Mouse Small Bowel Research Articles

Epithelial Mechanosensitive Ion Channel Piezo2 Contributes to Pressure‐Induced Epithelial Chloride Secretion in Mouse Colon

BACKGROUNDMechanical stimulation of the gastrointestinal (GI) epithelium causes changes in epithelial ion transport by an unclear mechanism. We recently discovered the mechanosensitive ion channel Piezo2 is specifically expressed by the enteroendocrine cells (EECs) and is necessary for EEC mechanosensitivity. Further, in mouse small bowel, the pharmacologic inhibition or genetic knockout of the epithelial Piezo2 led to an inhibition of the pressure‐induced short‐circuit current (Isc) in the Ussing chamber. These results suggested that EEC Piezo2 plays a role in pressure‐induced epithelial ion transport, but the mechanism and whether these responses are region‐specific remains uknown.AIMDetermine whether epithelial Piezo2‐mediated pressure‐induced Isc is due to Clsecretion in the mouse colon.METHODSFull‐thickness mid‐colon preparations of VilCre/+::Piezo2f/f (Piezo2CKO) or Vil+/+::Piezo2f/f (Piezo2WT) mice at 0.3 cm2 were mounted in 4mL Ussing chambers with normal Krebs solution. The epithelial side was intermittently pressure‐clamped at a range of pressures (5, 10, 20, 30, 40, 60 mmHg) for 10s each. Forskolin (FSK, 10μM) was applied at the end of the experiments, and FSK response was used to normalize the pressure‐induced responses (ΔIsc/ΔIsc,FSK).RESULTSPressure applied to the mucosa of Piezo2CKO and Piezo2WTcolons caused a pressure‐dependent increase in Isc. Responses in Piezo2CKO colons were smaller than Piezo2WT at 40mmHg (0.41±0.1 vs. 0.76±0.1) and 60mmHg (0.51±0.1 vs. 0.87±0.1) (n=9–11, P<0.05, two‐way ANOVA). To determine the nature of the pressure‐induced Isc we removed Cl− from the Krebs solutions. Cl‐removal resulted in XX. We found a nearloss in the mechanically induced ΔIsc in both Piezo2WT tissues and Piezo2CKO: 5mmHg (− 88.85±0.1% and −87.44±0.1% Piezo2WT), 10mmHg (−86.46±0.1% Piezo2CKO and − 90.30±0.1% Piezo2WT), 20mmHg (−88.50±0.01% Piezo2CKO and −89.86±0.1% Piezo2WT), 40mmHg (−91.78±0.1% Piezo2CKO and −91.35±0.04% Piezo2WT), and 60mmHg (−93.93±0.04% Piezo2CKO and −92.08±0.04% Piezo2WT (n= 5–10, P <0.05, unpaired t‐test). Replacement of Cl− led to full recovery of the mechanically induced ΔIsc in both Piezo2WT and Piezo2CKO tissues when tested at 30 mmHg (Piezo2WT: 2.51±1.4 vs. 73.52±33.8, n=6 and 3, P <0.05, unpaired t‐test), (Piezo2CKO: 1.80±1.0 vs. 45.14±12.4, n=5 and 4, P <0.05, unpaired t‐test).CONCLUSIONSHydrostatic pressure applied to the colon epithelium leads to an increase in epithelial secretion that depends chloride and Piezo2 contributes to this mechanosensitive chloride secretion.Support or Funding InformationSupported by AGA RSA, NIH DK106456, NIH DK119683, NIH DK123549

Biological Activity of Fenbendazole Solid Dispersion Obtained by Mechanochemical Technology with Various Components for Targeted Delivery

The purpose of the research is studying anthelmintic efficacy of fenbendazole solid dispersion obtained by mechanochemical technology with various components for targeted delivery. Materials and methods. The tests were conducted on 80 white mice infected experimentally with Trichinella spiralis and 145 sheep infected spontaneously with gastrointestinal nematodes. In case of each helminthosis, animals in different groups of 8–10 units each were given, orally, as a single dose, a fenbendazole solid dispersion (FSD) with polyvinylpyrrolidone (PVP), arabinogalactan (AG), glycyrrhizinic acid sodium salt (NaGA), dioctylsulphosuccinate sodium (NaDSS), licorice extract (LE) and hydroxyethylstarch (HES) in a dose of 2.0 mg/kg of the active ingredient as compared to fenbendazole drug at 2.0 mg/kg. A group of animals that were not given the drug was a control group. Anthelmintic activity of the preparations was considered based on dissection of the mouse small bowel and data from the sheep coproovoscopic examination carried out by the flotation technique before and 15 days after application of such drugs. Results and discussion. FSD with PVP, AG, NaGA, NaDSS, LE and HES in a dose of 2.0 mg/kg of the active ingredient (fenbendazole) showed 100; 98.7; 99.8; 100; 96.8 and 95.3% activity in mice against T. spiralis respectively; in sheep – 100; 96.3; 98.0; 100; 94.6 and 93.8% effectiveness against Nematodirus spp., and 100; 97.5; 98.6; 100; 97.0 and 96.5% activity against other species of the gastrointestinal Strongylata at 22.6–27.2% effectiveness of the main drug, namely, the active ingredient fenbendazole.

Mechanosensitive ion channel Piezo2 is important for enterochromaffin cell response to mechanical forces.

The gastrointestinal epithelial enterochromaffin (EC) cell synthesizes the vast majority of the body's serotonin. As a specialized mechanosensor, the EC cell releases this serotonin in response to mechanical forces. However, the molecular mechanism of EC cell mechanotransduction is unknown. In the present study, we show, for the first time, that the mechanosensitive ion channel Piezo2 is specifically expressed by the human and mouse EC cells. Activation of Piezo2 by mechanical forces results in a characteristic ionic current, the release of serotonin and stimulation of gastrointestinal secretion. Piezo2 inhibition by drugs or molecular knockdown decreases mechanosensitive currents, serotonin release and downstream physiological effects. The results of the present study suggest that the mechanosensitive ion channel Piezo2 is specifically expressed by the EC cells of the human and mouse small bowel and that it is important for EC cell mechanotransduction. The enterochromaffin (EC) cell in the gastrointestinal (GI) epithelium is the source of nearly all systemic serotonin (5-hydroxytryptamine; 5-HT), which is an important neurotransmitter and endocrine, autocrine and paracrine hormone. The EC cell is a specialized mechanosensor, and it is well known that it releases 5-HT in response to mechanical forces. However, the EC cell mechanotransduction mechanism is unknown. The present study aimed to determine whether Piezo2 is involved in EC cell mechanosensation. Piezo2 mRNA was expressed in human jejunum and mouse mucosa from all segments of the small bowel. Piezo2 immunoreactivity localized specifically within EC cells of human and mouse small bowel epithelium. The EC cell model released 5-HT in response to stretch, and had Piezo2 mRNA and protein, as well as a mechanically-sensitive inward non-selective cation current characteristic of Piezo2. Both inward currents and 5-HT release were inhibited by Piezo2 small interfering RNA and antagonists (Gd3+ and D-GsMTx4). Jejunum mucosal pressure increased 5-HT release and short-circuit current via submucosal 5-HT3 and 5-HT4 receptors. Pressure-induced secretion was inhibited by the mechanosensitive ion channel antagonists gadolinium, ruthenium red and D-GsMTx4. We conclude that the EC cells in the human and mouse small bowel GI epithelium selectively express the mechanosensitive ion channel Piezo2, and also that activation of Piezo2 by force leads to inward currents, 5-HT release and an increase in mucosal secretion. Therefore, Piezo2 is critical to EC cell mechanosensitivity and downstream physiological effects.

Hexose Transporter Expression and Function in Mouse Small Intestine: Role of Diurnal Rhythm

BackgroundExpression and function of hexose transporters vary diurnally in rat small intestine; however, this subject remains unexplored in mice. AimThe aim of the study was to investigate the diurnal expression and function of hexose transporters SGLT1, GLUT2, and GLUT5 in mouse small bowel. MethodsTwenty-four c57bl6 mice maintained in a 12-h light/dark room (6 am–6 pm) were sacrificed at 9 am, 3 pm, 9 pm, and 3 am (n = 6 each). In duodenal, jejunal, and ileal mucosa, total cellular mRNA and protein levels were quantitated by real-time PCR and semiquantitative Western blotting, respectively. The everted sleeve technique measured transporter-mediated glucose uptake at 9 am and 9 pm. ResultsmRNA expression of SGLT1, GLUT2, and GLUT5 varied diurnally in all three intestinal segments (p ≤ 0.03). SGLT1, GLUT2, and GLUT5 protein levels varied diurnally in duodenum and jejunum (p < 0.05) but not in ileum. Transporter-mediated glucose uptake was greater at 9 pm than 9 am (p ≤ 0.04) in all three segments. Vmax was greater in duodenum (10 vs 6 nmol/cm/s) and jejunum (8 vs 5 nmol/cm/s) at 9 pm compared to 9 am (p = 0.01); Km remained unchanged. SummarymRNA levels of intestinal hexose transporters varied diurnally. Protein levels peaked 6–12 h later during dark cycle when >70% of food intake occurred; glucose transport followed a similar pattern with increased uptake at 9 pm. ConclusionHexose transporter expression and function vary diurnally with nocturnal feeding patterns of mice.

Small Bowel Transplantation In Mice

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive medications, infection prophylaxis, and suitable patient selection helped improve actuarial graft and patient survival rates for all types of intestine transplantation. Patients with irreversible intestinal failure and complications of parenteral nutrition should now be routinely considered for small intestine transplantation. However, Survival rates for small intestinal transplantation have been slow to improve compares increasingly favorably with renal, liver, heart and lung. The small bowel transplantation is still unsatisfactory compared with other organs. Further progress may depend on better understanding of immunology and physiology of the graft and can be greatly facilitated by animal models. A wider use of mouse small bowel transplantation model is needed in the study of immunology and physiology of the transplantation gut as well as efficient methods in diagnosing early rejection. However, this model is limited to use because the techniques involved is an extremely technically challenging. We have developed a modified technique. When making anastomosis of portal vein and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s portal vein. The left wall of the inferior vena cava and donor s portal vein is closed with continuing sutures in the inside of the inferior vena cava after, after one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s portal vein are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis. In this article, we provide details of the technique to supplement the video.

Protection of Mouse Small Bowel Allografts by FTY720 and Costimulation Blockade

The clinical application of small bowel transplantation (SBTx) is hampered by its pronounced immunogenicity. We aimed to test the hypothesis that prolonged sequestration of lymphocytes in secondary lymphoid organs may enhance the alloprotective effect of costimulation blockade. For this purpose, recipients of intestinal allografts were treated with MR1, FTY720, combined FTY720 plus MR1, or were left untreated. Grafts were examined 6 and 14 days after transplantation by applying a histologic rejection score, multiparameter-immunofluorescent staining, and flow cytometry. FTY720 or MR1 monotherapy did not prevent the rejection of mouse intestinal allografts, whereas combined therapy with FTY720 plus MR1 profoundly inhibited rejection at day 6 and day 14 after transplantation. In FTY720-treated mice infiltration of host lymphocytes in graft mesenteric lymph nodes, Peyer's patches, intraepithelial lymphocytes, and lamina propria lymphocytes (LPLs) was reduced on day 6. Anti-CD40L antibody improved the rejection score at day 14 but had no effect at day 6. Importantly, host CD8 T-cell infiltration in graft LPLs was significantly reduced compared with all other groups. FTY720 plus MR1 effectively inhibited intestinal allograft rejection in mice, possibly by enhancing the alloprotective effects of costimulation blockade by prolonged sequestration of lymphocytes in secondary lymphoid organs.

A CELL CULTURE MODEL FOR SMALL INTESTINAL STEM CELL FUNCTION AND DIFFERENTIATION

Aim: The stem cells located in small intestinal crypts are responsible for the continuous renewal of the epithelium and thus have a crucial role in maintaining tissue homeostasis. Since at present there is no cell culture model for small bowel epithelial stem cell function and differentiation, our aim is to establish culture conditions that allow intestinal stem cell propagation and proliferation as well as differentiation into all four epithelial cell types present in the small bowel. Methods: Single crypt epithelial cells and small organoids were isolated from mouse small intestine and cultured under specific conditions on collagen with fibroblasts seeded within the gel as feeder layer. Cultures were observed daily and studied immunohistologically. Results: The isolated small intestinal epithelial cells form organized clusters that remained viable for at least 10 weeks. The clusters were characterized by a large central lumen and well-deposited basement membrane. A portion of the cells were proliferative as evinced by Ki-67 positivity. In addition the clusters also harboured polarized columnar cells that had obviously differentiated as indicated by alkaline phosphatase activity and mucin profile typical for small intestine. When these parental clusters were broken into single cells that were cultured under identical conditions they formed daughter clusters with similar characteristics as the parental ones. Summary: At this point we have cultured the clonogenic cells derived from mouse small bowel for 10 weeks - the longest period ever published. During this time the cells form clusters containing both differentiated cells and undifferentiated proliferating cells that can also be subcultured. Conclusion: We have developed a long-term cell culture system that models the in vivo small intestinal crypt villus axis in terms of stem cell proliferation and differentiation. If applicable to human, the development of this culture system will eventually allow the use of small intestinal stem cells in regenerative medicine as a source for transplantation in diseased patients to correct dysregulated or prolonged mucosal regeneration.

Cultured Nestin–Positive Cells from Postnatal Mouse Small Bowel Differentiate Ex Vivo into Neurons, Glia, and Smooth Muscle

Little is known about postnatal enteric nervous system (ENS) development, but some reports suggest that the postnatal bowel may contain neural stem cells. Therefore, we created an in vitro model of desegregation using an enzymatic and mechanical tissue technique. This approach yielded a group of cells from the small intestine of lactating and adult mice, which ex vivo attach to the culture dish; actively proliferate; and express nestin, vimentin, and the pro-neural transcription factors neurogenin-2 (ngn-2), Sox-10, and Mash-1. In the conditions grown, double immunostains suggest that they differentiate into various cell types, particularly neurons, smooth muscle, and glia including 04 protein-positive cells. They also express the neurotrophic-protein tyrosine kinase (Trk) receptors TrkA, TrkB, and TrkC; the low-affinity neurotrophin receptor p75NTR; and the glial-derived neurotrophic factor receptors (GFR)alpha-1, GFRalpha-2, and GFRalpha-3. The neurons expressed several sensory and motor neurotransmitters present in the central and enteric nervous systems, including calcitonin gene-related peptide, neuropeptideY, peptideYY, substance P, vasoactive intestinal polypeptide, and galanin; along with glia, these neurons formed elaborate intercellular connections. They also express c-KIT, CD34, CD20, and CD45RO, suggesting they either have a hematogenous origin or may differentiate toward hematogenous lines. These findings suggest that these cells may be enteric neural stem cells (ENSCs); may normally be present in the small intestine; and may have the capacity to proliferate and differentiate into neurons, glia, and smooth muscle. Further identification and purification of intestinal ENSCs will provide a means to study the regulation of their differentiation and should give insight into the mechanisms involved in development and remodeling of the ENS. The possible therapeutic application of postnatal stem cells such as ENSCs needs to be evaluated, including their use for transplantation in the central nervous system.

Graft mucosal blood flow is reduced prior to histologic evidence of rejection in mouse small bowel transplantation: a murine model

THE SUCCESS of small bowel transplantation is hampered by vigorous and early acute rejection responses. At present, the diagnosis of acute rejection is based mainly on histology and endoscopy. The late onset of morphologic changes and the initial patchy pattern makes it hard to discover early. Therefore efforts continue to identify novel and effective means for early diagnosis of acute rejection in intestinal transplantation. It has been demonstrated that one of the first targets in acute rejection of the small bowel allograft is the vascular endothelial cells in the lamina propria. This response may result in early mucosal microcirculatory disturbances and progressive ischemia that eventual lead to morphologic changes. We assumed that this attack on the microvasculature would be reflected in mucosal blood perfusion. Mucosal perfusion was measured by means of laser Doppler flowmetry at different time intervals and compared with synchronous histologic samples.

Modulation of Mouse Paneth Cell α-Defensin Secretion by mIKCa1, a Ca2+-activated, Intermediate Conductance Potassium Channel

Paneth cells in small intestinal crypts secrete microbicidal alpha-defensins in response to bacteria and bacterial antigens (Ayabe, T., Satchell, D. P., Wilson, C. L., Parks, W. C., Selsted, M. E., and Ouellette, A. J. (2000) Nat. Immunol. 1, 113- 38). We now report that the Ca(2+)-activated K(+) channel mIKCa1 modulates mouse Paneth cell secretion. mIKCa1 cDNA clones identified in a mouse small intestinal crypt library by hybridization to human IKCa1 cDNA probes were isolated, and DNA sequence analysis showed that they were identical to mIKCa1 cDNAs isolated from erythroid cells and liver. The genomic organization was found to be conserved between mouse and human IKCa1 as shown by comparisons of the respective cDNA and genomic sequences. Reverse transcriptase-PCR experiments using nested primers amplified mIKCa1 from the lower half of bisected crypts and from single Paneth cells, but not from the upper half of bisected crypts, villus epithelium, or undifferentiated crypt epithelial cells, suggesting a lineage-specific role for mIKCa1 in mouse small bowel epithelium. The cloned mIKCa1 channel was calcium-activated and was blocked by ten structurally diverse peptide and nonpeptide inhibitors with potencies spanning 9 orders of magnitude and indistinguishable from that of the human homologue. Consistent with channel blockade, charybdotoxin, clotrimazole, and the highly selective IKCa1 inhibitors, TRAM-34 and TRAM-39, inhibited (approximately 50%) Paneth cell secretion stimulated by bacteria or bacterial lipopolysaccharide, measured both as bactericidal activity and secreted cryptdin protein, but the inactive analog, TRAM-7, did not block secretion. These results demonstrate that mIKCa1 is modulator of Paneth cell alpha-defensin secretion and disclose an involvement in mucosal defense of the intestinal epithelium against ingested bacterial pathogens.

Apposition of enteric nerve fibers to plasma cells and immunoblasts in the mouse small bowel

Light (LM) and electron microscopy (EM) were used to investigate the structural relationship between enteric nerves and the population of immune cells in the mouse small bowel. By LM, the osmium-zinc iodide procedure was used for visualizing nerve fibers; the incidence of nerve-plasma cell contacts in the mucosa and submucosa was calculated to be ~4 times and, respectively, 3 times greater than would be expected by chance alone ( P<0.0001). EM revealed close, synaptic-like contacts between axonal varicosities and plasma cells or B immunoblasts. The results presented here provide systematic quantitative evidence that a structural foundation for communication between nerve fibers and B cells exists in the mouse small bowel.

AOM-induced mouse colon tumors do not express full-length APC protein.

While evidence in both sporadic and inherited human colorectal cancer and MIN mice implicate the tumor suppressor gene, APC, in the causation of colorectal carcinogenesis, this gene has not been confirmed to be involved in rodent chemically-induced colon cancer models (RCCM). These experimental models are widely used to elucidate mechanisms involved in colon carcinogenesis (initiation, promotion and progression) as well as studies on chemoprevention (dietary and other) and intervention. To validate the RCCM as relevant models for sporadic human colorectal cancer, and to facilitate research on the role of the APC gene in colon carcinogenesis, we investigated the role of APC in azoxymethane (AOM)-induced colorectal tumors in mice. Using an antibody that recognizes the carboxy terminus of APC, we have characterized the pattern of staining observed in normal mouse intestinal tissue, in MIN mouse intestinal adenomas and in AOM-induced mouse colon tumors. The APC protein was localized in the cytoplasm of normal colonic epithelial cells. In the small intestine there was APC immunoreactivity along the villous and staining of the Paneth cells at the base of the glands. In the proximal and distal colonic crypts there appeared to be a gradient of staining which increased towards the luminal surface. This gradient was not as apparent in the small intestinal villi. Nuclei and mucus in the goblet cells showed no immunoreactivity. MIN mouse small bowel and colonic adenomas, known to have lost APC, stained negatively for APC. AOM-induced adenomas and carcinomas also consistently stained negatively using this antibody. This study demonstrates for the first time the loss of wild-type APC protein in AOM-induced mouse colon tumors and suggests that alterations in expression of this tumor suppressor gene, which is so commonly mutated in human colon cancer, is also involved in this animal model of colon cancer.

Biological determinants of intestinotrophic properties of GLP-2 in vivo.

Glucagon-like peptide 2 (GLP-2) appears to regulate growth of the small bowel epithelium. The importance of dose, vehicle, and route of administration for GLP-2 bioactivity was examined in vivo. Mouse small bowel weight increased following subcutaneous injection of GLP-2, 0.25 to 5.0 microg twice daily, with an increment in jejunal crypt plus villus height detected with only 250 ng GLP-2 twice daily for 14 days (1.35-fold greater than control, P < 0.05). Small bowel weight was increased by 6 days of GLP-2 administration (1.4-fold greater than control, P < 0.05) and further increased after up to 14 days of GLP-2 treatment (P < 0.001, GLP-2 vs. control). GLP-2 increased small bowel weight in either 12% gelatin or phosphate-buffered saline (PBS) formulations (P < 0.01 vs. P < 0.05, gelatin vs. PBS, respectively), and GLP-2 was intestinotrophic following either intramuscular, intraperitoneal, or subcutaneous administration (P < 0.05 for im and ip, P < 0.001 for sc, GLP-2 vs. control). GLP-2 also induced bowel growth when administered daily or every other day. These results define important biological parameters for the activity of GLP-2 in vivo.

Positional specificity of defensin gene expression reveals Paneth cell heterogeneity in mouse small intestine.

Cryptdins are antimicrobial peptides of the defensin family that are expressed specifically by Paneth cells in small intestinal crypts (M.E. Selsted, S.I. Miller, A.H. Henschen, and A.J. Ouellette. J. Cell Biol. 118: 929-936, 1992), and at least 17 cryptdin isoforms have been reported in mouse small intestine (A.J. Ouellette, M.M. Hsieh, M.T. Nosek, D.F. Cano-Gauci, K.M. Huttner, R.N. Buick, and M.E. Selsted. Infect. Immun. 62: 5040-5047, 1994). Analysis of cryptdin gene expression in adult mouse small bowel revealed that the cryptdin-4 isoform is differentially expressed along the proximal-to-distal intestinal axis. By peptide-specific reverse transcriptase-polymerase chain reaction-based assays, cryptdin-4 mRNA was found to be absent from the proximal small bowel, increasing to maximal levels in the ileum. In contrast, intestinal content of cryptdin-1 and -5 mRNAs was equivalent in duodenum, jejunum, and ileum, and Northern blot hybridization experiments were consistent with both sets of data. Similarly, individual crypts isolated from duodenum contain cryptdin-1 mRNA but not cryptdin-4 mRNA. Taken together, the results show that Paneth cells are heterogeneous, depending on their position along the longitudinal axis of the small bowel. The positional specificity of defensin gene expression suggests that cryptdins may be useful markers for investigating the establishment and maintenance of this epithelial lineage in the mouse small intestine.

Deconjugation of bile acids by lactobacilli in the mouse small bowel

Conjugated and unconjugated bile acid concentrations in the small bowel contents and portal serum samples collected from mice with and without lactobacilli (RLFL and RLF, respectively) as gastrointestinal inhabitants were measured. The major portion of bile acids in the small bowel contents of RLFL mice was unconjugated (67.9%) in contrast to that of RFL animals in which a smaller portion of bile acids was unconjugated (23.5%). This study demonstrated that bile salt hydrolase produced by lactobacilli is active under the conditions prevailing in the proximal small bowels of mice.

The defensin-related murine CRS1C gene: Expression in paneth cells and linkage to Defcr, the cryptdin locus

The site of defensin-related CRS1C gene expression in mouse small bowel and the chromosomal location of the CRS1C locus, Defcr-rs1, have been determined. CRS1C (cryptdin-related sequence 1C) mRNA is an abundant small intestinal sequence that exhibits extensive similarity to the prepro-coding regions of defensin mRNAs yet does not encode a defensin (A. J. Ouellette and J. C. Lualdi, 1990, J. Biol. Chem. 265: 9831-9837). Using sequence-specific probes, CRS1C mRNA was detected in Paneth cells at the base of intestinal crypts by in situ hybridization. Southern blot analysis of genomic DNAs from inbred and recombinant inbred (RI) mouse strains, also conducted with probes specific for CRS1C, showed that the CRS1C locus maps to the proximal region of Chromosome 8. In 62 RI strains, no discordancies were found between Defcr-rs1 and Defcr, the cryptdin gene. Thus, both the Defcr-rs1 and the Defcr genes are expressed in Paneth cells and both are genetically inseparable within 1.58 cM on Chromosome 8. These studies identify a second defensin-related Paneth cell gene in mice.

Enteric defensins: antibiotic peptide components of intestinal host defense.

Five intestinal defensins, termed cryptdins 1-5, have been purified from mouse small bowel, sequenced, and localized to the epithelium by immunohistochemistry. Although identified as members of the defensin peptide family by peptide sequencing, enteric defensins are novel in that four cryptdins have amino termini which are three to six residues longer than those of leukocyte-derived defensins. A fifth cryptdin is the first defensin to diverge from the previously invariant spacing of cysteines in the peptide structure. The most abundant enteric defensin, cryptdin-1, had antimicrobial activity against an attenuated phoP mutant of Salmonella typhimurium but was not active against the virulent wild-type parent. Immunohistochemical localization demonstrated that cryptdin-1, and probably cryptdins 2 and 3, occur exclusively in Paneth cells, where the peptides appear to be associated with cytoplasmic granules. Biochemical and immunologic analysis of the luminal contents of the small intestine suggest that cryptdin peptides are secreted into the lumen, similar to Paneth cell secretion of lysozyme. The presence of several enteric defensins in the intestinal epithelium, evidence of their presence in the lumen, and the antibacterial activity of cryptdin-1 suggest that these peptides contribute to the antimicrobial barrier function of the small bowel mucosa.

A novel mouse gene family coding for cationic, cysteine-rich peptides. Regulation in small intestine and cells of myeloid origin.

Cryptdin is a Paneth cell corticostatin/defensin in the mouse small bowel. To help define the intestinal role of cryptdin, cryptdin-related sequence (CRS) mRNAs have been characterized with respect to developmental regulation, sequence homology, putative coding function, and occurrence in myeloid cells. Cryptdin, CRS1C, and CRS4C mRNAs are transcribed from separate genes, occur at equivalent abundance in small intestine, and appear in the small bowel in concert during the 2nd and 3rd weeks postpartum. Cryptdin and CRS1C mRNAs are not detectable in adult mouse bone marrow, but probes specific for the 5'- or the 3'-untranslated regions of CRS4C mRNA hybridize to a moderately abundant 1.05-kilobase bone marrow mRNA in contrast to a highly abundant 0.75-kilobase mRNA in small intestine. Nucleotide sequences corresponding to the deduced prepro-coding regions of cryptdin, CRS1C, and CRS4C mRNAs contain a highly conserved 200-base pair region of 92% sequence similarity (CSE.2), but the mRNAs are not homologous otherwise. The deduced CRS1C and CRS4C polypeptides are apparent precursors of secreted, cationic, proline- and cysteine-rich peptides that contain Cys-Pro-X repeats. Unlike cryptdin, however, the proposed CRS1C and CRS4C mature peptide regions lack the structural motif characteristic of defensins. Attempts to find homologies between the putative CRS peptides and existing protein sequences have been unsuccessful, leading us to speculate that CRS1C and CRS4C represent a new family of nondefensin antimicrobial peptides in the mouse small bowel.

Metallothionein mRNA induction in mouse small bowel by oral cadmium and zinc.

Induction of metallothionein mRNA was investigated in organs of mice receiving oral zinc and cadmium. The low molecular weight, heat stability, and 30% cysteine content of metallothionein facilitated its detection in cell-free translation products analyzed by Sephadex G-50 chromatography and by gel electrophoresis. Native and heat-denatured [35S]cysteine-labeled polypeptides directed by poly(A)+ mRNA from organs of mice exposed orally to 3 mg CdCl2 or 15 mg ZnCl2 were compared with those of control mice. Although parenteral administration of CdCl2 (30 micrograms) did not elicit a measurable metallothionein mRNA accumulation in gut, oral administration of this metal induced intestinal metallothionein mRNA to high levels; translatable metallothionein mRNA was not detected in control organs. Five hours after oral CD or Zn, metallothionein constituted 12-17% of [35S]cysteine directed into protein by gut mRNA, and metallothionein mRNA was induced to similar levels in liver, kidney, and spleen of the same mice. After oral Zn, metallothionein mRNA levels in proximal jejunum were twice those in other regions of intestine. Metallothionein mRNA accumulates rapidly in small bowel after oral Zn and Cd; simultaneous induction in other mouse organs indicates a systemic response.