Developmental Gastroenterology Laboratory, Combined Program in Pediatric Gastroenterology and Nutrition, Massachusetts General Hospital, 114 16th Street (114-3650), Charlestown, MA 02129 and Harvard Medical School, Boston, MA
Received on June 18, 2004; revised on October 6, 2004; accepted on October 7, 2004
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Abstract |
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Key words: germ-free mice / hormonal regulation / microflora / postnatal development
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Introduction |
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Intestinal maturation can be regulated by three major factors: an intrinsic timing mechanism, circulating hormones, and extrinsic factors, such as luminal microbes. An intrinsic timing mechanism coordinates the onset of the adult phenotype (Biol et al., 1987; Dai and Walker, 1999
; Henning, 1987
; Hooper et al., 1998
). In the case of sucrase induction, however, glucocorticoids accelerate the ontogeny, leading to a rapid transition to the adult phenotype. The in vivo importance of glucocorticoids was reaffirmed by the discovery of a developmental surge of the circulating level of corticosterone in the rat and mouse 2 days prior to the initiation of gut ontogeny (Martin and Henning, 1984
; Nanthakumar and Henning, 1993
). In addition, both exogenous and premature induction of endogenous glucocorticoids can precociously induce an adult phenotype of rodent gut during the first 2 weeks postpartum. During this induction period, glucocorticoids stimulate proliferative cells to initiate differentiation into mature villus epithelium (Nanthakumar and Henning, 1993
; Henning, 1987
). The kinetics of this effect in vitro and in explant cultures suggests that steroids may affect differentiated epithelium indirectly through gut mesenchyme (Simo et al., 1992
). This sensitivity to glucocorticoids is lost after the third postnatal week with the onset of an adult intestinal phenotype (Nanthakumar and Henning, 1993
) by a mechanism that is not completely understood. Sucrase and lactase have been used as tissue-specific markers to elucidate the effect of steroids and their interplay with intrinsic timing mechanisms that lead to the onset of the adult phenotype. However, they are insensitive to luminal factors, such as changing microflora (Reddy and Wostmann, 1966
). Thus disaccharidases alone are not useful as markers for studying the interplay between steroids and luminal microbes in establishing the adult phenotype in the gut.
Gut microflora may also influence intestinal ontogeny. In all mammals, rapid colonization of gut microflora begins at birth (Berg, 1996; Dai and Walker, 1999
), followed by an equally rapid transition to a new complex microbial ecosytem at weaning (Berg, 1996
; Falk et al., 1998
). Adult human microflora usually includes more than 500 species of bacteria. This complex and dynamic society of commensal (and symbiotic) microflora forms a stable niche by adhering to the surface of the gut lumen (Berg, 1996
; Falk et al., 1998
; Hooper et al., 1998
; Karlsson, 1995
). This adherence is mediated through binding to glycoconjugates on the brush border membrane (Dai and Walker, 1999
; Dai et al., 2000
; Hooper and Gordon, 2001
). The change in luminal microflora during weaning parallels other changes in intestinal glycoconjugates (Biol et al., 1987
; Dai and Walker, 1999
; Midtvedt et al., 1987
; Pang et al., 1987
). For example, the ratio of sialic acid to fucose in the nonreducing terminal residues of surface carbohydrates of the rodent intestinal epithelium reverses from a predominance of sialic acid in the neonate to a predominance of fucose after weaning (Dai et al., 2002
); this has been attributed to a decrease in intestinal sialyltransferase activity coincident with a reciprocal increase in fucosyltransferase activity (Bry et al., 1996
; Dai et al., 2002
; Hooper et al., 2001
).
The developmental induction of 1,2-fucosyltransferase and ontogenic decline of
2,3/6-sialyltransferase observed in conventional mice are not observed in germ-free (GF) mice. However, on inoculation of GF mice with luminal bacteria from adult mice, even after maturity, these two enzymes achieve adult patterns of expression within 2 weeks (Dai et al., 2002
; Hooper et al., 2001
; Nanthakumar et al., 2003
). The level of expression of these two glycosyltransferases in adults is primarily regulated by microbial colonization (Bry et al., 1996
; Dai et al., 2002
). Bacteroides thetaiotaomicron is a commensal bacterium that colonizes the gut lumen by adhering to glycoconjugates with terminal
1,2-linked fucose; when adult GF mice are colonized by this bacterium alone, the expression of
1,2-fucosyltransferase is induced to normal mature levels (Bry et al., 1996
; Hooper et al., 2001
). Thus microflora may be an important extrinsic factor controlling the ontogeny of glycosyltransferases, leading to the reciprocal changes in the surface glycoconjugate content during the suckling to weaning transition. However, the interaction between extrinsic and intrinsic regulators of the developing gut could not be investigated without using markers specific to each. ß1,4-Galactosyltransferase (ßGT), because it synthesizes part of the glycan core rather than the more variable nonreducing terminus, is potentially a marker for developmental changes in general glycan expression. Mice whose gene for ßGT had been inactivated displayed phenotypes that included early death at the third postnatal week, coinciding with the loss of sucrase induction in the intestine (Asano et al., 1997
; Lu et al., 1997
). Therefore, we investigated whether ßGT and trehalase were suitable intestinal markers for comparing the regulatory control of extrinsic factors (luminal microbes) and intrinsic regulators (glucocorticoids). These two markers could be used to study the relative contributions of extrinsic and intrinsic factors in regulating the transition from the suckling to the adult intestinal phenotype.
In this study, experiments were designed to address five major issues. (1) To determine whether expression of ßGT is a marker of maturation distinct from that of trehalase, their ontogeny was compared in conventional (CONV) mice. (2) To determine the influence of glucocorticoids on ontogeny of intestinal ßGT, ßGT expression was compared in steroid-treated versus untreated CONV mice. (3) To determine the role of microbes in this process, the ontogeny of these two enzymes was compared between CONV and GF mice. (4) To determine influence of glucocorticoid regulation on ßGT and trehalase in the absence of microbes, ontogeny of these two markers was compared between GF and CONV mice. (5) To determine whether the loss of glucocorticoid responsiveness is dependent on microbial colonization, both ßGT and trehalase gene expression were measured in developing GF mice.
To answer these questions, the ontogeny of ßGT (UDP-Gal: GlcNAc ßGal ß1,4-galactosyltransferase, EC 2.4.1.38) and trehalase (EC 3.2.1.28) expression was determined by measuring their enzyme activities, mRNA induction, and glycoconjugate expression in the mouse duodenum, jejunum, ileum, and colon during the first 4 weeks of postnatal life. These experiments were performed in GF, CONV, and GF mice conventionalized (XGF) by inoculating GF mice with normal intestinal flora obtained from age-matched CONV mice.
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Results |
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To test whether indigenous microflora affect the development of intestinal ßGT, GF mice were inoculated with microflora from suckling mice (XGF-suckling) or CONV 6-week-old mice (XGF-adult). The XGF mice were sacrificed 2 weeks after the introduction of microflora. In the absence of microflora, the levels of ßGT activity (Figure 4A) remained low, which is typical of immature suckling mice regardless of actual age. On the introduction of luminal bacteria from aged-matched CONV mice (data from inoculation at 6 weeks shown), the levels of ßGT activity increased to that of CONV mice in the duodenum (p < 0.01), jejunum (p < 0.01), ileum (p < 0.005), and colon (p < 0.001) (Figure 4A). In contrast, the introduction of gut microflora from the suckling mice had no effect on ßGT activity. Unlike ßGT, the levels of trehalase enzyme activity (Figure 4C) of CONV, GF, and XGF mice showed no significant difference in any region of the small intestine whether inoculated at 4 weeks or 6 weeks of age. The mRNA levels for ßGT and trehalase displayed parallel changes during these treatments: Levels of ßGT mRNA (Figure 4B) increased on inoculation of microbes from the adults, whereas trehalase mRNA levels were unchanged (Figure 4D), suggesting that changes in intestinal ßGT activity (but not trehalase) is controlled by microflora that is unique to the suckling mice and these effects are manifested at the level of transcription.
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GF mice were treated with CA on days 9 and 17 and sacrificed 5 days thereafter. When these GF pups were treated with CA on day 9, trehalase activity was induced from low levels to elevated adult levels by day 14 (p < 0.01). However, when these GF pups were treated with CA on day 17, there was no change in trehalase activity. Note that the trehalase activity had already reached adult levels by this time and probably could not be further induced by CA (Figure 6C). Induction of trehalase activities was accompanied by induction of mRNA levels (Figure 6D), suggesting that these changes are regulated at the level of transcription. In GF mice, ßGT activity and mRNA were also low on day 9 and were induced by CA treatment to adult levels by day 14 (p < 0.01) (Figure 6A and 6B). However, following treatment with CA on day 17, the ßGT expression remains at low suckling levels. Thus even in the absence of microflora, trehalase activities follow the normal developmental pattern, but in the absence of microflora ßGT activity remains at immature levels, irrespective of CA treatment, suggesting that the developmental induction of ßGT is primarily regulated by colonization by adult microflora. This observation suggests that the loss of glucocortiocoid responsiveness is an intrinsic property of intestinal ontogeny and is independent of any enzyme induction by microflora. Thus early postnatal development is primarily controlled by intrinsic mechanisms and is sensitive to steroids, while after weaning, some enzymes, such as glycosyltransferases, are modulated primarily by extrinsic factors (microbial colonization).
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Discussion |
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Traditionally, disaccharidases have been used as developmental markers for investigating intestinal maturation (Henning, 1987; Mircheff et al., 1985
; Nanthakumar and Henning, 1993
). The phenotype of the suckling gut is characterized by high lactase and low trehalase and sucrase activities (Buller et al., 1990
; Nanthakumar and Henning, 1993
, 1995
; Oesterreicher et al., 1998
). During the weaning period, both trehalase and sucrase rapidly increase to adult levels in the mouse intestine while lactase decreases. Glycosyltransferase activities also change rapidly during this period, with
2,3/6-sialyltransferase activity changing from an initial high activity to the lower activity of the adult gut, whereas fucosyltransferase activity increases from an initially low activity to the elevated activity of adult gut (Dai et al., 2002
; Nanthakumar et al., 2003
). In this study, we found that both trehalase and ßGT increase with the maturation of the gut, but trehalase, like sucrase, is controlled more by an intrinsic mechanism of gut ontogeny. In contrast, ßGT, like
1,2-fucosyltransferase (Bry et al., 1996
; Hooper et al., 2001
), is controlled more by extrinsic factors. Therefore, trehalase and ßGT are good markers for comparing the relative contributions of intrinsic and extrinsic factors in gut ontogeny.
In mature mice, neither glucocorticoids nor luminal microbes caused increases in levels of trehalase and sucrase activities (Midtvedt et al., 1987; Nanthakumar and Henning, 1993
). However, because these marker enzymes are already at maximal levels by the fourth postnatal week, the lack of glucocorticoid responsiveness could be due to true lack of intrinsic tissue responsiveness at this age or simply due to the use of markers that have already reached maximum activity. In GF mice, the continued low expression of ßGT levels into adulthood provides an opportunity to determine whether the lack of intestinal responsiveness to steroids is truly a characteristic feature of mature gut. Figure 6 shows that GF mouse intestine after day 18 of postnatal life is unresponsive to steroids, as measured by the lack of change in the immature levels of ßGT. In a similar manner, 4-week-old GF mice also failed to respond to steroids (data not shown). Thus the loss of steroid responsiveness during the third postnatal week is an independent intrinsic feature of mouse gut development and not affected by extrinsic factors. This may provide guidance in the timing of the prophylactic use of glucocorticoids in premature infants to prevent necrotizing enterocolitis (NEC), a destructive inflammatory disease of the immature gut.
NEC is a devastating gastrointestinal tract disease of premature infants whose etiology is unclear. When premature infants are treated with glucocorticoids to prevent respiratory distress syndrome (Jobe, 2004; Kattner et al., 1992
; MacKendrick and Caplan, 1993
; Neu, 1996
), a coincidental decrease in NEC is observed. The degree of prevention is much greater with prenatal than with postnatal treatment (Bauer et al., 1984
). NEC is typically associated with an underdeveloped gut (Furlano and Walker, 1998
; Kliegman et al., 1993
). Thus exogenous glucocorticoids may act to accelerate intestinal maturation to afford protection against inflammation. However, not all studies on the use of glucocorticoids resulted in a decrease of NEC incidence (Jobe, 2004
; Kosloske, 1994
) because the intestine is sensitive to glucocorticoids for a restricted period during development (Nanthakumar and Henning, 1993
, 1995
). The loss of tissue responsiveness to steroids observed herein is consistent with a greater reduction in the risk of NEC with prenatal steroid exposure than with neonatal steroid treatment; however, NEC is a multifactorial disease whose etiology includes an inappropriate response to bacterial colonization after the introduction of enteral feeding (Furlano and Walker, 1998
; Kattner et al., 1992
; Kliegman et al., 1993
; MacKendrick and Caplan, 1993
; Neu, 1996
). Therefore the interrelationship between steroid-induced intestinal maturation and bacterial colonization may be an important element in our understanding the pathophysiology of this inflammatory disease of the immature gut.
Bacteria colonize the luminal surface of the gut through binding to specific glycoconjugates (Dai and Walker, 1999; Karlsson, 1995
). The surface of the intestinal epithelium has abundant glycoproteins and glycolipids intrinsic to the apical brush border membrane, as well as secreted glycoproteins on the apical surface (such as digestive fluids and mucus) and on the basolateral surface (such as extracellular matrix components and signaling molecules) (Dudeja et al., 1988
; Mircheff et al., 1985
; Morita et al., 1986
; Mulivor et al., 1978
; Robbe et al., 2003
; Roth et al., 1985
, 1986
; Srivastava et al., 1987
). Many of the terminal glycans of these glycoconjugates serve as receptors for pathogenic microbes in the gut lumen. For example, galactose residues of O-linked glycoconjugates play an essential role in the early stages of Entamoeba histolytica attachment and invasion (Hughes et al., 2003
) and in enteroaggregative Escherichia coli adhesion (Grover et al., 2001
). A more detailed knowledge of the galactose-terminal glycoconjugate structures and their functions in gut and the role of ßGT in generating these receptors is needed to better understand the pathophysiology of galactose-binding organisms. Specifically, regional specific expression of galactose-containing glycans and their temporal changes in expression during development helps us understand the regional affinity and temporal susceptibility of gut to infection during development.
Changes in expression of glycosyltransferases can result in an alteration in the composition of the terminal moieties of glycoconjugates that control important developmental processes (Biol et al., 1987; Kotani et al., 2001
; Lu et al., 1997
). The importance of the ßGT is apparent by the disruption of normal development in its absence (Kido et al., 1999
; Kotani et al., 1999
; Love et al., 2001
). The targeted disruption (knock-out) of the mouse ßGT gene is semi-lethal during the weaning period. However, the cause of this lethality is not well understood (Asano et al., 1997
; Lu et al., 1997
). Curiously, in these mutant mice, the expression of an adult phenotype in the gut is apparent by the end of the first postnatal week (Asano et al., 1997
). These studies suggest that ßGT plays a critical role in epithelial differentiation in developing mouse intestine and may be necessary for handling the microbial colonization in the gut lumen associated with weaning (Asano et al., 1997
). Although endocrine insufficiency of the anterior pituitary gland has been postulated as a mechanism in the lethal phenotype of these mutant mice (Lu et al., 1997
), alteration in the gut physiology due to lack of ßGT during the weaning period may also contribute.
In CONV mice, the timing of the changes in intestinal ßGT coincides with a shift from microflora characteristic of suckling animals to that of adults. In this study we have attempted to begin addressing the question of whether changes in mucosal glycoconjugate expression select for the nature of colonizing microflora or whether a change in colonizing microflora induces changes in intestinal glycoconjugate expression (microbial-epithelial cross-talk). CONV suckling mice have a different microbial ecosystem than that of adult mice. When the adult GF mice were inoculated with microbes from immature animals, ßGT expression was not induced, but when inoculation occurred with adult microflora ßGT expression was induced to the levels comparable to CONV adult mice (Figure 4). These data imply that the elevated levels of intestinal ßGT that characterize the adult gut are dependent on colonization by specific bacteria at the time of weaning that are intrinsic constituents of the commensal flora in the adult intestine. This change in ßGT can be used as a marker for changes in activity of many glycosyltransferases that are induced during the general shift in microflora at weaning (Biol et al., 1987; Lenoir et al., 2000
; Srivastava et al., 1987
), which in turn cause a modification in the types of glycoconjugates expressed in gut during development. For example, the activity of N-acetylgalactosyltransferase also increases during postnatal development of the mouse gut (Dall'Olio et al., 1990
). One would expect the change in expressed glycoconjugates in the intestinal mucosa to influence the type of bacteria colonizing the gut. It is possible, however, that one subset of specific colonizing bacteria induced these shifts in glycoconjugate receptors through specific alteration in gene expression, which in turn select for those and other bacteria with this glycoconjugate as an adhesion factor to become part of the stable adult microflora (Midtvedt et al., 1987
; Umesaki et al., 1995
). These phenomena may represent a mutually beneficial cross-talk during intestinal ontogeny, that is, a reciprocal communication between colonizing bacteria and the gut in which bacteria influence the ontogeny of the gut, which in turn causes expression of glycoconjugates that help select and stabilize the colonizing microflora of the adult gut.
This study distinguishes between intrinsic and extrinsic regulation of intestinal ontogeny in mice using two markers of development: (e.g., trehalase as a marker for the general development of gut and ßGT as a marker of maturation of glycosylation subsystems in gut development). Glucocorticoids may modify the intrinsic genetic program of the developing intestine before the onset of weaning, as exemplified by disaccharidase expression, whereas microbial colonization appears to be a major extrinsic modifier of development from weaning to adulthood, as exemplified by glycosyltransferase expression. Extrinsic factors, circulating glucocorticoids, and the intrinsic genetic program of a tissue work synergistically and are responsible for the rapid development of the gut that occurs during the third postnatal week, thus accommodating the digestive function of the nutrients as well as accompanying changes in the resident microbial ecosystem and development of the mucosal defense. These mechanisms may underlie reciprocal control between resident bacteria and the intestinal mucosa (cross-talk) in which bacteria induce the production of glycoconjugates to which they bind, reinforcing their stable colonization of the gut to the mutual benefit of the human host and the microflora. We hypothesize that premature infants are unable to participate in such symbiosis and that their inappropriate colonization may lead to the onset of imbalance leading to diseases (e.g., NEC). In addition, the use of GF mice provides an approach to the understanding of the role of changes in intestinal glycoconjugates in elucidating the pathobiology of enteric infections.
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Materials and methods |
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Experimental animals
Black Swiss mice were purchased from Taconic Farms (Germantown, NY) as young adults (46 weeks) or as pregnant dams at 1618 days of gestation. Their pups' date of birth was designated as day 0. Pups were housed with their dams through 21 postnatal days under conventional conditions, whereupon they were weaned to mouse chow and water ad libitum in an animal room with a 12-h light/dark cycle and access.
Cortisone treatment
Each litter of suckling mice was divided randomly into two groups. At 10 days of age, one group was injected subcutaneously with a single dose (5 mg/100 g body weight) of cortisone acetate suspended in saline. The other group (controls) was injected with the same volume of normal saline (0.9% NaCl). The animals were maintained with their dams until they were sacrificed at 14 days of age. Cortisone-treated adult animals were injected on day 28 and sacrificed on 28, 32, 35, and 42 postnatal days.
GF animals and conventionalization
GF mice of the same strain were purchased from the same vendor at the desired ages. All GF animals were removed from the GF environment immediately before being sacrificed by cervical dislocation. XGF mice were produced by removing GF mice from their GF environment at the age of 4 weeks, inoculating them with intestinal contents from age-matched CONV mice as previously described (Dai et al., 2002; Midtvedt et al., 1987
), and keeping them in the same cage as CONV mice. They, along with age-matched GF and CONV mice, were sacrificed 2 weeks later by cervical dislocation. All animals were sacrificed between 12 noon to 3 PM to avoid circadian influences.
Preparation of enzyme fractions
The entire small intestine and colon were removed and thoroughly flushed with ice-cold 0.9% NaCl. The small intestine was divided into duodenum, jejunum, and ileum as follows: The small intestine from the stomach to the ligament of Treitz was defined as duodenum, and the proximal and distal halves of the remaining small intestine were defined as jejunum and ileum, respectively. The intestine was placed on a glass plate maintained at 4°C and cut open; the mucosa was harvested by scraping with a microscope glass slide. All subsequent procedures were performed at 4°C. A 10% mucosal homogenate in 0.1 M TrisHCl buffer (pH 7.4) was centrifuged at 1,000 x g for 15 min to remove nuclei and cellular debris. The supernatant was then centrifuged at 105,000 x g for 1 h in a Beckman L6-65 ultracentrifuge with 50.3 Ti rotor, leading to sedimentation of a microsomal fraction and to a soluble cell fluid. The enzyme activities were determined on the microsomal fraction. The resulting pellets were resuspended in the same buffer used for homogenization, aliquoted, frozen, and stored at 80°C, or used immediately for the enzyme assay.
Protein determination
Protein was determined using a BCA protein assay (Pierce, Rockford, IL) modified for use in 96-well microtiter plates according to the manufacture's protocol. To each protein sample of 50 µl, 200 µl of working reagent was added, followed by incubation at 37°C for 30 min. Absorbance at 560 nm was measured on a microtiter plate reader (BT 2000 Microkinetics Reader Spectrophotometer, Fisher Biotech, Pittsburgh, PA). The concentration of each protein sample was calculated using a standard curve produced with bovine serum albumin.
GT assay
The ßGT (UDP-Gal: GlcNAc ßGal ßGT) is the primary enzyme responsible for transferring terminal galactose from UDP-galactose to terminal N-acetylglucosamine of complex N-glycans. Asialo-agalactofetuin was the exogenous acceptor for the assay of intestinal ßGT (Lenoir et al., 2000; Ozaki et al., 1989
). The reaction mixture (total volume 0.1 ml) contained 0.4 mg asialo-agalactofetuin, 5 mM ATP, 100 mM cacodylate acetate (pH 6.3), 20 mM MgCl2, 0.5% Triton X-100, an appropriate amount of enzyme solution (50100 µg protein), and UDP-[14C]galactose (29.9 Ci/mmol, New England Nuclear) diluted with nonradioactive UDP-galactose (Sigma) to attain a concentration of 0.6 mM, 0.5 µCi. Incubation was carried out at 37°C for 1 h. The radioactive product formed was collected on Gelman GN-4 nitrocellulose filters and measured in an LKB scintillation beta counter (Dai and Walker, 1999
). Both UDP-galactose and the exogenous protein acceptor were present at saturating concentrations, and product formation was linear for 1 h of incubation time and up to 150 µg enzyme protein under these conditions. The activity of ßGT was stable in storage at 20°C for a year.
RNA preparation
Tissues were frozen in liquid nitrogen and stored at 80°C. The frozen tissue was homogenized, and total RNA was extracted using the TRIZOL reagent according to the manufacturer's protocol (Invitrogen, San Diego, CA). Total RNA was then dissolved in 50 µl of RNase-free water, and concentration was determined by measuring absorbance at 260/280 nm.
cDNA synthesis
RNA (1 µg) was used to generate cDNA using TaqMan Reverse Transcription Reagents according to the manufacturer's protocol (Applied Biosystems, Foster City, CA). Briefly, random hexamers were used to prime RNA samples for reverse transcription using MultiScribe Reverse Transcriptase.
qRT-PCR
Real-time quantitative PCR (qRT-PCR) was performed in a ABI 7700 sequence detection system using the TaqMan Cytokine Gene Expression Plate I (Applied Biosystems). The plate consists of a MicroAmp Optical 96-well reaction plate arranged into six columns for each mRNA in triplicate. Each column is made up of identical wells containing TaqMan primers and probes for ßGT or trehalase mRNA with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as endogenous control. PCR primers and probes for ßGT, trehalase and GAPDH were made as described previously (Dai et al., 2002; Lu et al., 1997
). The TaqMan probe was labeled with a reporter fluorescent dye, FAM (6-carboxyfluorescein), at the 5' end and a fluorescent dye quencher, TAMRA (6-carboxy-tetramethyl-rhodamine), at the 3' end. PCR primers and a TaqMan probe for GAPDH were obtained from Perkin-Elmer.
Detection of PCR products was measured by two dye layers to detect the presence of target and control sequences. The FAM dye layer yields the results for quantification of target mRNA, and the VIC dye layer yields the results for quantification of the GAPDH RNA endogenous control. Reaction mixtures for the qRT-PCR had a final volume of 50 µl containing 5 µl of cDNA and 25 µl of the master mix. Amplification conditions were: 2 min at 50°C, 10 min at 95°C, and 40 cycles of 15 s at 95°C and 1 min at 60°C. The endpoint used in RT-PCR quantification, CT, is defined as the PCR cycle number that crosses the signal threshold. CT values range from 0 to 40, with the latter number assumed to represent no product formation. Quantification of gene expression was performed using the comparative CT method (Sequence Detector User Bulletin 2; Applied Biosystems) and reported as the fold difference relative to the housekeeping gene. To calculate the fold change (increase or decrease), the CT of the housekeeping gene GAPDH was subtracted from the CT of the target gene to yield the net CT(CTn). Change in expression of the normalized target gene as a result of an experimental manipulation was expressed as 2 CTn where CTn = CT samples CT controls. PCR reactions lacking either cDNA, primers, or reverse transcriptase were run as controls.
Lectin-fluorescent staining with RCA-I
Analyses of ß1,4 galactosyl glycoconjugates was performed on frozen tissue sections using FITC-conjugated RCA-I agglutinin. One centimeter of tissue from each region was fixed for 2 h in 4% paraformaldehyde at 4°C, washed in ice-cold phosphate buffered saline (PBS) containing 30% sucrose overnight at 4°C, and embedded in OCT. Frozen sections (67 µm thick) were blocked with PBS containing 2% bovine serum albumin, then stained with FITC-RCA-I for 1 h ( 10 µg/ml). Sections were then washed three times in cold PBS, mounted using Anti-Fade (Vector Laboratories, Burlingame, CA), and analyzed by fluorescent and/or confocal microscopy.
Statistical treatment of results
Results are expressed as the mean ± SE. Effects of age and treatment on enzyme activities were analyzed by two-way ANOVA. After overall significance was confirmed, post hoc tests for individual variables were performed by a two-tailed unpaired t-test. Differences with a p value <0.05 were considered significant.
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Acknowledgements |
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Abbreviations |
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References |
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