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A J Ouellette

Publications and source records attributed to A J Ouellette.

At least 19 recordsLinked to original sources
PubMed1998

Amine binding and oxidation at the catalytic site for photosynthetic water oxidation.

Photosynthetic water oxidation occurs at the Mn-containing catalytic site of photosystem II (PSII). By the use of 14C-labeled amines and SDS-denaturing PAGE, covalent adducts derived from primary amines and the PSII subunits, CP47, D2/D1, and the Mn-stabilizing protein, can be observed. When PSII contains the 18- and 24-kDa extrinsic proteins, which restrict access to the active site, no 14C labeling is obtained. NaCl, but not Na2SO4, competes with 14C labeling in Mn-containing PSII preparations, and the concentration dependence of this competition parallels the activation of oxygen evolution. Formation of 14C-labeled adducts is observed in the presence or in the absence of a functional manganese cluster. However, no significant Cl- effect on 14C labeling is observed in the absence of the Mn cluster. Isolation and quantitation of the 14C-labeled aldehyde product, produced from [14C]benzylamine, gives yields of 1. 8 +/- 0.3 mol/mol PSII and 2.9 +/- 0.2 mol/mol in Mn-containing and Mn-depleted PSII, respectively. The corresponding specific activities are 0.40 +/- 0.07 micromol(micromol PSII-hr)-1 and 0.64 +/- 0.04 micromol(micromol PSII-hr)-1. Cl- suppresses the production of [14C]benzaldehyde in Mn-containing PSII, but does not suppress the production in Mn-depleted preparations. Control experiments show that these oxidation reactions do not involve the redox-active tyrosines, D and Z. Our results suggest the presence of one or more activated carbonyl groups in protein subunits that form the active site of PSII.

Amine Oxidase (Copper-Containing)↗
PubMed1997

Induction of epithelial chloride secretion by channel-forming cryptdins 2 and 3.

Salt and water secretion from intestinal epithelia requires enhancement of anion permeability across the apical membrane of Cl- secreting cells lining the crypt, the secretory gland of the intestine. Paneth cells located at the base of the small intestinal crypt release enteric defensins (cryptdins) apically into the lumen. Because cryptdins are homologs of molecules known to form anion conductive pores in phospholipid bilayers, we tested whether these endogenous antimicrobial peptides could act as soluble inducers of channel-like activity when applied to apical membranes of intestinal Cl- secreting epithelial cells in culture. Of the six peptides tested, cryptdins 2 and 3 stimulated Cl- secretion from polarized monolayers of human intestinal T84 cells. The response was reversible and dose dependent. In contrast, cryptdins 1, 4, 5, and 6 lacked this activity, demonstrating that Paneth cell defensins with very similar primary structures may exhibit a high degree of specificity in their capacity to elicit Cl- secretion. The secretory response was not inhibited by pretreatment with 8-phenyltheophyline (1 microM), or dependent on a concomitant rise in intracellular cAMP or cGMP, indicating that the apically located adenosine and guanylin receptors were not involved. On the other hand, cryptdin 3 elicited a secretory response that correlated with the establishment of an apically located anion conductive channel permeable to carboxyfluorescein. Thus cryptdins 2 and 3 can selectively permeabilize the apical cell membrane of epithelial cells in culture to elicit a physiologic Cl- secretory response. These data define the capability of cryptdins 2 and 3 to function as novel intestinal secretagogues, and suggest a previously undescribed mechanism of paracrine signaling that in vivo may involve the reversible formation of ion conductive channels by peptides released into the crypt microenvironment.

Amino Acid Sequence↗
PubMed1997

Paneth cells and innate immunity in the crypt microenvironment.

Paneth cells release granules into the lumen of the crypts of Lieberkuhn in the small intestine where their component proteins participate in mucosal immunity. The granules contain a number of proteins associated with roles in host defense, including lysozyme, secretory phospholipase A2, and alpha-defensins, termed cryptdins. Mouse cryptdins 1-6 and recombinant human Paneth cell alpha-defensin HD-5 are potent antimicrobial agents against certain microorganisms. As defensins, they kill microbes by disruption of the target cell membrane. The peptides are coded by individual, two-exon genes that map to homologous regions of chromosome 8 in mice and humans, and the differential expression of certain mouse cryptdin genes provides markers for studies of crypt ontogeny and epithelial cell differentiation and lineage determination. Neutrophil alpha-defensin peptides exhibit numerous biological activities in addition to antimicrobial function including regulation of cell volume, chemotaxis, mitogenicity, and inhibition of natural killer cell activity. When administered apically, mouse cryptdins 2 and 3 can reversibly stimulate human T-84 intestinal epithelial cells to secrete chloride ion, suggesting that alpha-defensins from Paneth cells also may be multifunctional. Thus, cryptdins and varied Paneth cell secretory products seem to contribute both to innate immunity of the crypt lumen and to defining the apical environment of neighboring cells.

Animals↗
PubMed1997

Cryptdin gene expression in developing mouse small intestine.

In rodents, the four intestinal epithelial cell lineages differentiate and become morphologically distinct during the first 2-3 postnatal wk. In studies reported here, reverse transcriptase-polymerase chain reaction (RT-PCR)-based assays detected Paneth cell defensin mRNAs in intestinal RNA from 1-day-old (P1) mice before crypt formation and maturation of the epithelium. Analysis of these defensin-coding RT-PCR products from P1 mice showed that 69% of clones sequenced coded for cryptdin-6, suggesting that it is the most abundant enteric defensin mRNA in the newborn. Paneth cell mRNAs, including cryptdins-4 and -5, lysozyme, matrilysin, and defensin-related sequences, also were detected in RNA from P1 mouse intestine. Unlike adult mice, where only Paneth cells are immunopositive for cryptdin, cryptdin-containing cells were distributed throughout the newborn intestinal epithelium and not in association with rudimentary crypts. Cryptdin immunoreactivity in the P1 mouse intestine was specific for intracellular granule contents, and immunofluorescent detection of cryptdins on mucosal surfaces suggested that the peptides are released into the intestinal lumen in P1 mice Defensin secretion may contribute to innate immunity of the neonatal intestine before the presence of distinguishable Paneth cells.

Aging↗
PubMed1996

Paneth cell defensins: endogenous peptide components of intestinal host defense.

Paneth cells are epithelial granulocytes at the base of the crypts of Lieberkühn in the small intestine of many mammalian species. These secretory cells contribute to mucosal barrier function by the apical release of granules containing a variety of antimicrobial products, including peptides termed cryptdins, for crypt defensins. In mice, six Paneth cell defensins have been characterized at the peptide level that have potent antimicrobial activities equivalent to or greater than that of rabbit neutrophil defensin NP-1. Cryptdin peptides that differ only by single amino acid substitutions have been shown to exhibit a high degree of specificity against certain target microorganisms. Cryptdins are coded by separate, two-exon genes that are located on chromosome 8 in both mice and humans. Human Paneth cells contain high levels of two different defensin mRNAs, but in mice at least 19 cryptdin isoforms are predicted from cDNA sequencing data. The mouse cryptdin-4 gene is expressed with positional specificity along the longitudinal intestinal axis, and cryptdin genes are active in the intestinal epithelium prior to Paneth cell differentiation. Accordingly, Paneth cell defensins are early markers of crypt ontogeny and are therefore useful in studies of lineage determination in the intestinal epithelium. Because cryptdins mediate innate immunity in the hostile environment of the intestinal lumen, it should be of interest to define biochemical and biophysical attributes that adapt these peptides to barrier function of mucosal surfaces.

Amino Acid Sequence↗
PubMed1996

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.

Animals↗
PubMed1995

Rat neutrophil defensins. Precursor structures and expression during neutrophilic myelopoiesis.

Defensins constitute a family of 3- to 4-kDa antimicrobial peptides that are stored in the cytoplasmic granules of neutrophils, some macrophages, and intestinal Paneth cells. We have assessed defensin gene expression during myeloid differentiation by first characterizing cDNAs for each of the four known rat neutrophil defensins (RatNP 1-4). The cDNA sequences revealed that the peptides are synthesized as 87-94 amino acid precursors, each containing signal, pro-, and mature peptide segments. RatNP-3 and -4 mRNAs, but not those for RatNP-1 and -2 or other myeloid defensins, contained unique polypurine tracts located close to the termination codon in the 3' untranslated region. By using cDNA probes and/or riboprobes, we evaluated defensin transcript levels in a variety of tissues and in the full spectrum of neutrophil precursors. By in situ hybridization, defensin mRNAs were localized to neutrophil precursors in the bone marrow, with the highest mRNA levels occurring in promyelocytes and somewhat lower signals occurring in myeloblasts and myelocytes. Defensin mRNAs were not detectable in bands or mature neutrophils, nor at significant levels in tissues other than bone marrow. The accumulation of defensin protein in bone marrow cells was assessed by immunohistochemical staining with anti-RatNP-1 Ab. RatNP 1-4 mRNAs and protein levels were then correlated for each stage of neutrophilic differentiation to reveal the maturational profile of myeloid defensin gene expression in the rat.

Amino Acid Sequence↗
PubMed1994

A family of defensin-like genes codes for diverse cysteine-rich peptides in mouse Paneth cells.

Cryptdins constitute a diverse population of defensins in Paneth cells of intestinal crypts. In mice, certain intestinal mRNAs, termed "CRSIC" and "CRS4C," are considered to be cryptdin-related sequences, because their prepro-coding sequences are 94% identical to those of cryptdin-1 mRNA; however, their predicted products, which are cationic, cysteine-rich peptides are not defensins (A. J. Ouellette and J. C. Lualdi, J. Biol. Chem. 265: 9831-9837, 1990). Here we describe several mouse small intestinal mRNAs and genes that code for CRS4C prepropeptides. The 10-kDa deduced CRS4C proteins consist of a prepro sequence, potential monobasic or dibasic peptide cleavage sites, a predicted 3.7-kDa peptide that contains 7 [C]-[X]-[Y] repeats, and a C(N/K)CNPK carboxyl-terminal consensus sequence. In situ hybridization experiments showed that CRS4C mRNAs are found in Paneth cells of adult small bowel. The CRS4C genes closely resemble cryptdin genes, having a two-exon structure with highly conserved transcription start sites, intron-exon junctions, and a single intron of approximately 550 bp. Like the cryptdin genes, exon 1 of CRS4C genes consists of 5' untranslated sequences (UTS) and the prepro-coding region, and exon 2 codes for the predicted mature peptide and 3' UTS. Despite the similarity of first exons in CRS4C and cryptdin genes, their introns exhibit very little homology, and their second exons code for unrelated peptides. Analysis of introns suggests that the ancestral cryptdin and CRS4C genes may have diverged from a common ancestor in the distant past and expanded only recently. We speculate that the cryptdin/CRS genes evolved so that prepro regions encoded by exon 1 were conserved to allow the varied peptides coded by exon 2 to be directed into Paneth cell secretory granules.

Amino Acid Sequence↗
PubMed1994

Structure and diversity of the murine cryptdin gene family.

Cryptdins are antimicrobial peptides of the defensin family produced by mouse intestinal Paneth cells. Characterization of genomic and cDNA clones of cryptdins 1-3, 5, and 6 revealed that each of these genes has a two-exon structure. The prepro- and mature peptide coding regions are found on different exons separated by an intron of approximately 550 bp. The 5' ends of cryptdin mRNAs are distinguished by a 45-nucleotide untranslated sequence (UTS) encoded completely by the first exon. This feature contrasts with the extended 5' UTS of myeloid defensin mRNAs, which are coded by a third exon that appears to be unique to defensin genes expressed in hematopoietic cells. Sequencing of cryptdin cDNAs from both C3H/HeJ and 129/SVJ mouse small intestine demonstrated the presence of at least 16 different mRNAs, identifying cryptdins as the largest known defensin family. Amplification of these two-exon crypt defensin genes, followed by mutation-induced divergence at a limited number of positions, may have played an important role in the development of a broad-spectrum enteric defense system in the mouse.

Amino Acid Sequence↗
PubMed1994

Mouse Paneth cell defensins: primary structures and antibacterial activities of numerous cryptdin isoforms.

Cryptdins are antimicrobial peptides of the defensin family that are produced by intestinal Paneth cells. mRNAs encoding 17 cryptdin isoforms have been characterized from a cDNA library generated from a single jejunal crypt. Six cryptdin cDNAs correspond to known peptides, and the remainder predict 11 novel Paneth cell defensins. Most cryptdin cDNAs have > or = 93% nucleotide sequence identity overall, except for cryptdin 4 and 5 cDNAs, whose respective mature peptide-encoding regions are only 74 and 78% identical to that of cryptdin 1. Cryptdin cDNAs differ at a small number of nucleotide positions: frequent substitutions were found in codons 38 and 52 of the propiece and in codons 68, 73, 76, 87, and 89 of the deduced peptides; cDNA clones with changes in codons 74, 83, and 88 were found, but there were fewer of these. The antimicrobial activities of cryptdins 1 to 6 were tested against Escherichia coli ML35 in two assays. In an agar diffusion assay, the potencies of cryptdins 1 to 3, 5, and 6 were approximately equivalent to that of rabbit neutrophil defensin NP-1 but cryptdin 4 was 30 times more active than NP-1. In a bactericidal assay system, cryptdins 1 and 3 to 6 were equally active at 10 micrograms/ml but cryptdin 2 and rabbit NP-1 were not active at this concentration. Since cryptdins 2 and 3 differ only at residue 10 (Thr and Lys, respectively), this amino acid appears to function in bactericidal interaction with E. coli. The demonstration that Paneth cells express a diverse population of microbicidal defensins further implicates cryptdins in restricting colonization or invasion of small intestinal epithelium by bacteria.

Amino Acid Sequence↗
PubMed1993

In vitro cDNA amplification from individual intestinal crypts: a novel approach to the study of differential gene expression along the crypt-villus axis.

The mouse small intestine is lined with a monolayer of continuously renewing epithelial cells. Cells of four distinct epithelial lineages are derived clonally from the stem cell zone, located near the crypt base, from which cells differentiate and migrate to the villus tip. The kinetics of the multilineage process are well understood. However, the molecular mechanisms underlying gene expression during lineage commitment and cell proliferation and differentiation remain obscure. A novel approach to the problem is presented here. Single intact epithelial crypts were isolated by incubation in ethylenediaminetetraacetic acid and mechanical vibration of everted mouse intestinal or colonic segments. Crypts isolated in this manner were suitable for mRNA-directed polymerase chain reaction, thus generating crypt epithelium-specific cDNA. The fidelity of transcript amplification was confirmed by Southern blot hybridization with cloned intestinal transcripts. To demonstrate the potential utility of crypt-specific cDNA, the amplified transcripts from a single jejunal crypt were used to construct a cDNA library, characterization of which revealed a high representation of cryptdin-1-related transcripts. This study presents a technique which will facilitate comprehensive analyses of gene expression in the differentiating mammalian intestine.

Animals↗
PubMed1993

Ontogeny of Ia messenger RNA in the mouse small intestinal epithelium is modulated by age of weaning and diet.

BACKGROUND: Exogenous antigenic peptides are presented to T cells by class II major histocompatibility complex (Ia) molecules on the surface of antigen-presenting cells. Class II-associated invariant chain (Ii) is also required for effective antigen presentation. Because messenger RNAs (mRNAs) for Ii chain and for class II I-A beta chain appear in the mouse intestinal epithelium after weaning, experiments were conducted to test the effect of age of weaning and diet on the appearance of Ia and Ii mRNA. METHODS: Four litters were split at day 17; one half was weaned and the other remained with the mother until day 24. On day 23, 25, 27, and 29, enterocytes were isolated from full-length small intestine by vascular perfusion with 30 mmol/L ethylenediaminetetraacetic acid, and the RNA was extracted. RESULTS: Appearance of Ii and I-A beta was significantly delayed by late weaning, as judged by RNA hybridization blots (Ii chain) and complementary DNA amplification (I-A beta chain). In mice on elemental diets, the appearance of Ii and I-A beta chain was delayed compared with littermates reared on standard chow. Ii mRNA failed to appear in mice maintained on the elemental diet by day 40, despite normal growth. CONCLUSIONS: Appearance of mRNA for both Ia and Ii depends on the introduction of a complex diet and not the "stress" of weaning or elimination of breast milk. Introduction of foreign dietary antigens or development of an altered intestinal flora may contribute to this process.

Age Factors↗
PubMed1993

Kid-1, a putative renal transcription factor: regulation during ontogeny and in response to ischemia and toxic injury.

We have identified a new putative transcription factor from the rat kidney, termed Kid-1 (for kidney, ischemia and developmentally regulated gene 1). Kid-1 belongs to the C2H2 class of zinc finger genes. Its mRNA accumulates with age in postnatal renal development and is detected predominantly in the kidney. Kid-1 mRNA levels decline after renal injury secondary to ischemia or folic acid administration, two insults which result in epithelial cell dedifferentiation, followed by regenerative hyperplasia and differentiation. The low expression of Kid-1 early in postnatal development, and when renal tissue is recovering after injury, suggests that the gene product is involved in establishment of a differentiated phenotype and/or regulation of the proliferative response. The deduced protein contains 13 C2H2 zinc fingers at the COOH end in groups of 4 and 9 separated by a 32-amino-acid spacer. There are consensus sites for phosphorylation in the NH2 terminus non-zinc finger region as well as in the spacer region between zinc fingers 4 and 5. A region of the deduced protein shares extensive homology with a catalytic region of Raf kinases, a feature shared only with TFIIE among transcription factors. To determine whether Kid-1 can modulate transcription, a chimeric construct encoding the Kid-1 non-zinc finger region (sense or antisense) and the DNA-binding region of GAL4 was transfected into COS and LLC-PK1 cells together with a chloramphenicol acetyltransferase (CAT) reporter plasmid containing GAL4 binding sites, driven by either a minimal promoter or a simian virus 40 enhancer. CAT activity was markedly inhibited in cells transfected with the sense construct compared with the activity in cells transfected with the antisense construct. To our knowledge, this pattern of developmental regulation, kidney expression, and regulation of transcription is unique among the C2H2 class of zinc finger-containing DNA-binding proteins.

Amino Acid Sequence↗
PubMed1993

Differential regulation of B7 mRNA in enterocytes and lymphoid cells.

To investigate the molecular details of antigen presentation by cells of lymphoid or epithelial origin, we compared B7 mRNA regulation in intestinal epithelium with that in spleen, since both cell types express class II major histocompatibility complex (MHC) and present antigen. As measured by cDNA amplification using sequence-specific primers, I-A beta mRNA content was found to be similar in mouse full-thickness small intestine, isolated intestinal epithelial cells and spleen. However, in contrast to I-A beta, B7 mRNA intestinal epithelial cell content was markedly lower than in spleen and whole small bowel; cardiac RNA was negative for both sequences. Administration of intraperitoneal interferon-gamma (IFN-gamma) (10(5) U daily for 2 days) to adult mice resulted in an increase in I-A beta mRNA in epithelial cells, but did not alter levels of B7 mRNA. In addition, exposure of the IEC-6 rat cell line to the IFN-gamma resulted in a dose-dependent increase in I-A beta mRNA without altering levels of B7 mRNA. Thus, an apparent dichotomy exists in regulation of B7 and I-A beta gene expression in rodent intestinal epithelial cells. Since maximal T-cell response to splenocytes depends on B7, the absence of B7 mRNA in intestinal epithelium may be a factor in determining why antigen-presenting enterocytes normally do not elicit damaging T-cell proliferative responses.

Animals↗
PubMed1992

Purification and primary structure of murine cryptdin-1, a Paneth cell defensin.

We have purified and determined the amino acid sequence of cryptdin-1, a murine Paneth cell defensin. The peptide corresponds to a previously characterized mRNA that accumulates to high abundance during postnatal ontogeny of the small bowel. Acid-extracted intestinal protein was fractionated by cation-exchange chromatography and fractions were assayed for antimicrobial activity. One peak of anti-Salmonella activity contained a putative defensin, based on its predicted electrophoretic migration in acid-urea PAGE. The peptide was purified to homogeneity by RP-HPLC and sequenced. These studies demonstrate defensin expression in non-myeloid tissue. The N-terminal extension of cryptdin-1 is a unique structural feature of this novel epithelial defensin.

Amino Acid Sequence↗
PubMed1992

Ontogeny of class II MHC mRNA in the mouse small intestinal epithelium.

MHC Class II (Ia) and invariant chain cooperate in the presentation of exogenous antigen by antigen presenting cells to T-helper cells. Both glycoproteins have been identified in the small intestine of the mature mouse. In this study, we examine the ontogeny of mRNA for three molecules; (Ii31, Ii41 and I-A beta) in whole intestine and in isolated epithelial cells. When RNA from whole intestine was analysed in northern blots using cDNA probe, Ii31 mRNA was present in Day 10 mice and at each 5 day time point thereafter; Ii41 and I-A beta were not detected by this technique. To examine ontogeny of Ii chain mRNA in enterocytes, RNA was purified from an enriched population of epithelial cells isolated after systemic perfusion with 30 mM EDTA in Day 21 and Day 28 and adult mice. Ii chain mRNA was not detected until Day 28 by blot hybridization. Reverse transcription of mRNA and amplification of the resultant cDNA by PCR revealed Ii41 and I-A beta as well as Ii31. RNA from Day 21 epithelial cells required five additional amplification cycles to attain cDNA levels equivalent to those found in Day 28 cells for Ii chain, and 10 additional cycles for I-A beta. In conclusion, Ii31, Ii41 and I-A beta mRNA increase rapidly in the enterocyte after weaning.

Aging↗