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PubMed1989

Different pattern of differentiation in two LLC-PK1 clones.

Two clones (LD3 and LC3) were isolated from the established renal cell line LLC-PK1. They differed with respect to the development of the Na+-dependent alpha-methyl-D-glucoside (AMG) uptake. The higher uptake capacity in LD3 as compared to LC3 was owing to the expression of a higher number of carrier molecules as was shown by the specific phlorizin binding. The intracellular cyclic AMP level, the D-glucose concentration in the growth medium and the cell density could be excluded as the causes of the difference between both clones. We found that the faster development of the Na+-dependent hexose carrier in LD3 was parallelled by a faster expression of trehalase in this clone. This suggests that the expression of both apical proteins is correlated. From the growth curves it was concluded that renewal of the undifferentiated population was roughly the same in both clones. The recruitment of cells from the undifferentiated to the growth arrested state seems also very similar as was deduced from the development of tight junctions and from the down-regulation of the alpha-methylaminoisobutyric acid (meAIB) uptake. The start of differentiation was identical as was shown by the similar rate of expression found for gamma-glutamyl transferase. The difference between both clones is most likely situated at the traverse to a fully differentiated cell. This process takes more time in LC3 than in LD3. Also the fully differentiated state seemed to be different in both clones. We conclude that the pattern of differentiated characteristics found in both clones is different and a model incorporating these differences is presented.

Cell Differentiation↗
PubMedDate not supplied

Amniotic fluid disaccharidases in the prenatal detection of cystic fibrosis.

Intestinal disaccharidases in amniotic fluid were studied in 41 pregnancies with a recurrence risk for cystic fibrosis (CF). In 11 out of 13 pregnancies with CF fetuses the maltase and sucrase activities were either below the control range (8 cases) or below the 10th percentile of control values (3 cases). Trehalase and lactase were slightly less informative indicators of CF. Of the other 28 pregnancies 3 had low amniotic fluid activities of several intestinal enzymes and were terminated, 12 resulted in the birth of a healthy child and 13 are continuing. The findings in fetal CF suggest an impairment of the defaecation of intestinal contents into the amniotic fluid. Reduced or low amniotic fluid disaccharidase activities were also found in other fetal disorders with demonstrated or presumed intestinal anomalies: e.g. anal atresia (2 cases), anencephaly (3 our of the 7 cases), trisomy 13 (5 cases), trisomy 18 (3 of the 5 cases) and trisomy 21 (19 of the 22 cases). Reduced amniotic fluid disaccharidase activities, although not specific for CF, are highly informative in pregnancies at high risk for CF. Using the 10th percentile of the normal range for amniotic fluid disaccharidase activities as an action line, the sensitivity of CF detection is estimated at 80 to 90 per cent, which could in high risk pregnancies reduce the risk of having another affected child from 1 in 4 to 1 in 20.

Amniocentesis↗
PubMedDate not supplied

Fetal intestinal microvilli in human amniotic fluid.

The intestinal microvilli of fetal origin in human amniotic fluid were purified by Ca2+ precipitation of contaminating organelles followed by differential centrifugation of microvillar membranes. In the purified preparation, the specific activity of the microvillar marker-enzymes maltase and sucrase increased about 77-fold over that in cell-free amniotic fluid. Significant contamination of the purified preparation by endoplasmic reticulum (microsomes) and lysosomes was ruled out on the basis of a low content of the marker enzymes glucose-6-phosphatase (microsomes) and acid phosphatase (lysosomes). Amniotic fluid microvilli contain typical enzymes of the fetal intestine including maltase, sucrase, trehalase, alkaline phosphatase and gamma-glutamyltransferase, and their morphology by electron microscopy resembles that of vesiculated intestinal microvilli. Prenatal detection of genetic diseases due to a deficiency of a protein expressed in these membranes or associated to abnormal microvilli seems feasible.

Acid Phosphatase↗
PubMed1993

The effects of castanospermine and swainsonine on the activity and synthesis of intestinal sucrase.

Castanospermine is an indolizidine alkaloid that is found in the seeds of the Australian tree Castanospermum australe. These seeds have been reported to be toxic to animals and to cause severe gastrointestinal upset. In order to determine whether castanospermine is responsible for this toxicity, the alkaloid was injected into young mice or rats, and its effects on various intestinal disaccharidases were determined. Another indolizidine alkaloid, the alpha-mannosidase inhibitor swainsonine, was also tested to compare its effects to those of castanospermine. Castanospermine strongly and rapidly inhibited the activity of the disaccharidases, sucrase, maltase, and trehalase, with sucrase being the most sensitive to inhibition. The loss of activity of these enzymes, especially sucrase, in injected animals appeared to be due to a direct inhibition of enzyme activity, rather than to a change in the structure of the glycan chains of the enzyme, since only minor alterations in carbohydrates were observed. On the other hand, swainsonine, when injected into animals, also profoundly decreased the activity of the sucrase, but this alkaloid had no direct effect on sucrase activity although it did markedly alter the carbohydrate nature of this glycoprotein. This change in oligosaccharide structure may affect protein conformation, stability, or targeting, any or all of which may in turn affect activity. In in vitro studies with the purified enzyme, castanospermine was found to be a competitive inhibitor of intestinal sucrase, but it was a noncompetitive inhibitor of intestinal maltase. A number of other glucosidase inhibitors that inhibit sucrase activity in vitro are also described.

Animals↗
PubMed1993

A dependable method for the synthesis of [14C]trehalose.

A new method for the preparation of [14C]trehalose was developed, based on the ability of yeast cells to accumulate trehalose under stress. The method is simple and reliable. It utilizes a yeast strain in which the gene that encodes for phosphoglucoisomerase has been deleted. Thus, exogenously supplied glucose is not metabolized, but is instead converted to trehalose. The [14C]-trehalose obtained is pure, it is hydrolyzed by trehalase, and it is not susceptible to the action of alpha-glucosidase. The yield of this method is in the order of 35% of the [14C]glucose supplied.

Carbon Radioisotopes↗
PubMed1995

Determination of intracellular trehalose and glycogen in Saccharomyces cerevisiae.

A simple, sensitive and nonlaborious enzyme-based method has been developed for determination of both trehalose and glycogen in yeast cells. The method is based on extraction of trehalose and glycogen into a 40 mM acetate buffer (pH 4.8) by mechanical disintegration of the cells in a bead mill. Subsequently, trehalose and glycogen can be hydrolyzed to glucose by the enzymes trehalase and amyloglycosidase, respectively. The formed glucose is quantified by a flow injection analyzer based on the enzyme glucose oxidase. The method gives results comparable to traditional methods but the simplicity of the analysis results in a much lower relative standard deviation. The excellent sensitivity of the glucose analyzer means that as little as 1 microgram trehalose or glycogen can be determined which reduces the required sample volume. This makes the method ideal for physiological studies, e.g., of transients in continuous cultures of Saccharomyces cerevisiae. In addition, a consistent procedure has been derived for pretreatment and storage of samples.

Glycogen↗
PubMed1997

Synthesis of the photoaffinity label [1'-14C]-6C-(azimethyl)octylglucoside and its reaction with isolated renal brush border membranes.

We describe the synthesis of the photolabile analog of n-octylglucoside (OG), 6C-(azimethyl)octylglucoside (diazirino-octylglucoside, DOG). This diazirino derivative (lambda max = 335 nm) can be activated with long-wavelength UV to generate a highly reactive carbene, an intermediate suitable for a covalent ligand-receptor linkage. In inhibitor studies on D-glucose uptake into rabbit renal brush border membrane vesicles (BBMV) DOG showed a competitive inhibition with regard to D-glucose with a Ki of 20 +/- 5 microM, which was not significantly different from that of the template compound OG (Ki = 30 +/- 10 microM). On irradiation (lambda max = 350 nm) of BBMV in the presence of [14C]DOG, proteins of 61, 72 to 78, and 95 kDa were labeled by photoinduction. A band of 73 kDa was also positive in Western blots using an anti-Na+/D-glucose cotransporter antibody. Further investigations revealed that the labeled proteins were presumably mainly glucosidases known to be present in high concentration in renal BBMV. By comparison with authentic material one of the labeled proteins (61 kDa) was identified as trehalase (EC 3.2.1.28). These studies suggest that labels such as DOG carrying the reactive group directly at the D-glucose moiety of the molecule can be used to label proteins with carbohydrate recognition sites.

Affinity Labels↗
PubMed1983

Organ culture of the small intestine of the suckling mouse in a serum-free medium.

Proximal and distal parts of the small intestine of 8-day-old suckling mice can best be maintained for 48 h in a serum-free organ culture system, Leibovitz L-15, at room air and room temperature. As determined by light and electron microscopy, the villous architecture was preserved as well as the classical ultrastructure of the enterocytes. Incorporation of 3H-thymidine and 3H-leucine continued during the culture period, reflecting a sustained synthesis of DNA and proteins for at least 48 h. The hydrolytic activities of the brushborder membrane, namely of lactase (L), trehalase (T), glucoamylase (GA) and alkaline phosphatase (AlPase) were measured in the explants as well as the culture medium. The overall enzymatic activities were increased as compared to the controls. In the tissue, L, GA and T activities remained stable or even increased during culture while in the medium an accumulation of enzymatic activities was noted especially for GA an AlPase. These results show that the morphological as well as the functional integrity of the mucosa is preserved for at least 48 h when small intestine of suckling mice is cultured in a serum-free medium.

Animal Population Groups↗
PubMed1986

Selective release of inner core proteins from intestinal microvillus membrane by lithium diiodosalicylate.

Lithium diiodosalicylate (LIS) was used to selectively solubilize proteins from purified intestinal brush border membrane vesicles. Incubation of the vesicles with increasing concentrations of LIS resulted in the progressive release of proteins with total disruption of the membranes being obtained at 200 mM. Maximum selectivity was observed at 20-30 mM LIS which preferentially released actin and other non-glycosylated proteins while all the glycoproteins remained associated with the membrane. Electron micrographs showed that, after LIS treatment, brush border vesicles are partially disrupted and have lost their inner core of microfilaments. Sucrase, trehalase, leucylnaphthylamide hydrolase, gamma-glutamyl transpeptidase and alkaline phosphatase all retained more than 70% of their activities and remained associated with the membrane fraction after LIS solubilization (30 mM). The results indicate that lithium diiodosalicylate treatment provides an efficient method for the separation of cytoskeletal proteins from intrinsic membrane glycoproteins and should be very useful for the purification of microvilli proteins and for the study of membrane-protein interactions.

Animals↗
PubMed1980

Characterization of isolated villus and crypt cells from the small intestine of the adult mouse.

Suspensions of sequentially isolated villus and crypt cells were obtained in order to study certain biochemical changes associated with differentiation of epithelial cells in the small intestine of the mouse. Microscopic observation of the various cell fractions reveals that the epithelial cells detach as individual cells or small sheets of epithelium from the tip to the base of the villus, whereas cells in the crypt regions are separated as entire crypt units. The isolated cells retain their ultrastructural integrity as judged by electron miscroscopy. Chemical characterization of the various fractions shows that the total cellular protein content, expressed in activity per cell, remains relatively constant throughout the villus region followed by a noticeable drop in the crypt zone. On the other hand, sharp variations in values of cell DNA content are observed in the crypt zone depending on the reference of activity being used. Activity profiles of several brush border enzymes confirm the biochemical changes that occur during the migration of cells from the crypt to the villus tip, as observed in other species, with maximum activity of sucrase in the mid-villus region, of glucoamylase, trehalase, lactase and maltase in the upper third region, and of alkaline phosphatase at the villus tip. Forty-eight-hour suspension cultures of cell fractions corresponding to cells at the base of the villus and crypt zones show a moderate decrease in protein and enzyme activities to approximately 70% of their original value, with DNA content remaining stable throughout the incubation period. The use of biochemical activities as indicators of cellular integrity during cell culture is discussed.

Animals↗
PubMed1987

Human fetal colon in organ culture.

Human fetal colon (14-16 weeks gestation) was cultured as explants for 15 days in serum-free Leibovitz L-15 medium at 37 degrees C. The overall morphology of the colonic explants was well maintained throughout the culture period and all epithelial cell types retained their ultrastructural characteristics. The incorporation of [3H]-leucine continued and even increased, reflecting sustained synthesis of proteins. Even though the incorporation of [3H]-thymidine into the total DNA decreased during culture, the synthesis of DNA continued. The sites of [3H]-thymidine incorporation into the different layers of the colonic wall were studied by radioautography. The incorporation of the radioactive precursor occurs mainly in the epithelium and to lesser degrees in the mesenchyme and the muscular layer. Labeled epithelial nuclei were located in the intervillous areas but not on the villi. The labeling index of the epithelial cells remained constant throughout the culture period indicating the preservation of the proliferative capacity of the epithelium. Brush-border hydrolytic activities, namely those of sucrase, maltase, lactase, trehalase, glucoamylase and alkaline phosphatase, were assayed in the colonic tissue. These enzymic activities generally decreased in the tissue and increased in the medium during the course of culture. These observations clearly demonstrate that fetal colon can be maintained viable for at least 15 days in a serum-free medium. Organ culture now provides the opportunity to study the normal function and metabolism of human colon during its development.

Autoradiography↗
PubMed1987

Spore activation by acetate, propionate and heat in Phycomyces mutants.

Exposure to heat, acetate, or propionate activates the spores of the fungus Phycomyces blakesleeanus and allows them to germinate. Using counterselection with the antibiotic N-glycosyl-polyfungin, seven mutants were isolated on the basis of decreased spore activation by propionate. The nine mutants showed decreased activation by both chemicals and by heat, increased heat lethality, and altered patterns of trehalase activation. These and other observations indicate that spore activation by the three agents and spore death by heat are mediated by the same cellular component(s), which is probably involved in the regulation of cyclic AMP concentration.

Acetates↗
PubMed1982

Regulation of energy metabolism in yeast. Inheritance of a pleiotropic mutation causing defects in metabolism of energy reserves, ethanol utilization and formation of cytochrome a.a3.

The recessive, nuclear gene mutation glc1, which causes glycogen deficiency in Saccharomyces cerevisiae, is highly pleiotropic. Studies of the inheritance of glc1 revealed two classes of phenotypic characteristics: I. Traits invariably associated with the mutant gene and II. Traits whose expressions require the presence of glc1 and one or more additional genes. Class I traits include glycogen deficiency and the loss of capacity to accumulate trehalose in nonproliferating conditions. Traits in the second class include a decreased rate of growth on ethanol medium, a deficiency in cytochrome a.a3 and an enhanced accumulation of pigment, probably a metalloporphyrin. Constructed strains containing both glc1 and the constitutive maltose fermentation gene MAL4c can accumulate trehalose but not glycogen during growth on glucose. However, accumulated trehalose is degraded when cells are exposed to nonproliferating conditions. It is proposed that the glc1 mutation affects a regulatory system, probably involving a protein kinase and/or protein phosphatase, which regulates glycogen synthase and trehalase. Independent regulation of trehalose synthesis by a system controlled by MAL4c is indicated.

Cytochromes↗
PubMed1976

[Systematic study of Saccharomyces: oxidases and vitamin requirements].

A study of the genus Saccharomyces by new biocharacters demonstrates indirectly the genotype of each species; intracellular osidases (lactose - maltose -cellobiase - melibiase - invertase - trehalase) and vitamin requirements (biotin-folic acid-inositol-niacine-pantothénate-para-aminobenzoic acid-pyridozine-thiamine) reveal some phylogenetic links between the species. It appears that Van der Walt's group I is very homogeneous and must be restricted to only 7 species. The torulospora group seems very similar to the genus Debaryomyces when vitamin needs and osidases are considered; the relations between the 2 groups are discussed. When other biocharacters are studied (nitrite reductase-immunological properties, etc. ...) the value in the GC content (4-5% lower in Debaryomyces) will be established. These results confirm that intracellular oxidases and vitamin needs are very valuable tools for systematic criteria.

Oxidoreductases↗
PubMed1986

Evidence for a possible regulatory gene (Suc-1) controlling sucrase expression in mouse intestine.

Assays for sucrase carried out on intestinal sonicates prepared from 18 different strains of mice revealed a threefold variation in specific activity, the values for CBA/Ca mice being significantly less than for any other strain. Further comparison of the CBA/Ca versus the C57BL/6J mouse showed this deficiency, which became established 2-4 weeks after birth, to apply to isomaltase as well as sucrase but not to maltase or trehalase. Backcross experiments indicated that this deficiency in sucrase activity was inherited as a single codominantly expressed genetic factor. The ability of the CBA/Ca mouse to regulate sucrase activity in response to changes in diet was also reduced compared to that of the C57BL/6J mouse. No difference could be detected in the affinity of sucrase for its substrate or in the ability of heat to denature sucrase prepared from CBA/Ca and C57BL/6J mice. It is suggested that part of the regulatory region of the gene coding for sucrase-isomaltase is modified in the CBA/Ca mouse and that this locus should be given the notation Suc-1 for future reference.

Animals↗
PubMed1980

Amphibian intestinal brush border enzymes during thyroxine-induced metamorphosis.

The development of intestinal brush border hydrolytic activities has been studied during thyroxine-induced metamorphosis of Rana catesbeiana. Alkaline phosphatase activity peaks at 3 and 10 days after the beginning of the thyroxine treatment. The cytochemical observations concerning alkaline phosphatase activity are in agreement with the biochemical data. At the ultrastructural level, alkaline phosphatase activity is particularly evident on the microvilli membranes of the enterocytes in the primary epithelium after 3 days and in the secondary epithelium after 10 days. gamma-Glutamyltranspeptidase exhibits an increase of activity between 7 and 10 days. On the other hand, glucoamylase, maltase, trehalase and leucylnapthylamidase activities decrease during thyroxine treatment, these enzymatic activities being lower than that normally observed after natural metamorphosis. The present study indicates that even though thyroxine is able to induce the morphological differentiation of the intestinal epithelium this hormone is unable to complete the enzymatic load of the new mucosa.

Alkaline Phosphatase↗
PubMed1978

Adequacy of endoscopic biopsy specimens for disaccharidase assays.

Intestinal mucosa from 40 patients obtained by fiber-endoscopic biopsy was assayed for disaccharidases to determine suitability of this tissue for assay. The combined specimens from each patient provided 4.7-38.7 mg of tissue, adequate in all instances for duplicate determinations of protein, lactase, sucrase, and maltase. Tissue remained for assays of palatinase in 39 instances, trehalase and cellobiase in 37, and alkaline phosphatase in 22 cases. Twenty-four subjects had normal lactose tolerance tests and normal sucrase/lactase ratios. Thirteen patients with abnormal oral lactose tolerance tests were identified as having a primary low lactase activity on the basis of elevated sucrase/lactase ratios. This ratio was most helpful in making the diagnosis of a primary low lactase, since the mucosal specimens were not obtained from comparable areas. Tissue from three subjects with an abnormally low maltase was unsuitable for diagnosis. Endoscopic biopsy of mucosa appears to be satisfactory for disaccharidase assays in most instances.

Adult↗
PubMed1989

Is the assay of disaccharidase activity in small bowel mucosal biopsy relevant for clinical gastroenterologists?

In a retrospective study, jejunal mucosal disaccharidase and alkaline phosphatase activities have been investigated in 40 controls and patients with proven celiac sprue (n = 26), lactase deficiency (n = 26), osteoporosis or osteomalacia (n = 16), chronic pancreatitis (n = 12), giardiasis (n = 7), or Crohn's disease (n = 7). Apart from a nonselective reduction of mucosal enzyme activities in the sprue syndrome and a selective reduction of lactase activity in the patients with primary lactase deficiency, assays of mucosal disaccharidases revealed only inconstant or slight deviations from the control group and were not of diagnostic significance for any of the above-mentioned disorders. Isolated forms of enzyme deficiencies other than lactase deficiency, such as sucrase-isomaltase or trehalase deficiency were not present among 168 investigations carried out from 1972-1982. It is concluded that assay of small intestinal disaccharidase or alkaline phosphatase activities does not expand the diagnostic impact of morphological examination of small bowel biopsy specimens and modern noninvasive methods for the detection of carbohydrate malabsorption. Thus, the method does not appear a necessary or relevant investigation in routine clinical practice.

Adolescent↗