Small Intestinal mRNA Research Articles

Effects of Rosa roxburghii & edible fungus fermentation broth on immune response and gut microbiota in immunosuppressed mice

With the rise of probiotics fermentation in food industry, fermented foods have attracted worldwide attention. In this study, protective effects of Rosa roxburghii & edible fungus fermentation broth (REFB) on immune function and gut health in Cyclophosphamide induced immunosuppressed mice were investigated. Results showed that REFB could improve the immune organ index, and promote the proliferation and differentiation of splenic T lymphocytes. In addition, it attenuated intestinal mucosal damage and improved intestinal cellular immunity. REFB administration also up-regulated the expression of IL-4, INF-γ, TNF-α, T-bet and GATA-3 mRNA in small intestine. Furthermore, administration of REFB modulated gut microbiota composition and increased the relative abundance of beneficial genus, such as Bacteroides. It also increased the production of fecal short-chain fatty acids. These indicate that REFB has the potential to improve immunity, alleviate intestinal injury and regulate gut microbiota in immunosuppressed mice.

Effects of dietary supplementation with Artemisia argyi alcohol extract on growth performance, blood biochemical properties and small intestinal immune markers of broilers challenged with lipopolysaccharide

Context Broilers are prone to immunological stress when subjected to unsuitable environmental conditions (such as virus attack, nutrient deficiency and high stocking density), lowering immunity and resulting in inflammatory bowel diseases. The herb-feed additive Artemisia argyi has been applied in poultry production, and its extract may improve small intestinal immune capacity. Aims This study was conducted to investigate the effect of A. argyi alcohol extract (AAAE) on growth performance and blood biochemical and small intestinal immune markers of broilers challenged with a proinflammatory substance, lipopolysaccharide (LPS). The study also examined possible mechanisms of action, and aimed to clarify whether AAAE could be applied as a feed additive. Methods In total, 192 one-day-old Arbor Acres broilers were allocated to four groups following a 2 × 2 factorial arrangement (including six replicates with eight birds per replicate) with two dietary AAAE rates (0 or 750 mg/kg) and two immune stress treatments (LPS or saline injection). Blood and small intestine were sampled on Days 21 and 35. Key results Dietary AAAE alleviated the LPS-induced decrease in bodyweight, average daily gain and average daily feed intake, and mitigated the elevated serum alanine aminotransferase, triglyceride, low-density lipoprotein cholesterol, adrenocorticotropic hormone and corticosterone concentrations at Day 21 and/or Day 35 in LPS-challenged broilers. AAAE significantly (P < 0.05) attenuated LPS-induced increases in intestinal immunoglobulin (IgA, IgG and IgM) and interleukin (IL-1β and IL-6) concentrations. Moreover, the small intestinal mRNA abundances of the genes TLR4, MyD88, NF-κBp65, IL-1β and IL-6 in LPS-challenged broilers were decreased (P < 0.05) in response to dietary AAAE treatment. Conclusions These results further demonstrated that AAAE at 750 mg/kg enhanced small intestinal tissue immune capacity of broilers, thereby alleviating LPS-induced immune stress damage in broilers. Its mechanism of action may be related to the mediating of TLR4/NF-κB pathways. Implications Dietary AAAE can be used to improve the immune function of broilers, and to provide a new scientific theoretical basis for the development of new anti-stress feed additives.

Influence of dietary fructose supplementation on visceral organ mass, carbohydrase activity, and mRNA expression of genes involved in small intestinal carbohydrate assimilation in neonatal calves

The hypothesis of this experiment was that dietary fructose would influence visceral organ mass, carbohydrase activity, and mRNA expression of carbohydrases and nutrient transporters in the small intestine in neonatal calves. Therefore, our objective was to use the neonatal calf as a model to evaluate the effects of postruminal fructose supply on small intestinal carbohydrate assimilation. Ten calves (<7 d of age; 41.2 ± 1.46 kg of body weight) were fed milk replacer at 2.0% of body weight daily (816 ± 90.5 g/d; 272 ± 30.1 g/L; dry-matter basis) in 2 equal portions and assigned to the following dietary treatment groups: (1) milk replacer (control; n = 6) or (2) milk replacer + 2.2 g of fructose/kg of body weight (fructose; n = 4). Calves were fed dietary treatments for 28 d, with jugular blood sampled every 7 d before and after the morning feeding. Calves were slaughtered, and visceral weights were recorded. Postruminal carbohydrase activities were assayed. Quantitative real-time PCR was conducted for small intestinal mRNA expression of nutrient transporters [solute carrier family 2 member 5 (GLUT5), solute carrier family 2 member 2 (GLUT2), and solute carrier family 5 member 1 (SGLT1)], carbohydrases (lactase, maltase-glucoamylase, and sucrase-isomaltase), and ketohexokinase (KHK). Data were analyzed using MIXED procedures in SAS version 9.4 (SAS Institute Inc, Cary, NC). Dietary fructose supplementation decreased serum glucose concentration. Small intestinal mass was greater in calves supplemented with fructose. Dietary fructose supplementation did not influence pancreatic α-amylase, small intestinal isomaltase, or maltase activities. Sucrase activity was undetected in the small intestine. Dietary fructose supplementation increased small intestinal glucoamylase activity per gram of tissue by 30% and increased maltase-glucoamylase mRNA expression by 6.8-fold. Dietary fructose supplementation did not influence mRNA expression of GLUT5, SGLT1, GLUT2, or KHK. Dietary fructose supplementation increased small intestinal lactase mRNA expression by 3.1-fold. Sucrase-isomaltase mRNA expression in the small intestine decreased 5.1-fold with dietary fructose supplementation. Dietary fructose supplementation does not induce sucrase activity in neonatal calves; however, sucrase-isomaltase may be transcriptionally regulated by dietary fructose in neonatal calves. More research is needed to compare glucose and fructose at isocaloric intakes to examine effects of dietary fructose at equal metabolizable energy intake.

Age-related chemokine alterations affect IgA secretion and gut immunity in female mice.

The chemokines CCL25 and CCL28, which promote immune cell migration, are primarily expressed in the small and large intestines and play critical roles in sustaining gut immunity. In particular, these chemokines are closely related to intestinal IgA secretion. However, there is no research regarding the effects of aging on CCL25 and CCL28 expression and function. Therefore, in the present study, we investigated the effects of aging on production of CCL25 and CCL28, and on gut immunity, especially IgA secretion, using young and aged female mice. By aging, the levels of small intestinal mRNA and protein of CCL25 lowered, while these levels of CCL28 in colon became higher. Moreover, the number of IgA-antibody secreting cells (IgA-ASCs) and total IgA concentration decreased in the small intestine due to the age-associated reduction of CCL25. In contrast, colonic IgA production was increased due to up-regulation of CCL28, while the number of colonic IgA-ASCs was unchanged with aging. These results clearly demonstrate that aging-associated decrease in small intestinal CCL25 production and increase in colonic CCL28 production c be involved in aging-associated deterioration of gut immunity.

Inhibitory Effect of Lactobacillus plantarum CQPC02 Isolated from Chinese Sichuan Pickles (Paocai) on Constipation in Mice

Sichuan pickles (Paocai) are a traditional Chinese fermented vegetable food that is rich in beneficial microorganisms. In this study, the inhibition of constipation by Lactobacillus plantarum CQPC02 (LP-CQPC02) isolated from Sichuan Paocai was studied by establishing a constipation model of mice using activated carbon. The constipation inhibition effect of LP-CQPC02 was determined by observing the defecation of mice, rate of active carbon propulsion, pathological section observation, serum index, and small intestinal mRNA expression. The results showed that LP-CQPC02 inhibited the decrease in body weight caused by constipation in mice. At the same time, LP-CQPC02 also increased the propulsion rate of activated carbon in the small intestine and shortened the time of discharge for the first black stool. LP-CQPC02 increased gastrin (Gas), motilin (MTL), endothelin (ET), and acetylcholinesterase (AChE) in constipated mice, and it also decreased the level of somatostatin (SS). qPCR results showed that LP-CQPC02 upregulated the expression of c-Kit, stem cell factor (SCF), and glial cell-derived neurotrophic factor (GDNF) and downregulated the expression of transient receptor potential cation channel subfamily V member 1 (TRPV1) and inducible nitric oxide synthase (iNOS) in mice with constipation, and a stronger effect was observed with a high concentration of LP-CQPC02. The experimental results showed that LP-CQPC02 has a stronger constipation inhibitory effect than the commonly used commercial Lactobacillus delbrueckii subsp. bulgaricus (LB), and the inhibitory effect of LP-CQPC02 on constipation increases with increasing concentration of LP-CQPC02. Therefore, LP-CQPC02 is a high-quality microbial resource with a good constipation inhibition effect and probiotic potential.

Salvianolate inhibits cytokine gene expression in small intestine of cirrhotic rats

To study the effect of salvianolate on expression of tumor necrosis factor (TNF)-α and interleukin (IL)-6 mRNA in small intestine of cirrhotic rats. Cirrhosis in rats was induced using CCl4 (0.3 mL/kg). Rats were randomly divided into non-treatment group, low-dose salvianolate (12 mg/kg) treatment group, medium-dose salvianolate (24 mg/kg) treatment group, and high-dose salvianolate (48 mg/kg) treatment group, and treated for 2 wk. Another 10 healthy rats served as a normal control group. Mortality of cirrhotic rats in each group was evaluated after treatment with salvianolate. Serum samples were taken from portal vein for the detection of endotoxin. Morphological changes in tissue samples from the ileocecum were observed under a light microscope. Expression of TNF-α and IL-6 mRNA in the small intestine of rats was analyzed by real-time reverse-transcriptase polymerase chain reaction. The mortality of cirrhotic rats in the non-treatment group was 37.5%. No cirrhotic rat died in the high-dose salvianolate treatment group. The serum endotoxin level was significantly higher in the non-treatment group than in the salvianolate treatment and normal control groups. The intestinal mucosal and villous atrophy, necrosis and shedding of the intestinal mucosal epithelium, observed in the non-treatment group, were reversed in different salvianolate treatment groups. The TNF-α and IL-6 mRNA expression levels in small intestine were significantly lower in different salvianolate treatment groups than in the non-treatment group. Salvianolate can reduce the endotoxin level, ameliorate the injury of intestinal mucosa, and inhibit the expression of TNF-α and IL-6 mRNA in small intestine of cirrhotic rats.

Study on the Expression of Organic Anion Transporting Polypeptide (oatp2a1) in Rat with Spleen Deficiency Syndrome and the Exploration of Clinical Significance

Objective: to explore the mechanism of transportation and transformation of dampness by the way of the expression of organic anion transporting polypeptide (oatp) superfamily member 2a1 (oatp2a1) mRNA in rat with spleen deficiency syndrome and the significance in transportation and transformation of dampness. Methods: 32 wistar male rats were divided randomly into four groups: normal group (n = 6), normal + AA group (n = 6), spleen deficiency group (n = 10), Spleen deficiency + AA group (n = 10). After reserpine-induced spleen deficiency model was made, intragastric administration of aristolochic acid (AA) was adopted for three days, the expression of oatp2a1 mRNA were detected in the tissues of lung, liver, kidney, stomach, small intestine and large intestine in four groups by using Fluorescent Quantitative-Polymerase Chain Reaction (FQ-PCR). Results: the expression of oatp2a1 mRNA in above six tissues could be detected. The ex-pression of oatp2a1 mRNA in liver tissue of rat with spleen deficiency syndrome was up-regulated compared to normal group (P = 0.035, P < 0.05), the expression of oatp2a1 mRNA in small intestinal tissue of rat with spleen deficiency syndrome was down-regulated compared to normal group (P = 0.004, P < 0.01), the expression of oatp2a1 in intestinal tissue in normal + AA group is down-regulated compared to normal group (P = 0.032, P < 0.05). Conclusions: oatp2a1 might be one of the material basis involved in transportation and transformation of dampness. The changes of expression of oatp2a1 mRNA in small intestine, liver tissue suggests that small intestine, liver might play an important role in the transportation and transformation of dampness in the state of spleen deficiency. We further concluded that the function of spleen’s governing transportation and transformation of dampness was not only including the function of the gastrointestinal, but also part of the liver function in some degree, which needs to be further studied.

Effect of Chitosan on Nitric Oxide Content and Inducible Nitric Oxide Synthase Activity in Serum and Expression of Inducible Nitric Oxide Synthase mRNA in Small Intestine of Broiler Chickens

The present study was conducted to determine the effects of chitosan on nitric oxide (NO) content and inducible nitric oxide synthase (iNOS) activity in serum, and relative expression of iNOS mRNA in the duodenum, jejunum, and ileum of broiler chickens. A total of 240 one-day-old Arbor Acre mixed-sex broiler chickens were randomly allotted to six dietary treatments with five replicates in each treatment and eight chickens in each replicate. The broiler chickens in the six treatments were fed the basal diet supplemented with 0 (control), 0.05, 0.2, 0.5, 1.0 or 2.0 g/kg chitosan. The trial lasted for 42 days. The results showed that dietary chitosan enhanced NO content and iNOS activity in serum as well as iNOS mRNA expression in the duodenum and ileum of broiler chickens in a quadratic dose-dependent manner (p<0.05), and improved jejunum iNOS mRNA expression in a quadratic dose-dependent manner (p<0.10) with increasing addition of chitosan. Chicks fed a diet containing 0.5-1.0 g/kg chitosan had higher NO content and iNOS activity in serum as well as small-intestinal iNOS mRNA expression compared with birds given the control diet, but positive effects of chitosan tended to be suppressed when addition of chitosan in the diet was increased to 2.0 g/kg. These results implied that there was a threshold level of chitosan inclusion beyond which progressive reductions in serum NO content and small intestinal iNOS expression occured, and the regulation of chitosan on immune functions in chickens is probably associated with activated expression of iNOS and NO secretion.

Cloning, chromosome mapping and expression pattern of porcine PLIN and M6PRBP1 genes

The PAT proteins, named after the three PLIN/ADRP/TIP47 (PAT) proteins, PLIN for perilipin, ADRP for adipose differentiation-related protein and TIP47 for tail-interacting protein of 47 kDa, now officially named M6PRBP1 for mannose-6-phosphate receptor binding protein 1, is a set of intracellular lipid droplet binding proteins. They are localized in the outer membrane monolayer enveloping lipid droplets and are involved in the metabolism of intracellular lipid. This work describes the cloning and sequencing of porcine PLIN and M6PRBP1 cDNAs, the chromosome mapping of these two genes, as well as the expression pattern of porcine PAT genes. Sequence analysis shows that the porcine PLIN cDNA contains an open reading frame of 1551 bp encoding 516 amino acids and that the porcine M6PRBP1 cDNA contains a coding region of 1320 bp encoding 439 amino acids. Comparison of PLIN and M6PRBP1 amino-acid sequences among various species reveals that porcine and bovine proteins are the most conserved. Porcine PLIN and M6PRBP1 genes have been mapped to pig chromosomes 7 and 2, respectively, by radiation hybrid analysis using the IMpRH panel. Expression analyses in pig showed a high expression of PLIN mRNA in adipose tissue, M6PRBP1 mRNA in small intestine, kidney and spleen and ADRP mRNA in adipose tissue, lung and spleen.

Molecular cloning, distribution and developmental regulation of porcine GLUT2 mRNA in small intestine

Molecular cloning, tissue distribution and developmental regulation of porcine GLUT2 (glucose transporter 2) mRNA were conducted in small intestine. GLUT2 cDNA is 2051 bp long, the predicted amino acid sequence revealed that it is highly conserved among vertebrate species. Animal tissue samples were collected from 35 pigs at different developmental stages: sucking (day 1, 7 and 26), postweaning (day 30) and growing (day 60, 90 and 150). The results showed that GLUT2 mRNA was detected at highest level in porcine liver, at moderate levels in the small intestine, kidney, and at low levels in the brain, lung, muscle and heart. In the small intestine, the highest level is in the jejunum. After weaning, the three small intestinal sements took on a similar pattern, GLUT2 mRNA increased through post‐weaning (d30, 2 days post‐weaning) and peaked in growing period (d90), and then decreased in finishing period (d150). In conclusion, the mRNA expression of GLUT2 was not only differentially regulated by age but also differentially distributed along the small intestine of piglets at different stages, which may be related to intestinal luminal substrate concentrations. This study was supported by the National Basic Research Program of China (Project No: 2004CB117501) and National Natural Science Foundation of China (Project No: 30671529).

A Targeted Mutation of Nkd1 Impairs Mouse Spermatogenesis

Nkd1 is an antagonist of the canonical Wnt/beta-catenin signaling pathway. The EF-hand motif of Nkd1 is required for its inhibitory function. Early studies suggested that Nkd1 might play important roles in mouse embryonic development and tumorigenesis. We constructed Nkd1(-/-) mice whose Nkd1 protein lacked the EF-hand and was unable to inhibit Wnt/beta-catenin signaling. The homozygotes were viable and grew normally, but their fertility in males was reduced. In wild-type adult testes, Nkd1 mRNA was expressed more abundantly in the elongating spermatids than in the round spermatids. Lack of EF-hand caused reductions in the testis weight and sperm count by 30 and 60%, respectively. During testis development, Nkd1 mRNA expression started at the 25th day after birth, coincident with the onset of Wnt1 expression. Nuclear localization of beta-catenin increased in the elongating spermatids, suggesting that the mutant Nkd1 failed to inhibit the Wnt/beta-catenin pathway. These results suggest that deletion of the EF-hand from Nkd1 reduces the number of the elongating spermatids at haploid stage. In contrast, the mutant Nkd1 did not affect intestinal polyposis in Apc(Delta716) mice.

Trefoil factors and human gastric cancer (Review)

TFF1/pS2, TFF2/SP and TFF3/ITF are soluble peptides with trefoil domain(s) and C-terminal dimerization domain, which are conserved among human, cow, mouse and rat. TFF1 mRNA is expressed in stomach (mucous cells in fundus and antrum), TFF2 mRNA in stomach (mucous neck cells in fundus and basal cells in antral and pyloric glands) and duodenum (Brunner's gland), TFF3 mRNA in small intestine and large intestine (goblet cells). Expression of TFF1, TFF2 and TFF3 mRNAs are differentially regulated by FGF2/bFGF, FGF7/KGF, estrogen, aspirin, arachidonic acid, X-ray irradiation, and hydrogen peroxide. Gastric cancer is classified into the intestinal type and the diffuse type. TFF mRNAs are preferentially expressed in diffuse-type gastric cancer cells. Custom-made microarray (TFF mRNAs) and ELISA (TFF proteins) might be applicable for screening methods of peritoneal and bone marrow dissemination from diffuse-type gastric cancer. TFF1 and TFF2 mRNAs are frequently down-regulated in intestinal-type gastric cancer. TFF1 gene, inactivated by deletion, missense mutation and promoter hypermethylation, is a tumor suppressor gene implicated in gastric cancer. TFF2 is a candidate tumor suppressor gene; however, genetic and epigenetic alterations of TFF2 gene in human gastric cancer remain unclear. TFF1, TFF2 and TFF3 play key roles in mucosal protection through mucous-barrier formation, and also in mucosal repair through promotion of restitution after injury. Patients with chronic atrophic gastritis and those with ulcerative colitis are at risk of gastric cancer and colorectal cancer, respectively. TFF1, TFF2 and TFF3 proteins might be applicable for chemoprevention of gastrointestinal cancer associated with chronic persistent inflammation.

Regulation of Intestinal Gene Expression by Dietary Zinc: Induction of Uroguanylin mRNA by Zinc Deficiency

The regulation of gene expression by nutrients plays an important role in the overall manifestations of nutritional deficiencies. Insufficient intakes of dietary micronutrients, such as zinc, produce profound effects in multiple organs and tissues. One of the major challenges, however, is to identify genes affected by changes in nutritional status. Differential display of mRNA has proved to be a valuable technique in meeting this challenge. In our ongoing search for genes responsive to dietary zinc, we compared small intestinal mRNA from rats that were fed zinc-deficient or -adequate diets using differential display to generate 3' anchored expressed sequence tags (EST). EST for intestinal mRNAs with altered expression due to zinc deficiency include two peptide hormones, intestinal fatty acid binding protein, intestinal alkaline phosphatase II, a proteasomal ATPase, cis-Golgi p28 and two subunits of the ubiquinone oxidoreductase. The EST for one of the hormones yielded the sequence for the 3' end of an mRNA encoding preprouroguanylin and was used to clone the remaining portion of the rat cDNA via 5' rapid amplification of cDNA ends. Northern blot analysis of RNA from rat intestine demonstrated that preprouroguanylin mRNA was 2.5-fold more abundant during zinc deficiency. Uroguanylin, a natriuretic peptide hormone, is an endogenous ligand for the same guanylate cyclase C that the Escherichia coli heat-stable enterotoxin (STa) binds when it causes secretory diarrhea by activating the cystic fibrosis transmembrane conductance regulator, thus altering fluid balance in the intestine. This suggests a mechanism whereby zinc deficiency could induce uroguanylin levels in the intestine and cause or potentiate diarrhea.

Effects of dietary protein and tumor necrosis factor on components of the insulin-like growth factor-I pathway in the colon and small intestine in protein-depleted rats

Intestinal cell growth is markedly affected by nutrient intake and the presence of cytokines. Since insulin-like growth factor-I (IGF-I) is an important hormone regulator of intestinal proliferation, this study examined the effects of dietary protein content and tumor necrosis factor (TNF) on mRNA levels of IGF-I, IGF-I receptor, IGF binding protein-3 (IGFBP-3), and IGFBP-4 and on the histology of the colon, jejunum, and ileum in protein-malnourished rats. After 2 weeks of feeding a 2% casein diet, rats continued on the 2% casein diet or were refed with a 20% casein diet and received daily intraperitoneal injections of either TNF (50 μg/kg) or saline for 4 days. The abundance of mRNA in the intestine was determined by RNA dot-blot analysis, and morphology measurements were performed by light microscopy. Simultaneous refeeding with the 20% casein diet and administration of TNF led to a modest increase in IGF-I and IGFBP-4 mRNA abundance in the colon. However, in the jejunum and ileum, refeeding had no effect but TNF caused a decrease in IGF-I and IGFBP-3 mRNA levels in malnourished rats. Refeeding with the 20% casein diet resulted in relatively modest histologic changes, which were greater in the colon versus the small intestine. The decreased magnitude of histologic changes in the order of the colon, ileum, and jejunum may reflect a response to a gradient of amino acid availability from intraluminal nutrients. These data demonstrate that TNF has distinct effects on colon and small intestine mRNA, but these mild changes had only a slight impact in the colon and did not translate into identifiable histologic changes in the small intestine. Combined protein restrictio and TNF administration had only a modest effect on intestinal mRNA levels and mucosal histology.

Transient expression of stromelysin-3 mRNA in the amphibian small intestine during metamorphosis.

It has been suggested that a matrix metalloproteinase, stromelysin-3 (ST3), is an important enzyme for epithelial transformation; the ST3 gene is known to be regulated by thyroid hormone during Xenopus metamorphosis. In this study, we have examined the distribution of ST3 mRNA in the small intestine of Xenopus during metamorphosis by using in situ hybridization. Around stage 58, ST3 mRNA is first detectable in larval fibroblasts near the muscular layer, and then increases in amount throughout the entire region of connective tissue. By stage 61, when connective tissue cells are rapidly increasing in number, ST3 mRNA is localized in fibroblasts just beneath the epithelium. This localization of ST3 mRNA is in good temporal accordance with modification of the basement membrane and epithelial transformation from the larval to adult form. Thereafter, ST3 mRNA gradually decreases and is no longer detected after stage 63 when the adult epithelium has completely replaced the larval type. The transient expression of ST3 mRNA in the fibroblasts of connective tissue during metamorphosis indicates that it plays an important role in epithelial transformation by changing the basement membrane.

Alternative mRNA Splicing and Differential Promoter Utilization Determine Tissue-specific Expression of the Apolipoprotein B mRNA-editing Protein (Apobec1) Gene in Mice.: STRUCTURE AND EVOLUTION OF Apobec1 AND RELATED NUCLEOSIDE/NUCLEOTIDE DEAMINASES

Apolipoprotein (apo) B mRNA editing consists of a C-->U conversion involving the first base of the codon CAA, encoding Gln 2153, to UAA, a stop codon. Editing occurs in the intestine only in most mammals, and in both the liver and intestine in a few mammalian species including mouse. We have cloned the cDNA for the mouse apoB mRNA editing protein, apobec1. Expression of mouse apobec1 cDNA in HepG2 cells results in the editing of the intracellular apoB mRNA. The cDNA predicts a 229-amino acid protein showing 92, 66, and 70% identity to the rat, rabbit, and human proteins, respectively. Based on the estimated values of divergence of apobec1 sequences in terms of the numbers of synonymous and non-synonymous suhstitutions per site, we found that apobec1 is a fairly rapidly evolving protein. Sequence comparison among mammalian apobec1 sequences has permitted the identification of seven conserved regions that may be functionally important for editing activity. We present a phylogenetic tree relating apobec1 sequences to double-stranded RNA adenosine deaminase and other nucleotide/nucleoside deaminases. Northern blot analysis indicates that apobec1 mRNA exists in two different sizes, a approximately 2.2-kilobase (kb) form in small intestine and a approximately 2.4-kb form in liver, spleen, kidney, lung, muscle, and heart. To study the molecular basis for the different sized apobec1 mRNAs, we cloned the apobec1 gene and characterized its exon-intron organization together with the sequences expressed in the hepatic and intestinal mRNA. The mouse apobec1 gene contains 8 exons and spans approximately 25 kb, and is located in chromosome 6. The major hepatic mRNA contains all 8 exons, whereas the major small intestinal mRNA misses the first 3 exons and its transcription is initiated in exon 4. The intestinal mRNA also contains at its 5' end a unique 102-nucleotide piece that is absent in the liver mRNA. We also identified two alternatively spliced hepatic apobec1 mRNAs with different acceptor sites in exon 4. Transient expression studies using promoter-reporter gene constructs in HeLa, Hepa, and Caco-2 cells indicate that the 5'-flanking sequences of the liver mRNA (i.e. upstream of exon 1) have predominantly hepatic promoter activity and the 5'-flanking sequences of the major small intestine mRNA (i.e. upstream of exon 4) have preferential intestinal promoter activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Epidermal growth factor and transforming growth factor-α mRNA in rat small intestine: In situ hybridization study

The expression of epidermal growth factor (EGF) and transforming growth factor-α (TGF-α) mRNA in the small intestine of suckling and adult rats was examined by in situ hybridization. EGF mRNA was found mainly in the intestinal crypts in adult rats. Adult rats also exhibited a considerably stronger signal for EGF mRNA in comparison to suckling rats, where the signal was very low or absent. In contrast to EGF, very strong expression of TGF-α mRNA was observed in the small intestine of both adult and suckling rats. These data suggest the differences between the expression of EGF and TGF-α in the developing small intestine.

Rabbit small intestinal trehalase. Purification, cDNA cloning, expression, and verification of glycosylphosphatidylinositol anchoring.

alpha,alpha-Trehalase (EC 3.2.1.28), an intrinsic protein of intestinal brush-border membranes, was purified to homogeneity from rabbits. Partial amino acid sequences were determined. Two degenerate oligonucleotides based on the sequence of a CNBr peptide were employed in a polymerase chain reaction to amplify a 71-base pair fragment of trehalase DNA with rabbit intestine cDNA as a starting template. This fragment was used as a hybridization probe to isolate full length trehalase clones from a rabbit intestine cDNA bank. Sequence analysis revealed that trehalase comprises 578 amino acids, contains at the amino terminus a typical cleavable signal sequence, at the carboxyl terminus a rather hydrophobic region typical of proteins anchored via glycosylphosphatidylinositol, and four potential N-glycosylation sites. Trehalase has no sequence homologies with other sequenced brush-border glycosidases. Northern blot analysis revealed a 1.9-kilobase trehalase mRNA in small intestine and kidney, smaller amounts in liver, and none in lung. Southern blot analysis indicated the gene has a length of 20 kilobase pairs or less. Injection into Xenopus laevis oocytes of mRNA synthesized in vitro from a trehalase template resulted in the expression of trehalase activity several hundredfold above background. The trehalase activity was membrane-bound and could be solubilized upon digestion with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis. This strongly suggests that rabbit small intestinal trehalase is anchored via glycosylphosphatidylinositol also when expressed in X. laevis oocytes.

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.

Expression of amino acid transport systems in Xenopus oocytes injected with mRNA of rat small intestine and kidney

Xenopus and Cynops oocytes were injected with exogenous mRNA prepared from rat small intestine and kidney and their electrical responses to amino acids were measured by both the current clamped and the voltage clamped methods. Oocytes injected with mRNA of rat small intestine showed a depolarization response to several neutral and basic amino acids, and almost no response to acidic amino acids. The responses to amino acids increased with incubation time after injection of mRNA, and followed Michaelis-Menten type kinetics. The responses were dependent on both Na + concentration and membrane potential, and were inactivated by a sulfhydryl reagent, 5,5-dithiobis(2-nitrobenzoate). These results are interpreted as due to the expression of Na +/amino acid cotransporter(s) in oocytes injected with rat small intestine mRNA. On the other hand, the oocyte injected with rat kidney mRNA showed a hyperpolarization response to neutral amino acids, a depolarization response to basic ones, and almost no response to acidic ones in frog Ringer solution. These responses were independent of Na + concentration and followed Michaelis-Menten type kinetics. These amino acid response characteristics in oocytes injected with rat kidney mRNA are interpreted as due to the expression of facilitated diffusion carrier protein(s) (uniporter) of amino acids in the oocyte.