Cloning of the novel gene intelectin, which is expressed in intestinal paneth cells in mice

OBJECTIVE: The purpose of our study was to assess the accuracy of CT-guided biopsy of musculoskeletal neoplasms with respect to technique, anatomic site, and histology. MATERIALS AND METHODS: During a 3-year period (January 1992 to December 1994), 176 core needle biopsies and 45 fine-needle aspirations were performed under CT guidance on patients with musculoskeletal neoplasms. To assess the accuracy of these procedures, we compared the diagnosis at biopsy with the final diagnosis as determined at the time of definitive treatment of the lesion. All biopsy findings were categorized as a primary malignancy (excluding round cell lesions), round cell lesion, local recurrence, or metastatic carcinoma. In addition, each lesion was analyzed according to which biopsy technique was used, whether frozen tissue section or rapid cytologic evaluation was used, and at which anatomic site the mass was found. RESULTS: The accuracy for needle biopsy was 93% and that for fine-needle aspiration was 80%. The complication rate for both techniques was less than 1%. Accuracy rates for the four categories of primary malignancy, round cell lesion, local recurrence, and metastatic carcinoma were 87%, 75%, 94%, and 100%, respectively. The mismatch rates were similar in soft-tissue lesions (5/52) and bone lesions (16/169). Diminished accuracy was associated with round cell lesions (20%) and lesions located in the spine or the perivertebral region (20%). Nondiagnostic and insufficient specimens were found in 18 (8%) of the 221 patients. CONCLUSION. CT-guided biopsy of musculoskeletal malignancies is a safe and effective procedure if performed by a team of clinicians, pathologists, and radiologists who possess subspecialty expertise.     

A new colorimetric method for the determination of free fatty acids with acyl-CoA synthetase and acyl-CoA oxidase

A rapid and sensitive spectrophotometric assay for free fatty acids using acyl-CoA synthetase and acyl-CoA oxidase is described. It is sensitive to as low as 5 nmol of free fatty acids, and the standard curve is linear up to 100 nmol. The assay consists of the measurement of H2O2 produced from free fatty acids by acyl-CoA synthetase and acyl-CoA oxidase. The quantity of H2O2 is determined by the absorbance at 550 nm in the presence of catalase and 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (AHMT). This method shows a broad specificity to long-chain fatty acids and the recoveries of added fatty acids (C12-C18) are more than 90%. The presence or absence of serum components or Escherichia coli cell-free extracts has no significant effect on the recovery of added palmitic acid.     

Cloning of BY55, a novel Ig superfamily member expressed on NK cells, CTL, and intestinal intraepithelial lymphocytes

Expression of the BY55 protein has been shown to be tightly associated with NK and CD8+ T lymphocytes with cytolytic effector activity. To determine the function of this protein, we molecularly cloned BY55 cDNA. The cDNA sequence predicts a cysteine-rich, glycosylphosphatidylinositol-anchored protein of 181 amino acids with a single Ig-like domain weakly homologous to killer inhibitory receptors. Reduction and carboxyamidomethylation of immunoprecipitated BY55 gave a band of 27 kDa, whereas reduction alone led to an 80-kDa species, suggesting that BY55 is a tightly disulfide-linked multimer. RNA blot analysis revealed BY55 mRNAs of 1.5 and 1.6 kb whose expression was highly restricted to NK and T cells. BY55 was expressed on the CD56dim, CD16+ subset of NK cells, which have high cytolytic activity, but was not expressed and was not induced on the CD56bright, CD16-subset of NK cells, a subset with high proliferative, but low cytolytic, capacity. In human tissues, BY55 mRNA was expressed only in spleen, PBL, and small intestine (in gut lymphocytes). BY55 was expressed on all intestinal intraepithelial lymphocytes, which were predominantly CD3+TCRalpha/beta+CD4-CD8+CD11b+CD28-CD45RO+C D56-CD101+CD103+ (alphaEbeta7 integrin). In addition, BY55 was expressed on most CD8+CD28- peripheral blood T cells. These phenotypic relationships suggest that CD8+CD28+ precursor CTL may terminally differentiate into CD8+CD28-BY55+ effector CTL and that some of the peripheral blood CD8+CD28- subset may represent recirculation from mucosal epithelial immune sites.     

Clostridium difficile toxin A binding to human intestinal epithelial cells

Clostridium difficile radiolabelled toxin A ([3H]-toxin A) bound to human duodenal and colonic epithelial cells isolated from endoscopic biopsies. Binding was greater at 4 degrees C than 37 degrees C, consistent with the thermal binding characteristic of toxin A to a carbohydrate moiety. At 37 degrees C colonic cells bound significantly more [3H]-toxin A than duodenal cells. The amount of [3H]-toxin A binding varied considerably between individuals. [3H]-toxin A was displaced by unlabelled toxin A by 50% for duodenal cells and 70% for colonic cells with 94.3 nM unlabelled toxin A. Low non-displacable binding was observed in some samples at 4 degrees C and 37 degrees C, suggesting that these cells came from individuals incapable of specifically binding toxin. Pre-treating cells with alpha- or beta-galactosidases to cleave terminal alpha- and beta-galactose residues reduced [3H]-toxin A binding. There was also a reduction in [3H]-toxin A binding after heat treating cells, which is suggestive of protein binding. The reduction in binding varied between individuals. The reduction of [3H]-toxin A binding, after the removal of beta-linked galactose units, implicates these as components of the receptor and adds credence to the idea that the Lewis X, Y and I antigens may be involved in toxin A binding to human intestinal epithelial cells. However, because the Lewis antigens do not possess terminal alpha-galactose units, the reduction in binding after alpha-galactosidase treatment suggests that other receptors may be involved in toxin A binding to some human intestinal cells. These data are the first demonstration of direct toxin A binding to human intestinal epithelial cells.     

Glutamine deprivation induces apoptosis in intestinal epithelial cells

BACKGROUND: Glutamine is the most abundant amino acid in the blood, and its deprivation leads to gut mucosal atrophy. The small intestinal mucosa is maintained by a balance between cell proliferation and cell death by apoptosis. We reported that glutamine is required for nitrogen-stimulated proliferation in intestinal epithelial cells. We do not know whether glutamine regulates apoptosis in the gut. The purpose of this study is to determine whether glutamine deprivation induces apoptosis in rat intestinal epithelial (RIE-1) cells and to compare the effect of glutamine starvation with that of methionine and cysteine (Met/Cys) starvation. METHODS: RIE-1 cells were deprived of either glutamine or Met/Cys for 24 hours. Cell numbers were determined by cell counting and tetrazolium enzymatic assay. Apoptosis was quantified by Annexin V assay and confirmed by DNA gel electrophoresis and Hoecsht nuclear staining. RESULTS: Deprivation of glutamine or Met/Cys resulted in decreased cell numbers. However, only the glutamine-deprived group showed significant induction of apoptosis with increased Annexin V staining, DNA laddering, and nuclear condensation. CONCLUSIONS: This study provides biochemical and morphologic evidence that glutamine deprivation induces apoptosis in rat intestinal epithelial cells. In contrast, Met/Cys starvation suppresses cell number without induction of apoptosis. These results suggest that glutamine serves as a specific survival factor in enterocytes.     

A sec1-related vesicle-transport protein that is expressed predominantly in epithelial cells

Sec1-related proteins are involved in docking and fusion of transport vesicles in eukaryotic cells. Here we report the cloning and molecular characterization of a Sec1-related protein expressed in the MDCK epithelial cell line. This protein represents a canine counterpart of the murine Munc-18-2/Munc-18b/muSec1 protein, displays 93% amino acid identity with these proteins, has a similar tissue mRNA expression pattern, and associates in vitro with syntaxins 1A, 2, and 3. In situ hybridization analysis of embryonic mouse tissues revealed prominent expression of the munc-18-2 mRNA in the epithelia of several tissues. Cell-fractionation studies demonstrated that the majority of Munc-18-2 is membrane associated. Most of the protein is washed off the membranes by sodium carbonate, pH 11.5. However, the protein is poorly solubilized by detergent treatment. The Munc-18-2 protein was localized, by immunofluorescence microscopy, to the plasma membrane of MDCK cells, and is apically distributed in the epithelial cells of mouse tissues. When overexpressed in COS-1 cells, the protein appeared to be largely cytosolic. However, upon expression with syntaxin 1A, it displayed a shift to the plasma membrane, where the two proteins colocalized. These results identified Munc-18-2 as a predominantly epithelial vesicle-transport protein with a polarized distribution and provided novel in vivo evidence for the association of Sec1-related proteins with members of the syntaxin family.     

Erythrocyte membrane reacylation in juvenile neuronal ceroid-lipofuscinosis: measurement of membrane-bound carnitine palmitoyl transferase, acyl-CoA synthetase, and lysophospholipid: acyl-CoA acyltransferase activities

In order to study the biochemical mechanisms responsible for the membrane fatty acid deficiency in juvenile neuronal ceroid-lipofuscinosis, we have analyzed the reacylation pathway in isolated erythrocyte membranes in 5 patients. We studied membrane carnitine palmitoyl transferase, and developed a combined assay to study acyl-CoA synthetase and lysophospholipid acyl-CoA acyltransferase activities. There were no significant differences between control and patient membranes, suggesting that abnormalities in these 3 putative candidate enzymes are not responsible for the disease.     

A mucosal intranet: intestinal epithelial cells down-regulate intraepithelial, but not peripheral, T lymphocytes

Epithelial cells and lymphocytes, including gammadelta and alphabeta T cells, in the gastrointestinal tract epithelium represent a major host defense intranet that is incompletely understood. Cell-to-cell interactions between intraepithelial lymphocytes (IELs) and intestinal epithelial cells (IECs) comprise this intranet, and we have assessed the role of IECs in the regulation of gammadelta and alphabeta T cell responses. When highly purified CD3+ IEL T cells were stimulated via the TCR-CD3 complex, high proliferative responses and cytokine synthesis were induced. However, the addition of viable IECs or purified IEC membranes (mIEC) down-regulated T cell proliferative and cytokine responses. Further, the inhibitory effect of mIEC was not restored by antibodies to TGF-beta, CD1d, E-cadherin, or MHC class I or II. This inhibitory effect was noted for both gammadelta and alphabeta T cell subsets from IELs, and mRNA levels were reduced for both Th1 (IL-2 and IFN-gamma) and Th2 (IL-4 and IL-5) cytokines in gammadelta and alphabeta IELs. In contrast, a purified membrane fraction obtained from thymocytes did not inhibit IEL proliferative responses. Further, mIEC did not inhibit splenic alphabeta T cell proliferative responses. These findings show that cell-to-cell interactions between intraepithelial gammadelta and alphabeta T cells and IECs occur via cell surface molecules, suggesting an intranet to prevent potential inflammatory responses at the intestinal mucosal surface.     

Differential membrane targeting and pharmacological characterization of chimeras of rat serotonin 5-HT1A and 5-HT1B receptors expressed in epithelial LLC-PK1 cells

The serotonin 5-HT1A and 5-HT1B receptors are two structurally related but pharmacologically distinguishable 5-HT receptor types. In brain, the 5-HT1A receptor is localized on the soma and dendrites of neurons, whereas the 5-HT1B receptor is targeted to the axon terminals. We previously showed that these two receptors are targeted in different membrane compartments when stably expressed in the epithelial LLC-PK1 cell line. Further investigations on the mechanisms responsible for their differential targeting were done by constructing chimeras of 5-HT1A and 5-HT1B receptors still able to bind specifically [3H]lysergic acid diethylamide and selective agonists and antagonists. Their cellular localization examined by confocal microscopy suggests that the third intracellular domain of the 5-HT1B receptor was responsible for its Golgi-like localization in transfected LLC-PK1 cells. In contrast, the third intracellular domain of the 5-HT1A receptor apparently allowed the sorting of the chimeras to the plasma membrane. Further inclusion of the C-terminal domain of the 5-HT1A receptor in their sequence led to a basolateral localization, whereas that of the 5-HT1B receptor allowed an apical targeting, suggesting the existence of a targeting signal in this portion of the receptor(s).     

A novel serine/threonine kinase gene, Gek1, is expressed in meiotic testicular germ cells and primordial germ cells

We have isolated a novel serine/ threonine kinase gene designated Gek1 from mouse primordial germ cell-derived embryonic germ cell. Gek1 is preferentially expressed in meiotic testicular germ cells and primordial germ cells. Gek1 mRNA is also detected in several other tissues, including hematopoietic organs in adult mice and central nervous system in embryos. The Gek1 cDNA encodes a protein with the consensus sequence of the catalytic domain of protein kinases in its N-terminal region. The deduced amino acid sequence of Gek1 in the kinase domain is related to those encoded by the Saccharomyces cerevisiae STE20, CDC15, and Drosophila melanogaster ninaC. The patterns of expression and the structural features of Gek1 suggest that the gene product is involved in signal transduction or nuclear division of germ cells and other proliferating cells. We also show that Gek1 locates on chromosome 11, near the wr locus, showing neuronal and reproductive defects.     

Adhesion and cytosolic dye transfer between macrophages and intestinal epithelial cells

Activated macrophages (M phi) found in the intestinal lesions of patients with inflammatory bowel disease (IBD) secrete many inflammatory mediators which can regulate intestinal epithelial cell (IEC) function. However, little is known about direct M phi-IEC interactions. Two potential mechanisms by which cells may interact are through specific receptor-ligand binding of adhesion molecules, such as integrins or cadherins, and by exchange of cytoplasmic substances through transmembraneous channels called gap junctions. We investigated whether M phi could adhere to epithelial cells in culture and form transmembrane communication channels as defined by dye transfer. Primary cultures of murine M phi and a M phi cell line, P388D1, adhered to Mode-K and IEC6, but not CMT-93 IEC. Antibody blocking studies determined that P388D1-Mode-K binding was partially dependent on beta 2 integrin (CD18) function, Mode-K constitutively expressed CD106 (VCAM-1) and cell associated fibronectin, while P388D1 expressed low levels of CD49d/CD29 (VLA4) but blocking antibodies to these surface molecules did not inhibit P388D1-Mode-K adherence. Transfer of calcein dye from M phi to IEC was quantitated by flow cytometry and was dependent on M phi-IEC adhesion. Dye transfer was concentration dependent in that the fluorescence intensity of Mode-K was proportional to the number of adherent P388D1 cells as well as the dye load of the M phi. These results indicate that M phi interact with IEC by adhesion and possibly through gap junctions and may thus regulate IEC function by direct cell-cell communication.     

Effect of glutamine on protein synthesis in isolated intestinal epithelial cells

The purpose of this study was to determine the etiologic factors of denture stomatitis. Fifteen subjects with clinical evidence of localized simple denture stomatitis, fifteen subjects without clinical signs of denture stomatitis, and forty-five subjects with clinical evidence of generalized simple denture stomatitis were investigated clinically and mycologically. Subjects were evaluated according to age, sex, duration of denture usage, smoking habits, frequency of denture brushing, overnight denture wearing, pH level of saliva and degree of candidal colonization and candidal formation. Salivary samples and swabs were taken from the palate and the mucosal surfaces of the dentures investigated mycologically in order to identify the yeast colonies. Smears were taken from the palate and investigated in order to identify candidal formation. No statistically significant relationship was found between denture stomatitis and age, sex, duration of denture usage, frequency of denture brushing, overnight denture wearing or pH level of saliva. There was however, a statistically significant relationship between denture stomatitis and denture hygiene, smoking habits, candidal colonization and candidal formation.     

Inhibition of Toxoplasma gondii replication in IFN-gamma-activated human intestinal epithelial cells

A therapeutic trial, involving 130 Schistosoma mansoni-infected children, with no previous history of antischistosomal treatment, was carried out to evaluate the efficacy of two different dose regimens of praziquantel. The study was carried out because low cure rates were described in this recently established (1990) S. mansoni focus in northern Senegal, following treatment with a standard dosage of 40 mg/kg. The subjects were randomly allocated into two groups: one group (1) received 40 mg/kg in one oral dose, the other group (2) was treated with two oral doses of 30 mg/kg at a 6-hr interval. Parasitologic examination and circulating anodic antigen (CAA) detection were performed before, 10 days, three, six, and 21 weeks after chemotherapy. No significant differences in cure rates were found between the two groups. Six weeks after treatment, 34% and 44% of the individuals were found to be stool negative in group 1 and group 2, respectively. However, only 10-15% became completely negative according to the serum CAA antigen assay. Mean egg counts were reduced by 99% in both groups. Antigen detection confirmed the parasitologic results. Fewer side effects were observed in the group treated with 2 x 30 mg/kg, which may be explained by split dosage administration. Our study shows that the low cure rates observed in this area could not be improved by using a higher dosage of praziquantel.     

A selective cyclooxygenase 2 inhibitor suppresses the growth of H-ras-transformed rat intestinal epithelial cells

BACKGROUND & AIMS: Constitutive expression of cyclooxygenase 2 (COX-2) has been found in 85% of colorectal cancers. Ras mutations are found in 50% of colorectal adenocarcinomas. The aim of this study was to determine the role of COX-2 in ras-induced transformation in rat intestinal epithelial (RIE) cells. METHODS: Cell growth was determined by cell counts. The expression of COX-2 was examined by Northern and Western analyses. For tumorigenicity assays, cells were inoculated into dorsal subcutaneous tissue of athymic nude mice. DNA-fragmentation assays were performed to detect apoptosis. RESULTS: The expression of COX-2 was increased in RIE-Ras cells at both messenger RNA (9-fold) and protein (12-fold) levels. Prostaglandin I2 levels were elevated 2.15-fold in RIE-Ras cells. Serum deprivation further increased COX-2 expression 3.8-fold in RIE-Ras cells. Treatment with a selective COX-2 antagonist (SC58125) inhibited the growth of RIE-Ras cells through inhibition of cell proliferation and by induction of apoptosis. SC-58125 treatment reduced the colony formation in Matrigel by 83.0%. Intraperitoneal administration of SC-58125 suppressed RIE-Ras tumor growth in nude mice by 60.3% in 4 weeks. SC-58125 treatment also induced apoptosis in RIE-Ras cells as indicated by increased DNA fragmentation. CONCLUSIONS: Overexpression of COX-2 may contribute to tumorigenicity of ras-transformed intestinal epithelial cells. Selective inhibition of COX-2 activity inhibits growth of ras-transformed intestinal epithelial cells and induces apoptosis.