased 3-fold in response to L-arginine. In vivo, rapamycin treatment of infected piglets increased gut permeability 2.5-fold, while ARG reversed the rapamycin effect, did not impact the diarrhea, and enhanced staining of phosphorylated p70s6k and ribosomal protein S6 over the entire villus epithelium. Conclusions: Rotavirus infection resulted in increased jejunal protein synthesis and activation of mTOR in the crypt. Intestinal activity of mTOR/p70s6k was not influenced by malnutrition, but was enhanced by ARG.

Ontogenetic expression and regulation of Na(+)-D-glucose cotransporter in jejunum of domestic chicken.

To evaluate the effect of age on sugar transport, we determined the uptake of methyl alpha-D-glucopyranoside and the abundance of the Na(+)-D-glucose cotransporter (SGLT1) in jejunal brush-border membrane (BBM) vesicles of 2-day- and 5-wk-old chickens. Methyl alpha-D-glucopyranoside transport per BBM protein was 40% lower in adults than in newly hatched chickens. This finding was matched by parallel declines in site density of SGLT1, which were detected by Western blot. The immunohistochemical study showed that SGLT1 was exclusively located in the BBM of enterocytes along the entire villus and was absent in the crypt in both age groups, and there was an 11-fold increase in the total absorptive area during development. Northern blot studies of the abundance of SGLT1 mRNA showed similar levels for the groups studied. We conclude that the age-related decline in Na(+)-dependent hexose transport per unit of BBM protein in the chicken jejunum is due to a reduction in the density of SGLT1 cotransporter and is regulated by a posttranscriptional mechanism.

Intestinal perfusion induces rapid activation of immediate-early genes in weaning rats.

C-fos and c-jun are immediate-early genes (IEGs) that are rapidly expressed after a variety of stimuli. Products of these genes subsequently bind to DNA regulatory elements of target genes to modulate their transcription. In rat small intestine, IEG mRNA expression increases dramatically after refeeding following a 48-h fast. We used an in vivo intestinal perfusion model to test the hypothesis that metabolism of absorbed nutrients stimulates the expression of IEGs. Compared with those of unperfused intestines, IEG mRNA levels increased up to 11 times after intestinal perfusion for 0.3-4 h with Ringer solutions containing high (100 mM) fructose (HF), glucose (HG), or mannitol (HM). Abundance of mRNA returned to preperfusion levels after 8 h. Levels of c-fos and c-jun mRNA and proteins were modest and evenly distributed among enterocytes lining the villi of unperfused intestines. HF and HM perfusion markedly enhanced IEG mRNA expression along the entire villus axis. The perfusion-induced increase in IEG expression was inhibited by actinomycin-D. Luminal perfusion induces transient but dramatic increases in c-fos and c-jun expression in villus enterocytes. Induction does not require metabolizable or absorbable nutrients but may involve de novo gene transcription in cells along the villus.

Morphological and functional abnormalities in the ileum of rats with spontaneous hypertension: studies on SGLT1 protein.

Na+-dependent D-glucose and D-galactose transport was studied in the ileal brush-border membrane vesicles from both spontaneously hypertensive rats (SHR) and their normotensive genetic control Wistar-Kyoto (WKY) rats. Initial rates and accumulation ratios of the transport of both monosaccharides were significantly lower in the hypertensive rats compared to the WKY rats. In order to determine whether such modifications are related to morphological abnormalities, ileal epithelium of SHR and WKY rats was examined by light and electron microscopy. In addition, immunohistochemical and immunocytochemical localization of Na+-glucose cotransporter (SGLT1) was performed. Light microscopy studies showed hypertrophy in the ileal villi of hypertensive rats, with an increase in the villus width when compared to those from normotensive rats. Immunohistochemical studies of SGLT1 showed the protein localized in the apical membrane of the absorptive epithelial cells, along the entire villus. No changes between SHR and WKY rats were noted in the intensity and distribution of the SGLT1 protein along the villus-crypt axis. Electron microscopy studies showed a patchy loss of microvilli in the ileal enterocytes of SHR, compared to those from WKY rats. Immunocytochemical studies of SGLT1 were carried out by the immunogold method. Colloidal gold particles were localized at the ileal microvilli of normotensive rats. No significant presence of SGLT1 was found in the smooth apical surface of ileum from hypertensive rats, although most adjacent microvilli were marked. Morphological changes were accompanied by modifications in the sugar transport and in the immunolocalization of SGLT1 in the ileal epithelium of SHR.

LI-cadherin gene expression during mouse intestinal development.

LI-cadherin (Liver-Intestine cadherin) is a member of a subclass (7-D cadherins) within the cadherin superfamily. Although its cellular function as a cell-cell adhesion molecule has been demonstrated in cell culture studies, its physiological function still needs to be explored in the intact organism. After isolating the cDNA for mouse LI-cadherin, we generated specific antibodies against the overexpressed protein and studied its expression pattern in adult mouse tissues and mouse embryos. The mouse LI-cadherin sequence is 91% identical to the sequence of rat LI-cadherin and exhibits the same structural features described for rat LI-cadherin. In mouse adult tissue, LI-cadherin is expressed in the intestine and in small amounts in the spleen. In contrast to rat, Mouse LI-cadherin was not expressed in liver. During mouse embryogenesis, LI-cadherin expression begins at embryonic day 12.5. With the exception of transient expression in the urogenital sinus and the common bile duct on day 13.5, LI-cadherin was found exclusively in the intestinal epithelium. Its expression coincides with the formation of intestinal villi, a developmental stage that includes major tissue remodeling, growth, and differentiation. LI-cadherin is expressed along the entire anterior-posterior axis of the developing intestine as well as along the entire villus axis once villi begin to form. LI-cadherin occupies all cell surfaces of the deeper layers of the epithelium, distributing to basolateral surfaces only in the cells of the outer epithelial layer. LI-cadherin was found to be always co-expressed with E-cadherin.

Diurnal rhythmicity in intestinal SGLT-1 function,Vmax, and mRNA expression topography

Mechanisms underlying the circadian rhythmicity in intestinal sugar absorption remain unclear. To test whether this rhythmicity is caused by changes in Na(+)-glucose cotransporter 1 (SGLT-1) function, we measured phloridzin-inhibitable sugar fluxes as an index of SGLT-1 activity. Jejunum obtained from rats killed at 6-h intervals during a 12-h light-dark cycle (CT0 is circadian time 0 h, time of light onset) were mounted in Ussing chambers, and 3-O-methylglucose (3-OMG) fluxes were calculated before and after addition of phloridzin. 3-OMG-induced change in short-circuit current and absorptive flux were significantly greater at CT9 than at CT3. This increase was phloridzin inhibitable. Kinetic studies indicated a significant increase in SGLT-1 maximal velocity (V(max)) at CT9. Food intake between CT3 and CT9 was <10% of the daily total, indicating that the increased SGLT-1 activity was anticipatory. Diurnicity of SGLT-1 mRNA was confirmed by Northern blotting. Expression topography analyzed by in situ hybridization revealed more intense labeling along the entire villus axis at CT9 and CT15 compared with CT3 and CT21. We conclude that diurnicity in intestinal sugar absorption is caused by periodicity in SGLT-1 V(max).

Morphological and Functional Abnormalities in the Ileum of Rats with Spontaneous Hypertension: Studies on SGLT1 Protein

Background: Na+-dependent D-glucose and D-galactose transport was studied in the ileal brush-border membrane vesicles from both spontaneously hypertensive rats (SHR) and their normotensive genetic control Wistar-Kyoto (WKY) rats. Initial rates and accumulation ratios of the transport of both monosaccharides were significantly lower in the hypertensive rats compared to the WKY rats. Methods: In order to determine whether such modifications are related to morphological abnormalities, ileal epithelium of SHR and WKY rats was examined by light and electron microscopy. In addition, immunohistochemical and immunocytochemical localization of Na+-glucose cotransporter (SGLT1) was performed. Results: Light microscopy studies showed hypertrophy in the ileal villi of hypertensive rats, with an increase in the villus width when compared to those from normotensive rats. Immunohistochemical studies of SGLT1 showed the protein localized in the apical membrane of the absorptive epithelial cells, along the entire villus. No changes between SHR and WKY rats were noted in the intensity and distribution of the SGLT1 protein along the villus-crypt axis. Electron microscopy studies showed a patchy loss of microvilli in the ileal enterocytes of SHR, compared to those from WKY rats. Immunocytochemical studies of SGLT1 were carried out by the immunogold method. Colloidal gold particles were localized at the ileal microvilli of normotensive rats. No significant presence of SGLT1 was found in the smooth apical surface of ileum from hypertensive rats, although most adjacent microvilli were marked. Conclusion: Morphological changes were accompanied by modifications in the sugar transport and in the immunolocalization of SGLT1 in the ileal epithelium of SHR.

Lactase-Phlorizin Hydrolase and Sucrase-Isomaltase Genes Are Expressed Differently Along the Villus-Crypt Axis of Rat Jejunum

Lactase-phlorizin hydrolase (LPH) and sucrase-isomaltase (SI) are two disaccharidases specifically expressed in small intestinal absorptive cells. We previously showed that the transcripts of both genes are elevated within 12 h of carbohydrate intake. To examine at which locus of villus-crypt axis this response to dietary carbohydrate occurs, 6-wk-old rats were fed a low-carbohydrate diet (5% energy) for 7 d, and then force-fed either the low-carbohydrate diet or a sucrose (40% energy) diet during the last 6 h. Cryostat sectioning of jejunal segments followed by RNA blot hybridizations of the transcripts revealed that, unlike SI mRNA which was expressed maximally in the lower villus, maximal LPH mRNA level was attained at the upper villus. The distribution of the respective immunoreactive protein and the enzymatic activity was shifted more toward the villus tips for LPH than for SI. Force-feeding the sucrose diet caused an abrupt increase in SI mRNA level in the lower villus within 3 h, while the rise in LPH mRNA level occurred in the mid- and upper-villus. The diet-induced increases in the LPH mRNA and SI mRNA levels were abolished along the entire villus by the administration of actinomycin D. These results suggest that LPH gene is maximally expressed in more apical villus cells than SI gene, and that dietary sucrose elicits enhancement of the gene expressions in the villus cells which are accumulating the respective transcripts.

Rat intestinal alkaline phosphatase II messenger RNA is present in duodenal crypt and villus cells

BACKGROUND & AIMS: Rats produce two messenger RNA (mRNA) isoforms encoding intestinal alkaline phosphatase that are regulated differentially by fat feeding but whose tissue distribution is not known. The aim of this study was to examine the distribution of these isoforms along the crypt/villus axis during fat feeding.METHODS: Nucleotide probes and polyclonal antibodies unique for each isoform were used for in situ hybridization and immunocytochemistry.RESULTS: mRNAs encoding isoforms I and II were distributed along the entire villus. The mRNA content of isoform II was more abundant. Quantitative grain counts showed that mRNA abundance of each isoform in the upper third of the villus was significantly (40%-65%) lower than in the remaining villus in the fasting duodenum. After fat feeding, the mRNA content of both isoforms increased over the villus. Five hours after feeding, the content of the mRNA encoding only isoform II was increased in the upper third of the villus relative to the rest of the villus. Only the mRNA encoding isoform II was found in the crypt cells. However, no immunoreactive enzyme was present in the crypt cells.CONCLUSIONS: The spatial expression of the two rat intestinal alkaline phosphatase isoforms is regulated differently in response to fat feeding.(Gastroenterology 1997 Feb;112(2):376-86)

Lactase gene expression during early development of rat small intestine

Expression of lactase messenger (m) RNA and protein in rat small intestine during fetal and postnatal development was analyzed using in situ hybridization and immunohistochemistry. Lactase mRNA was first identified at 18 days of development, and lactase protein was first detected at day 20. Lactase mRNA and protein were present along the entire villus. Lactase mRNA increased, reaching a maximum at day 20. Just before birth a decrease in lactase mRNA was observed. In newborn intestine, lactase mRNA was present only from the base of the villus up to the mid-villus region and was undetectable up to the villus tips. Lactase protein continued to be expressed along the entire villus. These data show that expression of lactase mRNA and protein do not parallel, indicating a posttranscriptional control in fetal development. Lactase gene transcription is initiated late in gestation concomitant with villus formation and is exclusively seen in villus epithelial cells. The restriction after birth of lactase mRNA expression to cells at the villus base suggests the occurrence of a previously unknown step in postnatal differentiation of the enterocyte.

Regional characteristics of intestinal iron absorption in the guinea-pig.

Features of iron absorption by the upper small intestine and ileum in the adult guinea-pig have been compared. Autoradiographic methods have revealed that whilst only upper villus enterocytes in the duodenum-jejunum are involved in brush-border uptake, the entire villus epithelium in the ileum displays this function. Consistent with these results was the finding that brush-border membrane vesicles prepared from ileal mucosa demonstrated a higher capacity for iron uptake (expressed per mg protein) compared to vesicles of duodenal origin. Iron transport to blood following 30 min exposure of mucosa to 59Fe-ascorbate was some elevenfold higher in upper, compared to lower, small intestine. The relevance of these findings to our knowledge of the cellular processes involved in intestinal iron absorption and its regional localization is discussed.

Effect of lactation on the decline of brush border lactase activity in neonatal pigs.

It has been shown that during the early phase of lactation porcine milk contains high concentrations of hormones and growth factors. The aim of the present investigation was to examine the hypothesis that the temporal coordination of intestinal maturation in piglets can be extrinsically regulated through changes in the composition of milk during the suckling period. Gut morphology and the ontogeny of brush border lactase activity were investigated in piglets reared on two suckling regimens designed to expose the animals to compositionally distinct milk. The first group of animals were cross-fostered onto postcolostrum sows and thereafter suckled normally for up to eight weeks. These normally suckled (N) animals consequently received both early and late lactation products. The second group of piglets were cross-fostered each week, for up to eight weeks, onto newly farrowed sows which were postcolostrum. As a result of this repeated cross-fostering (CF) these animals received only early lactation products. Animals were sacrificed at one, three, five, seven, and eight weeks postpartum. Biochemically active lactase decreased significantly (p less than 0.001) in both groups over eight weeks, but the rate of loss of activity was greater in the CF animals than in the N pigs by approximately 50% at week 3 and 25% at week 8. Quantitative histochemical analysis of lactase activity corroborated the biochemical data. At three weeks maximal enzyme activity was observed approximately 400 microns from the villus/crypt junction. Histochemically detected lactase decreased throughout the suckling period, but the intensity of reaction product was consistently weaker over the entire villus surface in the CF animals. Immunocytochemically detectable lactase was identified at the same sites as the histochemical reaction products. In addition, immunofluorescence microscopy showed the presence of histochemically undetectable enzyme on the basolateral and brush border membranes of both villus and crypt cells. Villus/crypt ratios were significantly lower (p<0.001) in the CF animals than in the N pigs between weeks 3 and 5. The results of this study suggest that lactation products can accelerate the loss of brush border lactase activity. The observed decline in biochemically and histochemically detected lactase was considered to be a consequence of reduced enterocyte lifespan, decreased synthesis of enzyme protein, or altered post-translational modification of enzyme protein, or a combination of there.