Loss of intestinal nuclei and intestinal integrity in aging C. elegans

The roundworm C. elegans is widely used as an aging model, with hundreds of genes identified that modulate aging (Kaeberlein et al., 2002. Mech. Ageing Dev. 123 , 1115–1119). The development and bodyplan of the 959 cells comprising the adult have been well described and established for more than 25 years ( Sulston & Horvitz, 1977 . Dev. Biol. 56 , 110–156; Sulston et al., 1983. Dev. Biol. 100 , 64–119.). However, morphological changes with age in this optically transparent animal are less well understood, with only a handful of studies investigating the pathobiology of aging. Age‐related changes in muscle ( Herndon et al., 2002 . Nature 419 , 808–814), neurons ( Herndon et al., 2002 ), intestine and yolk granules ( Garigan et al., 2002 . Genetics 161 , 1101–1112; Herndon et al., 2002 ), nuclear architecture ( Haithcock et al., 2005 . Proc. Natl Acad. Sci. USA 102 , 16690–16695), tail nuclei ( Golden et al., 2007 . Aging Cell 6 , 179–188), and the germline ( Golden et al., 2007 ) have been observed via a variety of traditional relatively low‐throughput methods. We report here a number of novel approaches to study the pathobiology of aging C. elegans. We combined histological staining of serial‐sectioned tissues, transmission electron microscopy, and confocal microscopy with 3D volumetric reconstructions and characterized age‐related morphological changes in multiple wild‐type individuals at different ages. This enabled us to identify several novel pathologies with age in the C. elegans intestine, including the loss of critical nuclei, the degradation of intestinal microvilli, changes in the size, shape, and cytoplasmic contents of the intestine, and altered morphologies caused by ingested bacteria. The three‐dimensional models we have created of tissues and cellular components from multiple individuals of different ages represent a unique resource to demonstrate global heterogeneity of a multicellular organism.     

Apoptotic process of porcine intestinal M cells

Membranous (M) cells of the follicle-associated epithelium (FAE) are believed to sample antigens from the gut lumen. However, the origin, differentiation mechanism, and cell death of M cells are still a matter of controversy. Therefore, we investigated the process of M cell differentiation and determined their fate in the intestine of three-way crossbred female pigs. We used anti-cytokeratin 18 and anti-PCNA antibodies to distinguish M cells and proliferative cells and performed immunohistochemistry, enzyme histochemistry, and scanning electron microscopy on fresh ileal Peyer’s patches. Cell migration and apoptotic cells were detected by BrdU labeling and the TUNEL method, respectively. The turnover of the FAE was similar to that of the villi. M cells were mostly observed from the FAE crypt to the FAE periphery, but not in the FAE apex. As proliferative M cells (cytokeratin 18⁺/PCNA⁺ cells) have previously been detected in the FAE crypt, porcine M cells may be directly derived from intestinal epithelial stem cells and committed as a distinct cell lineage in the crypts. M cells from the FAE periphery were unstained or only weakly stained for alkaline phosphatase, whereas cytokeratin 18⁺/alkaline phosphatase⁺ cells lying near to the FAE apex showed a columnar shape similar to that of adjacent enterocytes. These data suggest that the committed M cells differentiate to mature M cells by contact with lymphocytes at the FAE periphery, and that they trans-differentiate to enterocytes and are finally excluded near the FAE apex. This investigation was supported by a Grant-in-Aid for Scientific Research (16658107) from the Ministry of Education, Culture, Sports, Science and Technology, by two grants (Prion Project and Secure and Healthy Livestock Farming Project) from the Ministry of Agriculture, Forestry and Fisheries, and by a grant from the Naito Foundation.     

Decreased expression of the gut-enriched Krüppel-like factor gene in intestinal adenomas of multiple intestinal neoplasia mice and in colonic adenomas of familial adenomatous polyposis patients

Onconase((R)) (ONC) is a homolog of ribonuclease A (RNase A) that has unusually high conformational stability and is toxic to human cancer cells in vitro and in vivo. ONC and its amphibian homologs have a C-terminal disulfide bond, which is absent in RNase A. Replacing this cystine with a pair of alanine residues greatly decreases the conformational stability of ONC. In addition, the C87A/C104A variant is 10-fold less toxic to human leukemia cells. These data indicate that the synapomorphic disulfide bond of ONC is an important determinant of its cytotoxicity.     

The application of the “Lojda” method for intestinal lactase in intestinal biopsies from jaundiced newborn infants

Summary A new use of the histochemical method for intestinal lactase activity is described. Peroral intestinal biopsies from newborn light-treated infants with diarrhoea were investigated for brush-border lactase. The lactase activity found in these infants by the histochemical method correlated well with the infants ability to hydrolyze lactose judged by lactose tolerance test.     

Isolation and characterization of rat intestinal bacteria involved in biotransformation of (−)-epigallocatechin

Two intestinal bacterial strains MT4s-5 and MT42 involved in the degradation of (?)-epigallocatechin (EGC) were isolated from rat feces. Strain MT4s-5 was tentatively identified as Adlercreutzia equolifaciens. This strain converted EGC into not only 1-(3, 4, 5-trihydroxyphenyl)-3-(2, 4, 6-trihydroxyphenyl)propan-2-ol (1), but also 1-(3, 5-dihydroxyphenyl)-3-(2, 4, 6-trihydroxyphenyl)propan-2-ol (2), and 4′-dehydroxylated EGC (7). Type strain (JCM 9979) of Eggerthella lenta was also found to convert EGC into 1. Strain MT42 was identified as Flavonifractor plautii and converted 1 into 4-hydroxy-5-(3, 4, 5-trihydroxyphenyl)valeric acid (3) and 5-(3, 4, 5-trihydroxyphenyl)-γ-valerolactone (4) simultaneously. Strain MT42 also converted 2 into 4-hydroxy-5-(3, 5-dihydroxyphenyl)valeric acid (5), and 5-(3, 5-dihydroxyphenyl)-γ-valerolactone (6). Furthermore, F. plautii strains ATCC 29863 and ATCC 49531 were found to catalyze the same reactions as strain MT42. Interestingly, formation of 2 from EGC by strain MT4s-5 occurred rapidly in the presence of hydrogen supplied by syntrophic bacteria. Strain JCM 9979 also formed 2 in the presence of the hydrogen or formate. Strain MT4s-5 converted 1, 3, and 4 to 2, 5, and 6, respectively, and the conversion was stimulated by hydrogen, whereas strain JCM 9979 could catalyze the conversion only in the presence of hydrogen or formate. On the basis of the above results together with previous reports, the principal metabolic pathway of EGC and EGCg by catechin-degrading bacteria in gut tract is proposed.     

Modes of Cl− transport across the mucosal and serosal membranes of urodele intestinal cells

Summary The characteristics of Cl^– movement across luminal and basolateral membranes ofAmphiuma intestinal absorptive cells were studied using Cl^–-sensitive microelectrodes and tracer³⁶Cl techniques. Intracellular Cl^– activity (a _Cl ⁱ ) was unchanged when serosal Cl^– was replaced; when luminal Cl^– was replaced cell Cl^– was rapidly lost. Accordingly, the steady statea _Cl ⁱ could be varied by changing the luminal [Cl]. As luminal [Cl] was raised from 1 to 86mM,a _Cl ⁱ rose in a linear manner, the mucosal membrane hyperpolarized, and the transepithelial voltage became serosa negative. In contrast, the rate of Cl^– transport from the cell into the serosal medium, measured as the SITS-inhibitable portion of the Cl^– absorptive flux, attained a maximum whena _Cl ⁱ reached an apparent value of 17mm, indicating the presence of a saturable, serosal transport step. The stilbeneinsensitive absorptive flux was linear with luminal [Cl], suggestive of a paracellular route of movement. Intracellulara _Cl was near electrochemical equilibrium at all but the lowest values of luminal [Cl] after interference produced by other anions was taken into account.a _Cl ⁱ was unaffected by Na replacement, removal of medium K, or elevation of medium HCO ₃ ^– . Mucosae labeled with³⁶Cl lost isotope into both luminal and serosal media at the same rate and from compartments of equal capacity. Lowering luminal [Cl] or addition of theophylline enhanced luminal Cl^– efflux. It is concluded that a conductive Cl leak pathway is present in the luminal membrane. Serosal transfer is by a saturable, stilbene-inhibitable pathway. Luminal Cl^– entry appears to be passive, but an electrogenic uptake cannot be discounted.     

Histochemical demonstration of the intestinal hetero-β-galactosidase (glucosidase)

Summary The histochemical demonstration of hetero--galactosidase (glucosidase) has been attempted in sections and zymograms of rabbit, monkey and human intestine and of rat kidney.The leakage of this enzyme from unfixed sections was prevented by the use of cold microtome sections adherent to semipermeable membranes. Methods with -D-glucosides and galactosides of 6-Br-2-naphthol (postincubation azocoupling with Fast Blue B as well as simultaneous azocoupling with hexazonium-p-rosaniline), of -naphthol (simultaneous azocoupling with hexazonium-p-rosaniline) and of 4-Cl-5-Br-3-indolyl (with ferricyanide, phenazonium methosulfate or nitro BT and without any oxidation agent) were used an evaluated concerning the specificity, localization ability and inhibition of enzyme activity. Pretreatment of sections with distilled water or saline and inhibition by p-Cl-mercuribenzoate, glucono- and galactono-lactones were used for the characterization of the demonstrated enzyme activity.6-Br-2-naphthyl--D-glucoside is the most specific substrate for hetero--galactosidase. It is not split by lactase and acid -galactosidase. Only lysosomal -glucosidase can interfere. Because the latter enzyme is membrane-bound the difference in color intensity between untreated and prewashed sections are due to hetero--galactosidase. Only localization on the cellular (not intracellular) level can be achieved, however.The simultaneous azocoupling method with -naphthyl--D-glucoside and hexazonium-p-rosaniline enables a very good localization of hetero--galactosidase in the rabbit intestine. Due to a great inhibition exerted by hexazonium-p-rosaniline on the enzyme activity the method is unsuitable for the detection of hetero--galactosidase in zymograms and in the human intestine. Interference of lactase (or lactase-phlorizine hydrolase complex) is to be considered. The lysosomal -glucosidase does not seem to interfere.Indigogenic methods are not sensitive either. With ferricyanide as an oxidation agent it was not possible to detect the activity of hetero--galactosidase in zymograms and in sections. This is possibly due to overoxidation of indigo. The same holds true for phenazonium methosulfate used for the processing of zymograms. However, it was possible to reveal the activity of hetero--galactosidase in sections of the rabbit and monkey intestine with phenazonium methosulfate as oxidation agent. Nitro BT enhanced the coloration both in zymograms and in sections. In the latter case diffusion artifacts cannot be prevented, however. The interference of lactase, lysosomal -galactosidase and possibly of lysosomal -glucosidase (depending on the glycoside used) is always to be considered.Hetero--galactosidase was localized in the cytoplasm (particularly in the supranuclear region) of differentiated enterocytes covering the villi of the rabbit (the highest activity), monkey and human (the lowest activity) intestine. In crypt enterocytes and in cells of Brunner's glands the activity was lower. The occurrence of a low activity of hetero--galactosidase in the brush border of enterocytes of the rabbit intestine was also demonstrated.A proximodistal gradient was observed in the rabbit and monkey intestine, the upper jejunum displaying the highest activity.In jejunal biopsies of patients with celiac sprue (in the acute stage of the disease) the activity of hetero--galactosidase was lowered. No changes of activity were observed in jejunal biopsies of patients with isolated deficiencies of lactase or sucrase.In the rat kidney the enzyme was demonstrated particularly in the cytoplasm of cells of proximal convoluted tubules.     

Effect of short — chain fatty acids on electrical activity of the small intestinal mucosa of rat

Intravenous administration of short-chain fatty acid (SCFA), such as propionate, butyrate, valerate and caproate, caused a transient increase in transmural potential difference (p.d.) across the small intestine of rat $\text{in vivo}$. There was a sigmoid relationship between the change in the p.d. and the logarithm of the dose of SCFA. The median effective dose of propionate, n-butyrate, n-valerate and n-caproate, which was calculated from the each dose-response curve obtained from the terminal ileum, 1.31, 1.43, 0.83 and 0.81 μmole, respectively. Repeated administrations of the same dose of propionate evoked progressively smaller response. The dose-response curve of propionate was shifted to the left by neostigmine and to the right by atropine, suggesting that the action of SCFA may be mediated by acetylcholine, which was released from a nerve ending.     

Effects of α-tocopherol on the lipid peroxidation and fluidity of porcine intestinal brush-border membranes

The effect of α-tocopherol on the lipid fluidity of porcine intestinal brush-border membranes was studied using pyrene as a fluorescent probe. Addition of α-tocopherol to the medium decreased fluorescence intensity and lifetime, but increased the fluorescence polarization of pyrene-labeled membranes. β-, γ-, and δ-Tocopherols gave no appreciable effect on the fluorescence intensity and polarization of the complex. The apparent dissociation constant (3.1 ± 0.12 μM) of the interaction of α-tocopherol with the membranes, estimated from the change in the fluorescence intensity with varying concentrations of α-tocopherol, was in good agreement with the concentration required to cause the half-maximal inhibition of lipid peroxidation of the membranes performed by incubation with 100 μM ascorbic acid and 10 μM Fe²⁺. Decrease of the slope in the thermal Perrin plot of the polarization of pyrene-labeled membranes by α-tocopherol suggests that the movement of pyrene molecules in the membranes is restricted by binding of the tocopherol. This interpretation was confirmed by an increased harmonic mean of the rotational relaxation time of the dye molecules in the membranes from 10.9 ± 0.16 to 18.5 ± 0.51 μs after addition of 25 μM α-tocopherol to the medium. The perturbation of lipid phase in the membranes induced by α-tocopherol was also suggested from a decreased quenching rate constant of pyrene fluorescence in the membranes for Tl⁺. Based on these results, the effect of α-tocopherol on the lipid fluidity of the membranes is discussed.     

The stem cells of small intestinal crypts: where are they?

Recently, there has been resurgence of interest in the question of small intestinal stem cells, their precise location and numbers in the crypts. In this article, we attempt to re-assess the data, including historical information often omitted in recent studies on the subject. The conclusion we draw is that the evidence supports the concept that active murine small intestinal stem cells in steady state are few in number and are proliferative. There are two evolving, but divergent views on their location (which may be more related to scope of capability and reversibility than to location) several lineage labelling and stem cell self-renewing studies (based on Lgr5 expression) suggest a location intercalated between the Paneth cells (crypt base columnar cells (CBCCs)), or classical cell kinetic, label-retention and radiobiological evidence plus other recent studies, pointing to a location four cell positions luminally from the base of the crypt The latter is supported by recent lineage labelling of Bmi-1-expressing cells and by studies on expression of Wip-1 phosphatase. The situation in the human small intestine remains unclear, but recent mtDNA mutation studies suggest that the stem cells in humans are also located above the Paneth cell zone. There could be a distinct and as yet undiscovered relationship between these observed traits, with stem cell properties both in cells of the crypt base and those at cell position 4.     

Effect of chronic ethanol ingestion on intestinal metabolism and mutagenicity of benzo(α)pyrene

Chronic ethanol ingestion in male rats causes an increase in cytochrome P-450 content and in the activity of microsomal benzo(α)pyrene hydroxylase in the upper intestinal mucosa. Intestinal microsomes from ethanol fed rats also exhibit an enhanced capacity to activate the ubiquitous procarcinogen benzo(α)pyrene to a mutagen. These findings could be of relevance with respect to the increased incidence of cancer in the alcoholic.     

Protective effects of dietary EPA and DHA on ischemia–reperfusion-induced intestinal stress

The immunoregulatory effects of dietary omega-3 fatty acids are still not fully characterized. The aim of this study was to determine whether dietary eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) intake limits intestinal ischemia–reperfusion (IR) injury. To test this, rats were fed either control or EPA/DHA supplemented diet for 3 weeks following which they underwent either a sham or an IR surgical protocol. A significant reduction in mucosal damage was observed after EPA/DHA supplemented diet as reflected by maintenance of total protein content. To address the underlying mechanisms of protection, we measured parameters of oxidative stress, intestinal and serological cytokines and intestinal eicosanoids. Interestingly, EPA/DHA fed animals displayed a higher activity of oxidative stress enzyme machinery, i.e., superoxide dismutase and catalase in addition to a reduction in total nitrate/nitrite content. While no changes in cytokines were observed, eicosanoid analyses of intestinal tissue revealed an increase in metabolites of the 12-lipoxygenase pathway following IR. Further, IR in EPA/DHA fed animals was accompanied by a significant increase of 17,18-epoxyeicosatetraenoic acid, 8-Iso prostaglandin F_3α and thromboxane B₃, by more than 12-, 6-, 3-fold, respectively. Thus, the data indicate that EPA/DHA supplementation may be able to reduce early intestinal IR injury by anti-oxidative and anti-inflammatory mechanisms.     

Characterization of intestinal inflammation and identification of related gene expression changes in mdr1a−/− mice

Multidrug resistance targeted mutation (mdr1a (-/-) ) mice spontaneously develop intestinal inflammation. The aim of this study was to further characterize the intestinal inflammation in mdr1a (-/-) mice. Intestinal samples were collected to measure inflammation and gene expression changes over time. The first signs of inflammation occurred around 16 weeks of age and most mdr1a (-/-) mice developed inflammation between 16 and 27 weeks of age. The total histological injury score was the highest in the colon. The inflammatory lesions were transmural and discontinuous, revealing similarities to human inflammatory bowel diseases (IBD). Genes involved in inflammatory response pathways were up-regulated whereas genes involved in biotransformation and transport were down-regulated in colonic epithelial cell scrapings of inflamed mdra1 (-/-) mice at 25 weeks of age compared to non-inflamed FVB mice. These results show overlap to human IBD and strengthen the use of this in vivo model to study human IBD. The anti-inflammatory regenerating islet-derived genes were expressed at a lower level during inflammation initiation in non-inflamed colonic epithelial cell scrapings of mdr1a (-/-) mice at 12 weeks of age. This result suggests that an insufficiently suppressed immune response could be crucial to the initiation and development of intestinal inflammation in mdr1a (-/-) mice.     

(−)-Epicatechin mitigates radiation-induced intestinal injury and promotes intestinal regeneration via suppressing oxidative stress

Abstract

The high radiosensitivity of the intestinal epithelium limits the survival of victims by nuclear accidents or terrorism and limits effective radiotherapy against abdominal malignancies. Recently, we reported that (?)-epicatechin (EC) modulates oxidative stress and exerts neuroprotection. Here, we investigate the protective effects of EC against intestinal damage induced by radiation. The established model is acute moderate but reversible intestinal injury damage. We also set up the injured model of “minigut” ex vivo, which mimic the process of intestinal regeneration in vivo. We found that EC can repress oxidative stress by regulating SOD and MDA levels in serum and intestine tissue. Correspondingly, EC can decrease apoptosis of crypt cells in Lgr5-EGFP-IRES-creERT2 mice after radiation. Further studies demonstrated that EC can promote Nrf2 translocation from cytoplasm to nuclear and then activate the expression of HO1 and NQO1. Interestingly, EC can enhance the activity of intestine stem cells labelled by Lgr5 and promote intestinal epithelium regeneration determined by HE and immunofluorescence staining in vivo and in vitro. We also found that EC can activate the Wnt/β-catenin signal pathway confirmed by TCF/LEF luciferase reporter assay. Together, EC can provide the protective effect on intestine and promote intestinal regeneration after radiation through Nrf2 and Wnt/β-catenin signal pathway.     

Epac1 mediates protein kinase A–independent mechanism of forskolin-activated intestinal chloride secretion

Intestinal Cl⁻ secretion is stimulated by cyclic AMP (cAMP) and intracellular calcium ([Ca²⁺]_i). Recent studies show that protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac) are downstream targets of cAMP. Therefore, we tested whether both PKA and Epac are involved in forskolin (FSK)/cAMP-stimulated Cl⁻ secretion. Human intestinal T84 cells and mouse small intestine were used for short circuit current (I_sc) measurement in response to agonist-stimulated Cl⁻ secretion. FSK-stimulated Cl⁻ secretion was completely inhibited by the additive effects of the PKA inhibitor, H89 (1 µM), and the [Ca²⁺]_i chelator, 1,2-bis-(o-aminophenoxy)-ethane-N,N,N’,N’-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM; 25 µM). Both FSK and the Epac activator 8-pCPT-2’-O-Me-cAMP (50 µM) elevated [Ca²⁺]_i, activated Ras-related protein 2, and induced Cl⁻ secretion in intact or basolateral membrane–permeabilized T84 cells and mouse ileal sheets. The effects of 8-pCPT-2’-O-Me-cAMP were completely abolished by BAPTA-AM, but not by H89. In contrast, T84 cells with silenced Epac1 had a reduced I_sc response to FSK, and this response was completely inhibited by H89, but not by the phospholipase C inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 or BAPTA-AM. The stimulatory effect of 8-pCPT-2’-O-Me-cAMP on Cl⁻ secretion was not abolished by cystic fibrosis transmembrane conductance (CFTR) inhibitor 172 or glibenclamide, suggesting that CFTR channels are not involved. This was confirmed by lack of effect of 8-pCPT-2’-O-Me-cAMP on whole cell patch clamp recordings of CFTR currents in Chinese hamster ovary cells transiently expressing the human CFTR channel. Furthermore, biophysical characterization of the Epac1-dependent Cl⁻ conductance of T84 cells mounted in Ussing chambers suggested that this conductance was hyperpolarization activated, inwardly rectifying, and displayed a Cl⁻>Br⁻>I⁻ permeability sequence. These results led us to conclude that the Epac-Rap-PLC-[Ca²⁺]_i signaling pathway is involved in cAMP-stimulated Cl⁻ secretion, which is carried by a novel, previously undescribed Cl⁻ channel.