Maltase-glucoamylase and trehalase in the rabbit small intestine and kidney brush border membranes during postnatal development, the effects of hydrocortisone

Kidney and intestinal brush border membranes were isolated from 14-day-old rabbits and papa?n solubilized maltase-glucoamylase was purified to almost homogeneity from both membranes. Maltase-glucoamylase from kidney and intestine have the same molecular weight (669,000 daltons by AcA 22 gel filtration) and the same Km (4 mM, for maltose). Tris (Ki = 12.5 mM, for maltose) is a non-competitive inhibitor for both enzymes. In intestine, maltase and glucoamylase have low activity during the first two postnatal weeks and then undergo a sharp increase during the next 2 weeks. In contrast, for trehalase, adult levels are reached about 6 days after birth. Hydrocortisone injection to 10 days rabbits causes precocious increases in the specific activities of trehalase (3.6 x), maltase (5.2 x) and glucoamylase (7.4 x). Conversely, kidney maltase, glucoamylase and trehalase activities rise gradually from birth, reaching adult levels by the end of the third week. Administration of hydrocortisone to suckling rabbit does not affect either trehalase or maltase and glucoamylase in kidney brush border membrane.     

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.     

In vitro loss of hydrophobicity of trehalase from the brush border membrane of rabbit kidney cortex

Trehalase solubilized with 0.5% Triton X-100 and 0.5% deoxycholate from the brush border membrane of rabbit kidney cortex was all adsorbed on phenyl-Sepharose equilibrated with elution buffer containing no detergents, and all the adsorbed enzyme was eluted in one peak on the addition of 0.5% Triton X-100 to the elution buffer, in contrast to the results reported by Nakano and Sacktor (J. Biochem. 97, 1329-1335 (1985], who separated two forms of trehalase differing in hydrophobicity from rabbit kidney. On concentration of detergent-solubilized extracts, followed by incubation at 37 degrees C, however, there appeared trehalase nonadsorbable on phenyl-Sepharose, i.e. a hydrophilic trehalase. Various protease inhibitors added to the concentrated extracts did not inhibit this conversion at all. The concentration-incubation treatment also increased the proportion of trehalase that interacts with Con A-Sepharose. These results indicate that kidney trehalase that interacts with Con A-Sepharose. These results indicate that kidney trehalase is susceptible to some lytic action of a factor(s) intrinsic to the brush border membrane (limited autolysis), as seen with rabbit intestinal trehalase (Yokota et al., (1986) Biochim. Biophys. Acta 881, 405-414). Therefore, in studies of the molecular form of trehalase (and other proteins) in the brush border membrane of the kidney and intestine where a lot of hydrolases exist, it is very important to take account of limited autolysis which results in some chemical modifications without affecting enzymatic activity.     

Origin and developmental patterns of lactase and other glycosidases in sheep amniotic and allantoic fluid.

Intestinal lactase activity (with its associated cellobiase, 4-methylumbelliferyl-beta-galactosidase and -beta-glucosidase activities) was used as a specific intestinal marker enzyme to study the release of protein and enzymes of intestinal origin in sheep amniotic fluid during gestation. In amniotic fluid, intestinal lactase activity peaked at 66--85 days of gestation and then decreased with gestation. This enzyme activity was very low or absent in allantoic fluid throughout gestation suggesting that there is no important transfer of amniotic fluid lactase towards the allantoic cavity. Maltase and 4-methylumbelliferyl-alpha-glucosidase showed no statistically significant variation with gestation in both amniotic and allantoic fluid whereas alpha-galactosidase and N-acetyl-beta-hexosaminidase which were first higher in allantoic than in amniotic fluid increased in amniotic fluid to reach allantoic fluid levels near term. Such patterns are consistent with the suggestion that the fetal urine is a source of alpha-galactosidase and N-acety-beta-hexosaminidase activities and that sheep urine is first accumulated in the allantoic sac via the urachus up to 86--90 days of gestation and thereafter passes more and more into the amniotic sac.     

Temporal proteomic analysis reveals defects in small-intestinal development of porcine fetuses with intrauterine growth restriction

The fetus/neonate with intrauterine growth restriction (IUGR) has a high perinatal mortality and morbidity rate, as well as reduced efficiency for nutrients utilization. Our previous studies showed alterations of intestinal proteome in IUGR piglets both at birth and during the nursing period. Considering the potential long-term impacts of fetal programming and substantial increases in amounts of amniotic fluid nutrients from mid-gestation in pigs, the present study involved IUGR porcine fetuses from days 60 to 110 of gestation (mid to late gestation). We identified 59 differentially expressed proteins in the fetal small intestine that are related to intestinal growth, development and reprogramming. Our results further indicated increased abundances of proteins and enzymes associated with oxidative stress, apoptosis and protein degradation, as well as decreased abundances of proteins that are required for maintenance of cell structure and motility, absorption and transport of nutrients, energy metabolism, and protein synthesis in the fetal gut. Moreover, IUGR from middle to late gestation was associated with reduced expression of intestinal proteins that participate in regulation of gene expression and signal transduction. Collectively, these findings provide the first evidence for altered proteomes in the small intestine of IUGR fetuses, thereby predisposing the gut to metabolic defects during gestation and neonatal periods.     

Localization of renal and intestinal trehalase with immunofluorescence- and enzyme-labeled antibody techniques

The localization of trehalase with fluorescein isothiocyanate-conjugated and peroxidase-conjugated antibody techniques was examined. Antiserum against purified rabbit renal trehalase was produced against guinea pigs. Anti-renal trehalase immunoglobulin (Ig)G was isolated from the serum and used for the immunohistochemical localization of intestinal and renal trehalases. Specific fluorescence and peroxidase staining were observed in the brush borders of proximal tubules and of intestinal epithelial cells. These results are in good agreement with the biochemical results. Thus, it is concluded that trehalase is specifically localized in the renal and intestinal brush borders. Sections of rabbit intestine and of rabbit kidney treated with anti-rabbit renal trehalase IgG were observed to have a specific fluorescence at the brush borders. Sections of rat intestine treated with the same antibody, however, showed no specific fluorescence at the brush borders. From these results, it is strongly suggested that renal trehalase and intestinal trehalase from the rabbit have common antigenic determinants and that these differ from those in rat intestinal trehalase.     

The influence of experimentally produced oligohydramnios on lung growth and pulmonary surfactant content in fetal rabbits.

To study the effect of oligohydramnios on lung growth and biochemical lung development in fetal rabbits, amniotic fluid was drained through a tube inserted into the maternal peritoneal cavity on the 23 day of gestation. Littermate fetuses without an amniotic shunt were used as controls. The fetuses were delivered abdominally on the 28 day of gestation. In a total of 8 pregnant does, 17 fetuses underwent amniotic shunting and 22 fetuses were used as controls. The amniotic shunt produced a significant reduction in the amniotic fluid volume. There were no differences in the wet weights of the fetal body, liver or brain between the two groups. However, the amniotic shunt significantly decreased the wet weight of the fetal lung, fetal lung wet weight/body weight ratio, and protein concentration per lung as compared to the control fetuses. In the fetal liver and brain tissues, no changes were found in the concentrations of total phospholipids, phosphatidylcholine (PC) or disaturated phosphatidylcholine (DSPC, the main component of lung surfactant) per g of wet tissue and per mg of protein. However, the lungs of the fetuses with amniotic shunts contained significantly more PC and DSPC, and the L/S ratio was higher than in the control fetuses. These results suggest that the oligohydramnios produced by an amniotic shunt causes pulmonary hypoplasia, but raises the pulmonary surfactant content of fetal rabbit lung.     

Normal and glucocorticoid-induced development of the human small intestinal xenograft

The aim of this study was to determine whether intestinal xenografts could recapitulate human in utero development by using disaccharidases as markers. Twenty-week-old fetal intestine was transplanted into immunocompromised mice and was followed. At 20-wk of gestation, the fetal human intestine was morphologically developed with high sucrase and trehalase but had low lactase activities. By 9-wk posttransplantation, jejunal xenografts were morphologically and functionally developed and were then monitored for 相似文献   

Ontogenic and longitudinal activity of Na(+)-nucleoside transporters in the human intestine

The objectives of our study were to identify the types of nucleoside transporters present in the human fetal small intestine and to characterize their developmental activity, longitudinal distribution, and transport kinetics compared with those present in the adult intestine. Nucleoside uptake by intestinal brush-border membrane vesicles was measured by an inhibitor-stop rapid filtration technique. Only the purine-specific (N1; hCNT2) and the pyrimidine-specific (N2; hCNT1) Na(+)-dependent nucleoside transporters were found to be present on the brush-border membranes of the enterocytes along the entire length of the fetal and adult small intestines. The activity of these transporters was higher in the proximal than in the distal small intestine. Both the N1 and N2 transporters found in the fetal intestine shared similar kinetic properties (Michaelis-Menten constant and Na(+)-nucleoside stoichiometry) to those in the adult intestine. During the period of rapid morphogenesis (11-15 wk gestation), no temporal differences were apparent in the activity of the N1 and N2 transporters in the fetal small intestine. These findings have implications for the absorption of drugs from the amniotic fluid by the fetus after maternal drug administration of nucleoside drugs such as the antivirals zidovudine and didanosine.     

The origin and fate of hyaluronan in amniotic fluid

The mechanisms which regulate the steady-state concentration and molecular weight of hyaluronan in the amniotic fluid of sheep at different gestational ages have been investigated. An attempt to trace the origin of the polysaccharide has been made by analyses of various fetal fluids (amniotic fluid, allantoic fluid, tracheal fluid, urine, and serum). The fate has been studied by injection of radioactively labelled hyaluronan into the amniotic cavity and following the tracer in fetal tissues and fluids. The concentration of hyaluronan in amniotic fluid varies considerably but is in the order of 5 mg/l at mid-pregnancy and decreases to 1 mg/l in late pregnancy. The polysaccharide has a Mr-distribution with a weight-average in the order of 10(6) at 10 to 13 weeks of gestation which decreases to 10(5) closer to term. Calculations show that urine contributes 0.1 and 0.5 mg of low-molecular (Mr = 10(4) hyaluronan per day in mid- and late pregnancy, respectively, and the lung 10-20% of that amount in the form of high-molecular weight polymer (Mr greater than 10(6). The hyaluronan disappears from the amniotic cavity by bulk flow due to fetal swallowing. It is taken up and degraded in the fetal intestine. Molecules of Mr = 10(3) can pass the intestinal barrier. Calculations show that about 0.5 mg and 1.0 mg of hyaluronan is eliminated per day from the amniotic fluid at 12 and 17 weeks of gestation, respectively. Thus, the higher rate of elimination and the relatively high urinary contribution in more mature fetuses explain the low concentration and Mr of amniotic hyaluronan in late gestation, whereas a slower elimination combined with a relatively larger contribution of high molecular weight hyaluronan both from lung and urine and possibly from other sources are responsible for the higher concentration and Mr of the compound in early pregnancy.     

Androgen level in the sheep fetus during gestation.

The androgen content of amniotic fluid, plasma, and gonads from 107 fetal lambs was determined by radioimmunoassay in an attempt to understand the ontogeny of gonadal function. Testosterone (T) was too low to be reliably measured in the amniotic fluid from fetuses of either sex. Ovaries were without T activity at any of the stages of gestation studied. Testicular T-5alpha-dihydrotestosterone (T-DHT) concentration steadily decreased from 1.4/ml near term. It is suggested that nongonadal testosterone production increases during fetal life and that T secretion by the fetal testis may contribute steadily less to the plasma pool of T as gestation proceeds.     

Proteolytic enzymes in human fetal membranes and amniotic fluid. A comparison of normal and premature ruptured membranes

The amniotic and chorionic membranes obtained at term and term amniotic fluid contain a soluble protease activity which cleaves [14C]-labeled globin at acid pH. In contrast, a salt extract of the pellet fraction obtained from the fetal membranes displays only negligible protease activities at the pH range of 4-8. Specific activities of the proteases in the soluble and salt-extractable fractions of fetal membranes which were intact before onset of labor were not significantly different from the respective activities in cases of premature rupture of fetal membranes (PROM). However, the protease activity of the amniotic fluid was found to increase with advancing gestational age and to reach maximal activity at term. A heat-sensitive and nondializable protease inhibitory activity was found in term amniotic fluid. This inhibitory activity acted on the cytosolic protease of amniotic membranes from control and PROM cases, but not on the soluble protease of chorionic membranes, and had a similar potency in fluids from PROM cases or fluids collected at term. These results do not support a role for fetal membrane proteases, amniotic fluid proteases, or amniotic fluid protease inhibitory activities in the etiology of PROM. However, the observed changes in amniotic fluid protease activity with fetal age suggest a physiological role for the enzyme in normal fetal development.     

Enzyme-histochemically detectable glucose-6-phosphatase is present in chorion laeve trophoblasts of human fetal membranes

The purpose of the present study was to demonstrate the presence of glucose-6-phosphatase (G6Pase) in fetal membranes from various gestational ages (20-40 weeks of gestation). Ultrastructural enzyme-histochemical analysis of G6Pase was performed using cerium and lead as capturing agents. Precipitates indicating G6Pase activity were present mainly in the endoplasmic reticulum and partly in the nuclear envelope of chorion laeve trophoblasts, but absent in amniotic epithelial cells. Stringent histochemical control experiments performed ensured specific detection of G6Pase activity. The results indicate that histochemically detectable G6Pase is present in the chorion laeve trophoblasts of human fetal membranes. This enzyme may have some physiological significance in carbohydrate metabolism in human fetal membranes and regulation of amniotic fluid glucose concentration.     

Perturbations in the biochemical composition of fetal fluids are apparent in surviving bovine somatic cell nuclear transfer pregnancies in the first half of gestation

Amniotic and allantoic fluid volumes and composition change dynamically throughout gestation. Cattle that are pregnant with somatic cell nuclear transfer (NT) fetuses show a high incidence of abnormal fluid accumulation (particularly hydrallantois) and fetal mortality from approximately midgestation. To investigate fetal fluid homeostasis in these pregnancies, Na, K, Cl, urea, creatinine, Ca, Mg, total PO(4), glucose, fructose, lactate, total protein, and osmolalities were measured in amniotic and allantoic fluids collected at Days 50, 100, and 150 of gestation from NT pregnancies and those generated by the transfer of in vitro-produced embryos or by artificial insemination. Deviations in fetal fluid composition between NT and control pregnancies were apparent after placental and fetal organ development, even when no gross morphological abnormalities were observed. Individual NT fetuses were affected to varying degrees. Elevated allantoic Na was associated with lower K and increased allantoic fluid volume or edema of the fetal membranes. Total PO(4) levels in NT allantoic and amniotic fluid were elevated at Days 100 and 150. This was not accompanied by hypophosphatemia at Day 150, suggesting that PO(4) acquisition by NT fetuses was adequate but that its readsorption by the kidneys may be impaired. Excessive NT placental weight was associated with low allantoic glucose and fructose as well as high lactate levels. However, the fructogenic ability of the NT placenta appeared to be normal. The osmolality of the fetal fluids was maintained within a narrow range, suggesting that the regulation of fluid composition, but not osmolality, was impaired in NT pregnancies.     

Interactions among pulmonary surfactant, vernix caseosa, and intestinal enterocytes: intra-amniotic administration of fluorescently liposomes to pregnant rabbits

Although vernix caseosa is known to be a natural biofilm at birth, human pulmonary surfactant commences to remove the vernix from fetal skin into the amniotic fluid at gestational week 34, i.e., well before delivery. To explain this paradox, we first produced two types of fluorescently labeled liposomes displaying morphology similar to that of pulmonary surfactant and vernix caseosa complexes. We then continuously administered these liposomes into the amniotic fluid space of pregnant rabbits. In addition, we produced pulmonary surfactant and vernix caseosa complexes and administered them into the amniotic fluid space of pregnant rabbits. The intra-amniotic infused fluorescently labeled liposomes were absorbed into the fetal intestinal epithelium. However, the liposomes were not transported to the livers of fetal rabbits. We also revealed that continuous administration of micelles derived from pulmonary surfactants and vernix caseosa protected the small intestine of the rabbit fetus from damage due to surgical intervention. Our results indicate that pulmonary surfactant and vernix caseosa complexes in swallowed amniotic fluid might locally influence fetal intestinal enterocytes. Although the present studies are primarily observational and further studies are needed, our findings elucidate the physiological interactions among pulmonary, dermal-epidermal, and gastrointestinal developmental processes.     

Decline in lung liquid volume and secretion rate during oligohydramnios in fetal sheep

Reduced amniotic fluid volume often results in fetal lung hypoplasia. Our aim was to examine the effects of prolonged drainage of amniotic and allantoic fluids on lung liquid volume (Vl), secretion rate (Vs), and tracheal flow rate (Vtr) in fetal sheep. In five experimental animals, amniotic and allantoic fluids were drained from 107 to 135 days of gestation. The volume of fluid drained from the experimental animals was 411.8 +/- 24.4 ml/day (n = 140). In six control animals, amniotic fluid volume was 747.7 +/- 89.7 ml (n = 15). Wet and dry lung weights were 20-25% lower in experimental fetuses than in control fetuses. Fetal hemoglobin, O2 saturation, arterial PO2, pH, and hematocrit were unchanged by drainage. During the drainage period, Vl was up to 65% lower, Vs was up to 35% lower, and Vtr was up to 40% lower in experimental fetuses than in control fetuses. We conclude that prolonged drainage of amniotic and allantoic fluids decreases Vl, Vs, and Vtr in fetal sheep. These findings indicate that fetal lung hypoplasia associated with oligohydramnios may be the result of a prolonged reduction in Vl.     

Studies on L-arginase in developing rat small intestine, brain, and kidney. I. Ontogenic evolution of arginase isoenzymes

The adult patterns of arginase isoenzymes in rat intestine, kidney, and brain are nearly identical and consist of two forms, cationic A1 and anionic A4. In this paper, the organ-specific maturation of the enzyme equipment in these tissues is reported. The activity of arginase in all tissues studied could be detected on the 13th to 16th days of gestation. In fetal intestine and kidney the arginase activity is low, and persists up to the weaning time when the rapid, 10-fold rise of the enzyme activity occurs. However, the adult pattern of arginase isoenzymes in these tissues is accomplished in different ways. In the intestine, arginase A1 appears in fetal life and is the only form of the enzyme till the 19th to 21st days of postnatal life when the second form of arginase, A4, appears and rapidly accumulates, being exclusively responsible for the rise of the total enzyme activity at the time of weaning. In kidney, arginase A1 alone is present in the early fetal period. Arginase A4 appears 3-4 days before birth and its activity persists unchanged within the first 2 weeks of postnatal life. The intensive rise in total specific activity of kidney arginase at weaning is due to the accumulation of preexisting arginase A4. In brain, the adult pattern of arginase isoenzymes is achieved earlier than in other tissues. Both forms, A1 and A4, occur on Days 13-14 of gestation.     

Changes in acethylcholinesterase activity and total protein in the developing fetal brain

Summary Pre-natal changes in the physiological development of the porcine conceptus indexed by acetylcholinesterase (AChE) activity and total protein content of the fetal brain and amniotic fluid were determined from 4 to 12 weeks of gestation at intervals of two weeks. Marked brain and body development was observed between four and six weeks of gestation. AChE activity in the amniotic fluid declined non-significantly with gestation length while fetal brain AChE activity increased with advancing gestation. Total protein levels in both the amniotic fluid and fetal brain were relatively steady and no significant changes were observed. Changes in AChE activity of the fetal brain may therefore be related to growth changes in the fetus.     

Ontogeny of 11beta-hydroxysteroid dehydrogenase: activity in the placenta, kidney, colon of fetal rats and rabbits.

Mechanisms to regulate closely fetal GC exposure are of considerable importance, as certain organs (kidney, brain) are adversely affected by excess GCs. 11beta-Hydroxysteroid dehydrogenase type 2 (11beta-HSD2) reduces transplacental passage of maternal GCs to the fetus. We hypothesized that 11beta-HSD2, if active in fetal kidney and colon, might allow local tissue modulation of GC access during the critical last trimester. We determined the presence, ontogeny and functionality of 11beta-HSD in the placenta and fetal, neonatal and adult kidney and colon in rats and rabbits and the cortisol:cortisone ratio in human amniotic fluid, which represents fetal urine. There was clear a 11beta-HSD2 expression in last trimester fetal colon, kidney and placenta in both rats and rabbits. This appeared of functional importance, since the potency of cortisol on fetal rabbit colonic sodium flux in the Ussing chamber was increased by 11beta-HSD inhibition. In human amniotic fluid, we found a decreasing ratio of cortisol:cortisone across the last trimester, suggesting an analogous onset of renal 11beta-HSD2 activity in the human fetal kidney. Local fetal tissue 11beta-HSD2 may modulate exposure to the deleterious effects of GCs upon target tissue maturation during sensitive periods of late gestation when fetal GC levels rise to prepare other organs (lung) for adaptations at birth.     

The fetal and postnatal development of small intestinal disaccharidases in the rabbit

The development of small intestinal enzymes (lactase, acid- and hetero beta-galactosidases, cellobiase, maltase, trehalase, and sucrase) was studied from 18 days after conception until birth in 24 rabbit fetuses, and during the postnatal period in 15 newborn, juvenile, and adult rabbits. Lactase, acid- and hetero beta-galactosidases, cellobiase, and trehalase activities increased significantly during the fetal stage, while changes in sucrase and maltase activities were not substantial. In the postnatal period, lactase and cellobiase activities decreased significantly whereas maltase, sucrase, and trehalase activities increased significantly to reach adult values by 30 days of age. The acid- and hetero beta-galactosidases remained unchanged.