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D H Alpers

Publications and source records attributed to D H Alpers.

At least 19 recordsLinked to original sources
PubMed1992

The intrinsic factor (IF)-cobalamin receptor binding site is located in the amino-terminal portion of IF.

Intrinsic factor has two binding sites, one each for cobalamin and for the ileal receptor recognizing the intrinsic factor-cobalamin complex. To obtain initial functional mapping of these domains, cDNAs encoding intact rat and human intrinsic factor or fragments thereof were expressed transiently in COS-1 cells or in an in vitro transcription/translation system. Deletion of as little as 12% of the amino acids from the carboxyl terminus resulted in loss of cobalamin binding activity. On the other hand, the receptor binding region of intrinsic factor appears localized to a restricted region in the amino-terminal portion of the protein. Only those transcription/translation fragments of rat or human intrinsic factor tested that contained amino acid residues 25 to 62 (out of 399) showed calcium-dependent binding to isolated kidney brush borders, the shortest sequence corresponding with 20 consecutive amino acids. In contrast, a 232-amino acid carboxyl-terminal fragment of rat intrinsic factor and 243- and 338-amino acid carboxyl-terminal fragments of human intrinsic factor showed no receptor binding activity.

Animals↗
PubMed1992

In vitro expression and secretion of functional mammalian intrinsic factor using recombinant baculovirus.

Intrinsic factor was produced at levels of 1-2 mg per 1 (0.25 micrograms per 10(6) cells) by growth of recombinant baculovirus-infected Sf9 cells in spinner culture. The recombinant IF showed a binding affinity for cobalamin (2.6.10(-10) M) and for the intrinsic factor-cobalamin receptor (3.5.10(-10) M) nearly identical with native IF. Purification of the recombinant intrinsic factor could be accomplished by affinity chromatography, but final purification by gel chromatography (FPLC) was necessary to separate intrinsic factor from a 62 kDa protein secreted from uninfected Sf9 cells. This protein binds selectively to the cobalamin-Sepharose column, but demonstrates no cobalamin binding activity after elution. Microgram quantities of radiolabelled protein could be produced for metabolic and autoradiographic studies. The stability of intrinsic factor to pancreatic proteinases was nearly identical with human gastric intrinsic factor, both native and recombinant as produced in mammalian cells. Glycosylation of the intrinsic factor was demonstrated by lectin binding to the recombinant protein separated on SDS-PAGE, and by a shift in apparent molecular mass from 47 kDa to 43 kDa following treatment of Sf9 cells with tunicamycin. Most of the recombinant IF was produced by Sf9 cells in the first 48 h post infection.

Animals↗
PubMed1992

Pericrypt eosinophilic enterocolitis and chronic diarrhea.

An unusual pattern of eosinophilic infiltration around intestinal crypts was detected in mucosal biopsy specimens of 10 patients with chronic diarrhea, half of whom had evidence of systemic connective tissue disease. The median duration of symptoms was 11 months, and no other explanation for diarrhea could be determined in any case. The cellular infiltrate on biopsy specimens was present deep in the mucosa of small and large intestinal specimens, separating crypt bases from the muscularis mucosae and penetrating the latter. Consistent with the microscopic findings, surface mucosal appearance by endoscopy was uniformly normal. These histological features of colonic biopsy specimens were statistically differentiated from those of asymptomatic subjects (n = 8), subjects with diarrhea-predominant irritable bowel syndrome (n = 6), and subjects with collagenous colitis (n = 7) or lymphocytic colitis (n = 5). Diarrhea improved in five of seven subjects treated with oral prednisone or prednisone in conjunction with azathioprine (median follow-up period, 2.2 years). Histological changes on subsequent biopsy specimens correlated closely with symptomatic status. These findings strongly suggest that chronic diarrhea is related to this pericrypt eosinophilic enterocolitis, a pathological lesion often associated with features of systemic connective tissue disease. The disorder appears responsive to corticosteroid therapy in some cases.

Adrenal Cortex Hormones↗
PubMed1992

Apolipoprotein expression and cellular differentiation in Caco-2 intestinal cells.

Caco-2 cells, cultured for 18 days on porous filter supports and conventional plastic culture dishes, were used to study the effects of cellular differentiation on the expression of apolipoprotein (apo) genes. Media of filter-grown cells accumulated more apo B as apo B-48 and contained three times the amount of edited apo B mRNA compared with plastic-grown cells. The accumulation of apo A-I by media of plastic-grown cells was higher than accumulation by filter-grown cells, despite similar concentrations of apo A-I mRNA. The apo A-IV was detectable in the culture media earlier with filter-grown cells compared with plastic-grown cells, despite similar apo A-IV mRNA concentrations. Plastic-grown cells contained more apo E mRNA, and their media accumulated more apo E than filter-grown cells. With the exception of apo A-I, apo gene expression changed with Caco-2 cell differentiation to resemble more closely the patterns seen in adult enterocytes. There were no effects or minimal effects of added retinoic acid, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], or thyroid hormone on apo accumulation in media of filter-grown cultures of Caco-2 cells. However, 1,25(OH)2D3 and thyroid hormone increased apo B, apo A-IV, and apo A-I mRNA concentrations, retinoic acid increased apo B mRNA concentrations alone, and all three reduced apo E mRNA concentrations. Ratios of edited to unedited apo B mRNA were unaffected. In conclusion, culture substratum importantly influences Caco-2 cell differentiation. Soluble factors that influence cellular differentiation may affect apo gene expression over and above effects mediated by the culture substratum.(ABSTRACT TRUNCATED AT 250 WORDS)

Apolipoprotein A-I↗
PubMed1992

Caco-2 cell transfection by rat intestinal alkaline phosphatase cDNA increases surfactant-like particles.

The rat enterocyte produces a particle with surfactant-like properties (including a whorled appearance, enrichment for dipalmitoyl phosphatidylcholine, and ability to lower surface tension) that also is enriched for intestinal alkaline phosphatase. Human Caco-2 cells grown on polycarbonate filters were utilized to study the secretion of these particles and exhibited whorls and strands of unilamellar membranes, particularly concentrated at the apical pole or near junctional complexes. Concentrated culture medium from these cells separated on continuous NaBr gradients revealed a fraction at density = 1.07 g/l enriched for phosphatidylcholine and intestinal alkaline phosphatase. This fraction contained membranous sheets containing alkaline phosphatase, detected by immunolocalization. Phosphatidylcholine comprised 54% of phospholipid in this fraction, compared with 20% in brush borders. When Caco-2 cells were transfected with cDNA encoding rat intestinal alkaline phosphatase, cellular phosphatase activity increased twofold, but activity in the medium increased 14-fold to > 200 (average 32)-fold. Ultrastructurally, compared with mock-transfected cells or cells transfected with human placental alkaline phosphatase, transfection with rat intestinal alkaline phosphatase cDNA led to intracellular and extracellular accumulation of surfactant-like particles. We conclude that surfactant-like particles are produced by Caco-2 cells, and their production can be enhanced by transfection with a cDNA encoding a protein known to be associated with such particles.

Alkaline Phosphatase↗
PubMed1992

The two mRNAs encoding rat intestinal alkaline phosphatase represent two distinct nucleotide sequences.

Rat duodenal mucosa contains two mRNAs (2.7 and 3.0 kb) encoding intestinal alkaline phosphatase (IAP), but the mechanism for their production has not been clear. By means of the polymerase chain reaction (PCR), we isolated a fragment that identifies a second rat IAP (rIAP-II), differing from the rIAP-I sequence in the coding and 3' untranslated regions and encoding a COOH-terminal sequence predicted to be hydrophilic. By means of probes unique to each sequence, rIAP-I identified the 2.7-kb mRNA, and rIAP-II the 3.0-kb mRNA. The different structures and differential regulation of the mRNAs after fat feeding demonstrate the presence of two rat IAP transcripts.

Alkaline Phosphatase↗
PubMed1992

Mast cell granules within endothelial cells: a possible signal in the inflammatory process?

Mast cell-endothelial cell interactions have long been suspected to be important in inflammation. The detail of the pathways of communication have yet to be elucidated. In this report we describe, for the first time, mast cell granules within endothelial cells of colonic capillaries in a patient with florid colitis, as well as rats with experimentally induced mast cell degranulation. We postulate that this phenomenon may occur more generally during immunologically mediated inflammatory cell degranulation, and may be one mechanism of communication to endothelium.

Adult↗
PubMed1991

Do life events or depression exacerbate inflammatory bowel disease? A prospective study.

OBJECTIVE: To determine whether depressed mood or life events are associated with an exacerbation of inflammatory bowel disease. DESIGN: A prospective study of a consecutive sample of patients with relapsing inflammatory bowel disease, followed by monthly questionnaires and periodic office visits. SETTING: A referral-based gastroenterology clinic at a medical school. PATIENTS: A consecutive sample of 32 patients with inflammatory bowel disease who had had at least one relapse in a 2-year period after entry into the study. MEASUREMENTS AND MAIN RESULTS: The Social Readjustment Rating Scale (measuring life events), the Beck Depression Inventory (a visual analog scale for depressed mood) and an inventory of intestinal symptoms were completed monthly by each subject with a 78% rate of compliance. A mean of 2.2 exacerbations was seen per subject during the study period. Life events were not temporally associated with changes in intestinal symptoms. Significant associations were found between intestinal symptoms and the two mood scales (P less than 0.05 for each), but no directionality in symptom occurrence could be detected in a time-lagged analysis. The results were similar when the months preceding exacerbations of inflammatory bowel disease were analyzed separately. CONCLUSIONS: Although these findings suggest that mood changed concurrently with exacerbation of inflammatory bowel disease, no evidence indicated that stressful life events or depressed mood precipitated exacerbations in this study group.

Colitis, Ulcerative↗
PubMed1991

Rat intestinal alkaline phosphatase secretion into lumen and serum is coordinately regulated.

We have reported the presence of intestinal alkaline phosphatase on particles with surfactant-like properties within enterocytes, on the luminal surface (light mucosal scrapings) and in the lumen of adult fat-fed rat intestines ((1989) J. Clin. Invest. 84, 1355). To test the physiological role of these particles, we compared the effect on particle secretion of a known inducer of luminal and serum alkaline phosphatase secretion (fat), with the effect of pharmacological stimulators (cholecystokinin and bethanecol). Fat induced a 2-3-fold increase in membrane-free phosphatase activity in serum, and in particle-bound alkaline phosphatase activity in proximal luminal washings and light mucosal scrapings, reaching a peak in both compartments 7 h after a corn oil feed. Bethanecol given subcutaneously induced a quantitatively similar increase in serum alkaline phosphatase activity and in particle-bound phosphatase activity in proximal light mucosal scrapings, reaching a peak 7.5 min after injection. Cholecystokinin also had a 2-3-fold stimulatory effect, 30 min after injection, on particle-bound phosphatase activity in proximal intestinal light mucosal scrapings and distal intestinal luminal washings. The increase in alkaline phosphatase activity in serum samples reached a peak 60 min after cholecystokinin injection. Thus, three independent stimuli increase both luminal and serum appearance of intestinal alkaline phosphatase. These data support the earlier findings that intestinal alkaline phosphatase secretion into the lumen is mediated by a secreted particle, further show that secretion into serum and lumen is coordinately regulated, and are consistent with the hypothesis that the rise in serum alkaline phosphatase activity could be related to extracellular release of the enzyme from the particles.

Alkaline Phosphatase↗
PubMed1991

The minor components of the rat asialoglycoprotein receptor are apically located in neonatal enterocytes.

Because specific uptake of the asialoglycoprotein haptocorrin has been reported in suckling distal intestine, evidence of the asialoglycoprotein receptor in rat ileum was sought. On Western blot, two different polyclonal antisera against purified rat holoreceptor recognized only proteins of the size of the minor receptor components (51 and 55 kilodaltons) in both suckling and adult rat intestine. Messenger RNA encoding the minor component (RHL-2/3) was detected in total RNA extracted from rat ileum. Calcium-specific binding of porcine or rat haptocorrin-[57Co]cobalamin complexes was detected in the brush border membranes of distal suckling rat and porcine small intestine. This binding was almost completely blocked by another asialoglycoprotein, asialofetuin. The pH optimum for binding was 6-9, with a Ka of 0.25 nmol/L and a capacity of 4.6 fmol/mg protein. These properties, with the exception of the low capacity, are all consistent with those of the intact receptor on hepatocytes. The intestinal receptor was localized not to the basolateral membrane, as in the liver, but to the apical brush border, as suggested by the binding data. Furthermore, immunoperoxidase stains of paraffin-embedded tissue detected strong binding to the brush border and apical phagolysosome of mid and distal suckling rat intestine. These data show that, contrary to expectations, the minor components of the asialoglycoprotein receptor are functional and expressed in the apical membrane of the suckling intestine. The abundance of the protein by Western blot suggests possible roles for it in the neonatal gut other than haptocorrin binding.

Animals↗
PubMed1991

Human gastric intrinsic factor: characterization of cDNA and genomic clones and localization to human chromosome 11.

A human gastric intrinsic factor (IF) cDNA clone was isolated using a rat cDNA clone as a probe. Comparison of the predicted amino acid sequence revealed 80% identity of human IF with rat IF. These cDNA clones were used to isolate and map two overlapping clones encoding the human IF gene. The first exon of the cloned region (exon 2) contains 30 bp of the 5' untranslated region, the signal peptide, and the first 8 amino acids of the mature protein. Exons 3-10 encode the remainder of the coding and 3' noncoding regions. Southern analysis of genomic DNA indicated the presence of a single human IF gene and also revealed the presence of strong hybridizing sequences in genomic DNA from monkey, rat, mouse, cow, and human, suggesting that the IF gene is well conserved. The IF gene was localized to human chromosome 11 by concurrent cytogenetic and cDNA probe analysis of a panel of human X mouse somatic cell hybrids. Southern analysis of genomic DNA from patients with congenital pernicious anemia (lacking intrinsic factor) revealed normal restriction fragment patterns, suggesting that a sizable gene deletion was not responsible for the deficiency.

Amino Acid Sequence↗
PubMed1991

Undergraduate teaching project of the American gastroenterological Association: a pathophysiology resource.

The Undergraduate Teaching Project was founded as a committee of the American Gastroenterological Association to improve medical school teaching of the pathophysiological basis of gastrointestinal and liver diseases. Twenty-six units of colored illustrations have been produced in 18 years, which are designed to be used by instructors to supplement their own material. The slides are the result of extensive reviews and revisions, a process that has created a unique repository of educational concepts utilized by most US medical schools and over 50 foreign schools. This brief summary of the genesis and operation of this venture may be of interest to other professional societies concerned with the development of teaching materials.

Audiovisual Aids↗
PubMed1991

Glycosylation is not required for ligand or receptor binding by expressed rat intrinsic factor.

A cDNA clone encoding rat intrinsic factor (IF), pIFQ, has been inserted into the eukaryotic expression vector pSVL and used to transfect COS-1 cells. The IF produced by the transfected cells was secreted nearly exclusively into the medium at concentrations of 0.1-0.2 micrograms/ml. Tunicamycin treatment (1-10 micrograms/ml) completely blocked N-linked glycosylation but had no effect on IF secretion. The secreted glycosylated IF retained all the properties of native IF, i.e., high affinity for cobalamin (Cbl) and for the IF-Cbl receptor and relative resistance to low pH and to proteolysis. The nonglycosylated IF also retained these properties except that it was more protease sensitive. The protease degradation was prevented by the presence of the ligand Cbl. The presence of carbohydrate may play a role in protecting IF from digestion by pancreatic proteases in the intestinal lumen.

Animals↗
PubMed1991

Developmental expression of intestinal surfactant-like particles in rats.

We have reported the appearance of surfactant-like particles enriched for intestinal alkaline phosphatase and phosphatidylcholine within enterocytes and in the lumen of adult fat-fed rat intestine. Because rat pulmonary surfactant decreases in abundance during the first postnatal days, we examined the developmental expression of these intestinal particles in suckling rats. Electron microscopy revealed abundant particles in 1-day-old rats within and surrounding the villus enterocytes, declining in frequency by day 14. Phosphatidylcholine content, alkaline phosphatase, sucrase-isomaltase, and lactase activity in particles peaked 1 day after birth, declining rapidly to adult levels by day 3 of life, except for sucrase, which peaked again after weaning. The postnatal developmental profile of the same brush-border-associated enzymes was totally different. Membrane fractions enriched for alkaline phosphatase and of similar density to rat surfactant-like particles were isolated from the small intestine of an amphibian (Xenopus laevis) and a fish (grass carp). Electron microscopy of the Xenopus membranes revealed unilamellar structures similar to the rat particles, but the carp membranes were of dissimilar morphology. We conclude that particles with surfactant-like properties in the rat intestine are ontogenically expressed like pulmonary surfactant; similar particles are evident only in animals with lungs.

Alkaline Phosphatase↗
PubMed1991

A novel intracellular pathway for rat intestinal digestive enzymes (alkaline phosphatase and sucrase) via a lamellar particle.

To identify the mechanism of intestinal alkaline phosphatase (IAP) release into serum that is known to be associated with fat feeding, both luminal and serum IAP as well as sucrase-isomaltase levels were monitored by rocket electroimmunoassays and by immunogold labeling electron microscopy. Luminal and serum IAP, and to a much lesser extent sucrase-isomaltase, peaked at 7 hours after fat feeding. Analysis of the luminal IAP by isoelectric focusing showed that the enzyme had a slightly different pI than brush border IAP, but was still partially membrane bound, whereas serum IAP was no longer membrane bound. In parallel, by morphology, IAP and sucrase-isomaltase localized to intra- and extracellular lamellar membranous particles, most conspicuous at 7 hours after fat feeding, while very scarce in nonfat-fed animals. The membranous particles bearing the enzymes were commonly associated with fat droplets. These data are consistent with the existence of a novel pathway, possibly secretory, for IAP and sucrase-isomaltase via lamellar bodies, leading to appearance of the enzymes in lumen and serum, with subsequent release from the membrane extracellularly. They also offer an explanation for the known association of the increased secreted IAP after fat feeding.

Alkaline Phosphatase↗
PubMed1990

Isolation and characterization of a cDNA encoding porcine gastric haptocorrin.

A cDNA encoding the gastric haptocorrin was isolated from a porcine gastric mucosal lambda gt11 cDNA library using oligonucleotide probes. The 1.4-kb cDNA contains a 1.25-kb open reading frame and 178 nucleotides of 3' noncoding region. Although no initiator methionine is present, primer extension analysis indicated that the transcription initiation site is only 100 bp upstream of the 5' end of this clone. Northern blot analysis showed that a single mRNA species of 1.6 kb exists in hog gastric mucosa. There was no cross-hybridization between this cDNA and the mRNA for haptocorrin in rat submaxillary gland or gastric RNA by Northern blot analysis. This lack of cross-reactivity was also seen on Southern blots, where cow, sheep and dog but neither rat nor mouse genomic DNAs showed cross-hybridizing bands. Comparison of the deduced amino acid sequence of this cDNA with that previously reported for rat intrinsic factor [Dieckgraefe, B.K. et al. (1988) Proc. Natl Acad. Sci. USA 85, 46-50] showed considerable similarity, suggesting that these cobalamin-binding proteins may have a common evolutionary origin.

Amino Acid Sequence↗