Search PubMed⌕ Search

Biomedical subjects

D H Alpers

Publications and source records attributed to D H Alpers.

At least 127 records · Page 7Linked to original sources
PubMed1986

Expression of different-sized placental alkaline phosphatase mRNAs in placenta and choriocarcinoma cells.

The expression of human placental-type alkaline phosphatase (ALPase) in the placenta and in three choriocarcinoma cell lines was examined by translation in vitro and RNA blot analysis using a cDNA for placental ALPase. Placental RNA directed the synthesis of two polypeptides that could be immunoprecipitated with antiserum to placental ALPase. Translation of RNA from the choriocarcinoma cell lines, with or without sodium butyrate treatment, yielded a single immunoprecipitable product of molecular weight intermediate between those of the products from the placenta mRNA. Two cDNA clones for placental ALPase were isolated by antibody screening of a placental cDNA library constructed in lambda gt11. The overlapping cDNAs include 462 nucleotides of coding sequence. RNA blot analysis has confirmed that induction of placental-type ALPase levels during placental development is accompanied by an increase in steady-state placental ALPase mRNA concentrations. Examination of the mRNAs revealed a placental ALPase mRNA of 3.0 kilobases (kb) and a distinct choriocarcinoma placental-type ALPase mRNA of 2.6 kb, implying that transformation of normal to malignant trophoblast is associated with the expression of a distinct placental-type ALPase gene transcript and its protein product.

Alkaline Phosphatase↗
PubMed1986

The relationship of psychiatric disorder to gastrointestinal illness.

The definition of psychiatric illness by specific criteria has provided a new method for examining the relationship of certain gastrointestinal (GI) diseases to psychiatric disorder. Using this technique, researchers have found that rates of psychiatric diagnosis in patients with certain functional GI syndromes and Crohn's disease exceed those found in the general population and in comparably ill control groups. Timing of onset of both sets of symptoms (GI and psychiatric) suggests that the functional syndromes generally follow or coincide with the onset of psychiatric symptoms, whereas the onset of Crohn's disease is more independent. A relationship of psychiatric illness to other GI disease is suspected based on results of psychometric testing or on less structured attempts at psychiatric diagnosis employed in earlier studies. Psychiatric diagnosis by criteria definition may play an important role in more clearly understanding any relationship of psychiatric disorder to somatic GI disease as the applications of this scientific technique are expanded.

Adult↗
PubMed1986

Uptake and fate of absorbed amino acids and peptides in the mammalian intestine.

Intraluminal and brush-border digestion of proteins results in a mixture of amino acids and small peptides. Thirteen brush-border peptidases have been described. Despite all of these enzymes, some peptides escape digestion and are absorbed intact. The assimilated products of protein digestion can follow multiple paths: absorption into the blood as amino acids or small peptides, metabolism within the enterocyte, incorporation into proteins of the enterocyte, and incorporation into proteins to be secreted into plasma. Unlike other tissues, the intestinal mucosa is not very responsive to metabolic regulation as regards amino acid uptake or regulation of protein synthesis. Most effects after dietary manipulation or drug or hormonal stimulation are modes (two-to fivefold increases). This constitutive metabolism of amino acids in the intestinal mucosa is consistent with its essential role in absorption. The mucosa also is a major contributor to apolipoproteins, which are probably the quantitatively most important proteins secreted from the intestine. Alterations in apoprotein secretion have been noted after fat feeding, and are both transcriptionally and translationally regulated. Although the fractional renewal rate of protein in the intestine is the highest of any tissue in the body, the quantitative importance of alterations in protein synthesis or secretion to the fate of intracellular amino acids is not known.

Amino Acids↗
PubMed1986

Light microscopic immunocytochemical localization of hepatic and intestinal types of fatty acid-binding proteins in rat small intestine.

Monospecific antisera to purified hepatic fatty acid-binding protein (hFABP) and gut fatty acid-binding protein (gFABP) have been used to localize these two proteins in the small intestine of fed rats at the light microscopic level. Pieces of duodenum, jejunum, and ileum were removed from 4-, 10-, 20-, 22-, and 60-day-old Sprague-Dawley rats. Both cryostat and paraffin sections were studied for the presence of hFABP or gFABP by the avidin-biotin immunoperoxidase method. Slides were graded blind for the intensity of staining. Despite the structural and immunological differences between these two proteins, we showed no major differences between their staining patterns or their staining intensity throughout the intestine during postnatal development. The staining for both fatty acid-binding proteins was cytoplasmic. No brush border staining was found. Staining was more intense in the proximal rather than distal intestine, in the villus rather than crypt cells, and in the apex rather than the base of intestinal cells. Shifts in staining patterns, and staining intensity occurring during development may be related to variations in dietary fat intake, rates of cell proliferation, intestinal anatomy, and mechanisms for fat absorption.

Animals↗
PubMed1985

Membrane interactions of rat intestinal alkaline phosphatase: role of polar head groups.

Lipid-protein interactions with purified membranous intestinal alkaline phosphatase have been studied by using rat intestine. The enzyme was incorporated equally well into neutral lecithin and anionic liposomes, including those made from phosphatidic acid alone. It could not be solubilized with chaotropic salts nor by phospholipases C and D from either native membranes or phospholipid vesicles. Detergents effected nearly complete release of enzyme from the vesicles. Phosphatase activity was lost upon treatment with phospholipase D alone. The activity was restored with free choline, or choline containing phospholipids, but not by the addition of other phospholipids or amines. The catalytic activity was also lower when the enzyme was bound to a phosphatidylcholine vesicle containing additional phosphatidic acid. Neither phosphatidylserine nor phosphatidylinositol addition altered enzyme activity. These results show that the enzyme binds to the membrane by a primary hydrophobic interaction with membrane phospholipids without requiring the polar head group and that the enzyme activity is affected via a secondary interaction with choline. We suggest that choline protects the active site of brush border alkaline phosphatase from inhibition by endogenous membrane phosphate groups.

Alkaline Phosphatase↗
PubMed1985

Tissue specific expression and developmental regulation of two genes coding for rat fatty acid binding proteins.

We have examined the tissue distribution and developmental regulation of two low molecular weight cytosolic fatty acid binding proteins. Based on their initial site of isolation, they have been referred to as liver and intestinal fatty acid binding proteins (FABP). Cloned cDNAs were used to probe blots of RNAs extracted from a wide variety of adult rat tissues as well as small intestine and liver RNA obtained from fetal, suckling, and weaning animals. The highest concentrations of "liver" FABP mRNA were found in small intestine and liver. "Intestinal" FABP mRNA is most abundant in small bowel RNA while only trace amounts were encountered in liver. Both mRNAs were detectable in stomach, colon, pancreas, spleen, lung, heart, testes, adrenal, and brain RNA at 1-8% the concentrations observed in small intestine. Accumulation of both mRNAs in the small intestinal epithelium increases during development. The mRNAs are first detectable between the 19th and 21st day of gestation. They undergo a coordinated 3-4-fold increase in concentration within the first 24 h after birth. Thereafter, gut levels of intestinal FABP mRNA remain constant during the suckling period while liver FABP mRNA increases an additional 2-fold. Liver FABP mRNA levels are also induced in hepatocytes during the first postnatal day but subsequently do not change during the suckling and weaning phase, despite marked alterations in hepatic fatty acid metabolism. These observations support the concept that the major role of these proteins is to facilitate the entry of lipids into cells and/or their subsequent intracellular transport and compartmentalization. The data also raise questions about the identity of extragastrointestinal FABPs.

Aging↗
PubMed1985

Regulation of the biosynthesis of two distinct fatty acid-binding proteins in rat liver and intestine. Influences of sex difference and of clofibrate.

Two distinct fatty acid-binding proteins (FABPs) have been identified in rat intestine, gFABP (15,063 Da) which is confined to intestinal epithelium and hFABP (14,184 Da) which is found in both liver and intestine. We have examined the influence of sex difference and the effect of clofibrate, both of which affect cellular fatty acid metabolism and hFABP levels, on the concentration, and mRNA levels of both hepatic and intestinal FABPs. In the liver, hFABP concentration was approximately 2-fold greater in females and in clofibrate-treated males than in untreated male rats. These differences were not accompanied by changes in the fractional turnover of the polypeptide but rather by parallel increases in hFABP mRNA. In the intestine, the two FABPs exhibited different regulatory responses. Intestinal hFABP turnover was 33% greater in females than in males, whereas mRNA concentration was 50% greater. Thus, unlike hFABP in liver, there was no sex-related difference in the steady-state level of hFABP in intestine. However, clofibrate treatment, similar to its effects in the liver, doubled intestinal hFABP protein and mRNA concentration. In contrast to hFABP, neither gFABP protein nor mRNA concentration were sex dependent, whereas clofibrate produced only a modest increase in gFABP concentration without significantly changing gFABP mRNA levels. The results indicate that the influence of sex difference and the effect of clofibrate on hepatic fatty acid metabolism are both associated with changes in hFABP synthesis mediated pretranslationally. The differential response of hFABP and gFABP in intestine suggests that these proteins play distinct roles in the cellular metabolism of fatty acids.

Animals↗
PubMed1985

Gender differences in antacid-induced phosphate deprivation in rats.

To determine whether an animal model could be used to study the susceptibility of women to antacid-induced phosphate deficiency, 6-wk-old male and female rats were given basic aluminum carbonate gel (Basaljel) (1 ml/100 g body wt) or distilled water by gastric intubation daily for 3 wk. Rats were fed either ad libitum (group 1) or by pair-feeding (group 2) with pelleted rat food containing 0.74% phosphorus. In group 1, baseline, 1-wk, and 3-wk values for serum phosphorus in Basaljel-treated females were 7.7 +/- 0.2, 6.3 +/- 0.2, and 6.2 +/- 0.2 mg/dl, respectively. Corresponding values for control females were 7.8 +/- 0.3, 7.0 +/- 0.2, and 7.3 +/- 0.2 mg/dl. Values for treated females were significantly lower (p less than 0.02) than values for control females by 1 wk of treatment. Basaljel-treated males did not differ from controls. The pattern for group 2 was similar. Intestinal absorption and intramuscular stores of phosphate were assessed in group 1. After 3 wk of treatment, [32P]phosphate assimilation from the duodenum into the body was lower in Basaljel-treated females than in controls (33.8% +/- 1.9% vs. 49.8% +/- 6.2% of the luminally administered dose, p less than 0.05). This was due to increased retention of [32P]phosphate in the intestine of treated females (19.9% +/- 2.0% vs. 11.9% +/- 2.4% for control females, p less than 0.02). Results in jejunum were similar. Total intramuscular phosphate in females was significantly lower (p less than 0.005) than in males both before and after antacid treatment. Thus hypophosphatemia in the female rat during antacid administration is probably secondary to the additive effects of decreased assimilation and decreased soft tissue stores of phosphate.

Aluminum Hydroxide↗
PubMed1985

Interaction of intestinal disaccharidases with phospholipids: effect of cholesterol.

Although the rat intestinal brush border disaccharidases are the most easily solubilized protein components, the nature of the lipid-protein interactions in the membrane is incompletely understood. Phospholipid vesicles were prepared using the lecithin fraction from brush border membranes and synthetic lecithins. Addition of cholesterol to brush border lecithins enhanced the binding of disaccharidases, but not of alkaline phosphatase. The addition of cholesterol to synthetic lecithin vesicles enhanced the binding of disaccharidases only when added above the transition temperature of the lecithin used. The maximal effect occurred at an equimolar ratio of lecithin to cholesterol. Binding of disaccharidases to phospholipid vesicles was independent of charge or the nature of the polar head group, and the enzyme was inserted so that the catalytic domain was excluded from the lipid matrix. These results demonstrate that membrane attachment of disaccharidases is hydrophobic, involving primarily fatty acyl chains and an interaction with cholesterol. The membrane interaction does not seem to affect enzyme activity.

Animals↗
PubMed1985

Intestinal uptake and release of cobalamin complexed with rat intrinsic factor.

The mechanism of uptake of intrinsic factor (IF) and cobalamin (Cbl) by enterocytes and their subsequent fate have been uncertain. To examine this problem double-labeled IF X Cbl was added to small intestinal organ cultures. When 125I-IF X [57Co]Cbl was added to rabbit ileal explants, binding and internalization increased linearly for 24 h. After an 18-h chase with nonlabeled IF X Cbl, no 125I-IF returned to the cell surface. An amount of 35-45% of the internalized Cbl was found free, not bound to IF or any other protein. About 60% of both internalized ligands was bound to membranes but by a non-Ca2+-dependent bond, suggesting binding to a protein other than the brush-border receptor. Cobalamin was released from IF at pH 5.0 to the same degree (30%) as free Cbl was found inside the cell (35-45%). Neither pancreatic proteases nor ileal homogenates effected release of Cbl from IF. When cathepsins were added, the Cbl released was no greater than could be attributed to pH 5.0 alone. Chloroquine added to tissue explants did not alter the percentage of free intracellular Cbl. From these results we suggest that IF X Cbl is internalized and detached from the receptor within the enterocyte. The mechanism of release is not known but seems to require an acid pH (5.0). The Cbl is released in the mucosa, perhaps when the IF X Cbl complex enters a nonlysosomal cellular compartment with an acidic environment. There is no substantial recycling of IF to the brush-border membrane.

Animals↗
PubMed1985

Distribution of apolipoproteins A-I and B among intestinal lipoproteins.

Chylomicrons and very low density lipoproteins (VLDL) are produced by the intestine and these nascent particles are thought to be similar to their counterparts in intestinal lymph. To study the relationship between these lipoproteins within the cell and those secreted into the lamina propria and lymph, we have isolated enterocytes, lamina propria, and mesenteric lymph from rats while fasted and after corn oil feeding. Apolipoprotein A-I and B content were measured by radioimmunoassay in cell, lamina propria, and lymph fractions separated by Sepharose 6B and 10% agarose chromatography, and by KBr isopycnic density centrifugation. ApoA-I in the cell and the underlying lamina propria was found partly in those fractions in which chylomicron and very low density lipoproteins (chylo-VLDL) and high density lipoproteins (HDL) elute, but more abundantly where unassociated 125I-labeled apoA-I was eluted. In the lymph, however, 74% of apoA-I eluted in the HDL region and no peak of free apoA-I was found. ApoB and apoC-III within the enterocyte were found distributed in the position of particles eluting not only with chylomicrons and VLDL, but also in the regions corresponding to LDL and HDL. In the lamina propria and lymph, on the other hand, most of the apoB was found in the region of VLDL and chylomicrons. These results indicate that the patterns in lymph lipoproteins and the lamina propria do not exactly mirror the distribution of apoA-I and B among lipoproteins inside the cell. This may be because intracellular apoproteins may be unassociated with lipoproteins, or they could be associated with lipoproteins in various stages of assembly of protein with lipids. Furthermore, the apoprotein composition of intestinal lipoproteins is altered after secretion from the enterocyte. Finally, not all apoproteins seem to be secreted in association with identifiable lipoprotein particles from the enterocyte.

Animals↗
PubMed1984

Biosynthesis and compartmentalization of rat-intestinal vitamin-D-dependent calcium-binding protein.

We have purified the primary translation product of rat intestinal vitamin-D-dependent calcium-binding protein mRNA from wheat germ and ascites cell-free systems. We show that calcium-binding protein is neither synthesized as a larger percursor nor likely to be exported from the intestinal epithelium. Our conclusions are based on the following observations. (1) The primary translation product, NH2-terminally labeled with formyl[35S]methionine, comigrates with the mature cytoplasmic protein during electrophoresis through denaturing gels. (2) It does not possess a cleavable signal peptide sequence or internal signal equivalent as judged by co- and post-translational cleavage assays in vitro. (3) The NH2 terminus of the cell-free product is acetylated. (4) Comparison of the NH2-terminal amino acid sequences of the primary translation product and cyanogen bromide peptides obtained from the blocked, purified cytoplasmic protein. The kinetics of calcium-binding protein mRNA accumulation and decay in rachitic intestinal epithelium after primary and secondary stimulation with 1,25-dihydroxycholecalciferol (calcitriol) were studied using the cell-free translation system. The results are reminiscent of other steroid-hormone-inducible systems. Both the rate of mRNA accumulation and the peak response were greater after secondary stimulation.

Amino Acid Sequence↗
PubMed1984

Biosynthesis of apolipoprotein C-III in rat liver and small intestinal mucosa.

We have examined the biosynthesis of rat apolipoprotein C-III in the small intestine and liver. The primary translation product of its mRNA was recovered from wheat germ and ascites cell-free systems. Comparison of its NH2-terminal sequence with the NH2 terminus of plasma high density lipoprotein-associated apolipoprotein C-III showed that apo-C-III was initially synthesized as a preprotein with a 20 amino acid long NH2-terminal extension: Met-X-X-X-Met-Leu-Leu-X-X-Ala-Leu-X-Ala-Leu-Leu-Ala-X-Ala-X-Ala. Co-translational cleavage of the cell-free translation product by signal peptidase generated a polypeptide with the same NH2 terminus as the mature protein (X-Glu-X-Glu-Gly-Ser-Leu-Leu-Leu-Gly-Ser-Met). Therefore, this apolipoprotein does not undergo post-translational proteolytic processing like two other high density lipoprotein-affiliated proteins, proapo-A-I and proapo-A-II. The mRNA encoding apolipoprotein C-III comprises 0.4% of the translatable RNA species in adult rat liver and 0.14% of the translatable RNA species in small intestinal epithelium. Acute fat feeding with a triglyceride meal resulted in a 2-fold increase in intestinal preapo-C-III mRNA accumulation but no change in the levels of preproapo-A-I mRNA. Thus, the acute response of the apo-A-I and C-III genes to triacylglycerol absorption differs.

Amino Acid Sequence↗
PubMed1984

Metabolism of glycosylated human salivary amylase: in vivo plasma clearance by rat hepatic endothelial cells and in vitro receptor mediated pinocytosis by rat macrophages.

Salivary-type amylase normally comprises about 60% of the amylase activity in human serum, but only a small fraction is a glycosylated isoenzyme (amylase A). In contrast, 1/3 of amylase in human saliva is glycosylated. Since glycosylation can affect circulatory clearance, we studied the clearance of amylase A in rats and its uptake by rat alveolar macrophages. Following intravenous injection, 125I-labeled amylase A disappeared rapidly from plasma (t 1/2 = 9 min) and accumulated in the liver. Simultaneous injection of mannose-albumin slowed its clearance to a rate comparable to that of 125I-labeled nonglycosylated salivary amylase (t 1/2 = 45 min). In contrast, galactose-albumin had no effect. Electron microscope autoradiography of the liver following injection of 125I-labeled amylase A revealed a localization of grains over the hepatic endothelial cells. In vitro studies indicated that amylase A is taken up by alveolar macrophages via receptor-mediated pinocytosis. Uptake was linear over time, saturable, and inhibited by mannan and mannose-albumin, but not by galactose-albumin. We conclude that amylase A, which is a naturally occurring human glycoprotein with at most three terminal L-fucose residues per molecule, is recognized in rats by a mannose receptor located on hepatic endothelial cells. We speculate that this receptor, by rapidly clearing circulating amylase A, may be responsible for the low level of amylase A in human serum.

Amylases↗
PubMed1984

Quantitation of human intestinal and liver/bone alkaline phosphatase in serum by rocket electroimmunoassay.

The separation and quantitation of the various human serum alkaline phosphatases has been largely by indirect and/or imprecise methods. A rocket electroimmunoassay method has been developed for the quantitation of the alkaline phosphatases, which uses enzymatic activity to detect the rocket and bromochloroindolylphosphate as substrate. The assay for intestinal phosphatase requires only one-dimensional electrophoresis, since the antibody to the intestinal enzyme does not cross-react with the other phosphatases. Both liver and bone enzymes give a line of identity when tested against antibody directed against the liver phosphatases, and these phosphatases require separation first by acrylamide gel electrophoresis before quantitation by crossed immunoelectrophoresis. Liver/bone enzyme levels in serum are easily detected within the linear range of the assay without concentration of the serum. The assay is 10-fold more sensitive than quantitation by acrylamide gel electrophoresis. The inability to detect circulating intestinal alkaline phosphatase in the serum of most fasting hospitalized patients has been documented by this sensitive assay, and the predominance of the liver isoenzyme has been confirmed. The assay should prove useful for determining the tissue of origin of serum alkaline phosphatases, and in providing quantitative data for physiological studies. It is not adaptable for automation and will not prove useful in the routine clinical laboratory.

Alkaline Phosphatase↗
PubMed1984

Cloning of a cDNA encoding rat intestinal fatty acid binding protein.

Intestinal fatty acid binding protein mRNA is one of the most abundant mRNA species in the rat small intestinal epithelium. RNA transfer blot analyses disclosed that the mRNA encoding intestinal fatty acid binding protein is approximately equal to 900 nucleotides long and not represented in liver RNA. We have identified 564 nucleotides of this mRNA, including 12 nucleotides of the 5' nontranslated region, the coding portion, and 155 nucleotides of the 3' nontranslated domain. The primary translation product encoded by this mRNA contains 132 amino acids and has a Mr of 15,062. The derived protein sequence was verified by automated sequential Edman degradation of the intact polypeptide isolated from a wheat germ cell-free system. The in vitro product is NH2-terminally acetylated, a finding that is consistent with its ultimate cytoplasmic destination. Comparison of the amino acid sequence of this protein with liver fatty acid binding protein, a polypeptide specified by the most abundant small intestinal epithelial mRNA, revealed significant homology and similarity in the predicted secondary structures of their NH2-terminal domains.

Amino Acid Sequence↗
PubMed1984

Immunocytochemical localization of the intrinsic factor-cobalamin receptor in dog-ileum: distribution of intracellular receptor during cell maturation.

Absorption of cobalamin is facilitated by the binding of the intrinsic factor-cobalamin complex (IF-cbl) to specific receptors in the ileum. The physical and biochemical characteristics of this ligand-receptor binding reaction have been extensively studied, but little is known about the cellular mechanisms or receptor synthesis, intracellular transport, and expression on the microvillus surface membrane. We attempted to delineate these mechanisms by using ultrastructural immunocytochemistry to localize the IF-cbl receptor in the crypt, mid-villus, and villus tip regions of mucosal biopsies obtained from the ileum of anesthetized dogs. Prior to initiating the ileal localization studies, the antisera to purified canine IF-cbl receptor that was employed in our studies was shown to have specificity for site (e.g., ileal enterocytes vs. other cells within the gastrointestinal tract) and immunohistochemical specificity. Receptor synthesis in endoplasmic reticulum begins in crypt enterocytes, but continues in cells throughout the villus. In the mid-villus region synthesized receptor translocates vectorially to the microvillus surface associated with membranous vesicles and then inserts into the microvillus pit. Receptor remains fixed to the microvillus pit and does not distribute uniformly over the brush border membrane. All villus tip enterocytes contained IF-cbl receptor in microvillus pits, vesicles, and endoplasmic reticulum, but in addition extensive perinuclear membrane staining was evident as well as re-internalized receptor associated with multivesicular bodies. Basolateral membranes contained no receptor at any level of the villus. These observations suggest that the IF-cbl receptor (a) translocates to the apical cell surface at the mid-villus region by transport in vesicles, (b) directly inserts into and then remains fixed in microvillus pits, (c) is elaborated on the luminal surface most extensively in villus tip cells, and (d) although reinternalized, does not move IF and/or cbl to the basolateral cell surface.

Animals↗