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M Verhave

Publications and source records attributed to M Verhave.

16 recordsLinked to original sources
PubMed1998

Three distinct messenger RNA distribution patterns in human jejunal enterocytes.

BACKGROUND & AIMS: The importance of messenger RNA (mRNA) localization in human enterocytes is poorly understood. Previous studies from our laboratory have indicated that mRNAs are asymmetrically distributed in human intestinal epithelial cells, but in general colocalized with their encoded proteins. The aim of this study was to characterize, in human enterocytes, mRNA localization patterns of three genes with distinctly different functions. METHODS: mRNA distribution was determined by in situ hybridization with digoxigenin-labeled RNA probes in tissue sections of human jejunum. RESULTS: The mRNA for villin, a well-characterized microvillus cytoskeletal protein, was sorted to the basal region of the enterocyte. The mRNA for human sodium glucose cotransporter 1 was localized to the apical region, and the mRNA for human liver fatty acid-binding protein was distributed diffusely in the cytoplasm. CONCLUSIONS: The three distinct mRNA distribution patterns suggest that active mRNA sorting mechanisms exist in human enterocytes. This study also reveals for the first time that dichotomies may occur between the distribution patterns of sorted mRNAs and their encoded proteins.

Adult↗
PubMed1998

Asymmetrical localization of mRNAs in enterocytes of human jejunum.

Intracellular localization of specific mRNAs is known to be a mechanism for targeting proteins to specific sites within the cell. Previous studies from this laboratory have demonstrated co-localization of mRNAs and proteins for a number of genes in absorptive enterocytes of fetal rat intestine. The present study was undertaken to examine in human enterocytes the intracellular localization patterns of mRNAs for the microvillous membrane proteins lactase-phlorizin hydrolase (LPH), sucrase-isomaltase (SI), and intestinal alkaline phosphatase (IAP), and the cytoskeletal protein beta-actin. In sections of human jejunum, mRNAs were localized by in situ hybridization using digoxigenin-labeled anti-sense RNA probes. Both LPH and SI mRNAs were localized to the apical region of villous enterocytes, whereas IAP and beta-actin mRNAs were detected both apically and basally relative to the nucleus. Therefore, in contrast to LPH, SI, and beta-actin mRNAs, which co-localize with their encoded proteins, that of IAP is present in the basal region of the cell where IAP protein has not directly been demonstrated to be present. Absorptive enterocytes from humans possess the mechanisms for intracellular mRNA localization, but not all mRNAs co-localize with their encoded proteins.

Actins↗
PubMed1997

Rat lactase-phlorizin hydrolase/human growth hormone transgene is expressed on small intestinal villi in transgenic mice.

BACKGROUND & AIMS: Lactase-phlorizin hydrolase (LPH) is an absorptive enterocyte-specific gene that is expressed in a well-characterized pattern along the cryptvillus (vertical), proximal-distal (horizontal), and developmental (temporal) gradients. The aim of this study was to characterize the capacity of regulatory elements within the rat LPH gene to direct appropriate cell lineage and topographical patterns of expression in vivo in transgenic mice. METHODS: Transgenic mouse lines were established using a construction containing bases -2038 to +15 of the rat LPH gene fused to a human growth hormone reporter gene. RESULTS: In one line, the transgene was expressed only in small intestine and was localized to absorptive enterocytes on villi. The transgene was not expressed in goblet or enteroendocrine cells or in crypts. Transgene expression along horizontal and developmental gradients was different from that of the native mouse LPH gene. CONCLUSIONS: The results suggest that the region from -2038 to +15 of the rat LPH gene contains regulatory elements that direct correct tissue, cell, and vertical expression but may not contain all the elements necessary for appropriate horizontal and temporal control. This investigation provides further insight into the complexities of the molecular control of intestinal gene expression.

Aging↗
PubMed1996

Combined use of cyclosporine and azathioprine or 6-mercaptopurine in pediatric inflammatory bowel disease.

The aim of this study was to assess whether in steroid-resistant patients with pediatric inflammatory bowel disease (IBD) a combination of cyclosporine and azathioprine (or 6-mercaptopurine) could induce remission and subsequently permit maintenance on azathioprine/6-mercaptopurine as the sole immunosuppressive agent. Two boys and six girls (six with ulcerative colitis and two with Crohn's disease; ages 3-17 years) received 100-200 micrograms/kg/day cyclosporine intravenously and then 4-10 mg/kg/day orally. Doses were adjusted to achieve trough serum cyclosporine levels of 100-200 mu/L (Abbott's TDX assay). Seven of the eight patients received azathioprine/6-mercaptopurine, and all were given a 5-aminosalicylate preparation and corticosteroids. The latter drugs were continued and then tapered off as clinical status allowed. Cyclosporine was continued for 3-10 months in those who responded. In seven of eight patients, there was a rapid clinical response; one patient showed a transient response, but recurrent bleeding led to total colectomy 9 days after starting cyclosporine. Of the seven responders, three were able to discontinue prednisone and cyclosporine and are doing well on azathioprine at long-term follow-up (2-5 years). One who did not receive azathioprine/6-mercaptopurine maintained remission for 2 years after cyclosporine was stopped, one experienced a disease flare-up 5 months after start of cyclosporine treatment and required colectomy, one who did not tolerate 6-mercaptopurine had a flare-up during cyclosporine tapering and underwent surgery at 6 months, and one started to flare up with cyclosporine tapering at 6 months and was scheduled for surgery. No significant complications of treatment were observed. Seven patients had an initial response and four of them have so far not required surgery. These preliminary findings suggest that azathioprine/6-mercaptopurine can be used safely to maintain cyclosporine-induced remission in children with IBD.

Adolescent↗
PubMed1995

Further characterization of the 5'-flanking region of the rat lactase-phlorizin hydrolase gene.

The lactase-phlorizin hydrolase gene is widely used as a marker of intestinal differentiation. Recent evidence demonstrating that transcription plays a major role in the regulation of this gene suggests that study of the 5'-flanking region will allow an understanding of how the expression of this gene is controlled. However, sequence, restriction, and primer extension analysis of a rat genomic clone has revealed that previously published data are incomplete. In the present study, we used a directed sequencing strategy to carefully analyze this region. Our expanded analysis of the 5'-flanking region of the lactase-phlorizin hydrolase gene should facilitate future studies of its structure and function.

Animals↗
PubMed1995

Verification of the lactase site of rat lactase-phlorizin hydrolase by site-directed mutagenesis.

BACKGROUND & AIMS: Lactase-phlorizin hydrolase (LPH) is an intestinal microvillus membrane glycoprotein that hydrolyzes lactose and phlorizin. These enzymatic activities have been assigned to glutamic acid (E) residues 1271 and 1747 in rabbit LPH. The aim of this study was to determine directly if this assignment was correct and if these two amino acids are the only nucleophiles required for LPH enzyme activity. METHODS: Site-directed mutagenesis of a full-length rat LPH complementary DNA was used to convert the rat homologues E1274 and E1750 to aspartic acid or glycine. Mutants were analyzed by enzyme activity assays. RESULTS: All tested activities of E1274D and E1274G were virtually unaffected. In contrast, mutations E1750D and E1750G resulted in total loss of lactase and cellobiose activities, leaving only low ONP-glc and ONP-gal hydrolase activities detectable. A double mutant containing both E1274G and E1750G had no activity. CONCLUSIONS: These studies directly confirm that the two previously identified glutamic acids are essential to the enzymatic activity of rat LPH. Rat lactase activity is not associated with the E1274 site. This study provides the first evidence that rat LPH has its major catalytic site at E1750, representing all of the lactase and the majority of the phlorizin hydrolase activity.

Amino Acid Sequence↗
PubMed1994

Transcriptional regulation of intestinal hydrolase biosynthesis during postnatal development in rats.

Lactase-phlorizin hydrolase (LPH) and sucrase-isomaltase (SI) are intestine-specific microvillus membrane hydrolases whose specific activities demonstrate reciprocal regulation during development but whose mechanisms of regulation have not been fully defined. To investigate transcriptional control of these two proteins, the rat LPH and SI genes were cloned, and antisense probes for preprocessed mRNAs (pre-mRNAs) were developed from intron sequence. LPH mRNA, as measured by quantitative ribonuclease (RNase) protection assays, was abundant before weaning and decreased two- to fourfold during weaning, whereas SI mRNA was first detected 14 days after birth and increased rapidly to abundant levels by age 28 days. LPH and SI pre-mRNA levels paralleled those of their respective mRNAs. LPH transcriptional rate declined during weaning, whereas that of SI increased during this time as determined by RNase protection assays of pre-mRNAs and nuclear run-on assays. In the adult rat, LPH mRNA was restricted to the jejunum and proximal ileum, whereas SI mRNA was detected throughout the small intestine, a pattern regulated by transcriptional rate as confirmed by nuclear run-on assays. Lactase and sucrase specific activities correlated well with their respective protein and mRNA concentrations in all experiments. We conclude that gene transcription plays a major role in the developmental and horizontal regulation of LPH and SI biosynthesis and that these two genes are regulated differently in rat small intestine.

Aging↗
PubMed1993

Identification of nonsteroidal antiinflammatory drug-induced gastroduodenal injury in children with juvenile rheumatoid arthritis.

In a cohort of children with juvenile rheumatoid arthritis treated with nonsteroidal antiinflammatory drugs and referred for gastrointestinal complaints, more than 75% had gastritis, antral erosions, or ulcers. Epigastric pain strongly correlated with documented gastroduodenal injury. Therapy with ranitidine or misoprostol led to clinical improvement. Nonsteroidal antiinflammatory drugs are associated with significant gastrointestinal abnormalities in children.

Abdominal Pain↗
PubMed1991

Cyclic AMP- and phorbol ester-induced transcriptional activation are mediated by the same enhancer element in the human vasoactive intestinal peptide gene.

Transcription of the human vasoactive intestinal peptide (VIP) gene is regulated by both cyclic AMP and phorbol esters. A 17-nucleotide enhancer element within the human VIP gene mediates transcriptional activation by both phorbol esters and forskolin. Mutations of this element decrease responses to both agents, suggesting that the trans-acting proteins that mediate both modes of transcriptional regulation have similar DNA-binding characteristics. The response of the VIP enhancer element to forskolin, but not to 12-O-tetradecanoylphorbol-13-acetate, was attenuated by treatment with a recombinant inhibitor of the cAMP-dependent protein kinase, suggesting that the cAMP-dependent protein kinase and protein kinase C second messenger pathways that converge on this single enhancer element are distinct. The transcriptional activator cAMP-responsive element-binding (CREB) proteins and the c-fos.c-Jun complex interact with the VIP enhancer. The dual second messenger responses of the VIP gene may result from the interaction of this second messenger enhancer with different transcriptional activator proteins.

Base Sequence↗
PubMed1990

Somatostatin gene regulation: an overview.

The somatostatinergic system has proven to be one of the best models of neuropeptide biology. Originally characterized as a hypothalamic regulator of growth hormone secretion, somatostatin also regulates the secretion of several other pituitary, pancreatic, and gastrointestinal (GI) hormones including thyrotropin-stimulating hormone, insulin, glucagon, and gastrin. Disorders in somatostatin metabolism have been proposed to contribute to the pathogenesis of Alzheimer's disease, epilepsy, GI motility disorders, and diabetes. On a more basic level, studies of somatostatin action have integrated divergent concepts of intracellular signal transduction. Advances in the understanding of somatostatin biosynthesis have had an impact on areas outside the field of endocrinology by providing new concepts of eukaryotic gene regulation. This report focuses on the transcriptional regulation of somatostatin gene expression. Two aspects of somatostatin gene transcription will be considered--regulated expression by second messengers and tissue-specific basal expression.

Amino Acid Sequence↗
PubMed1990

Azathioprine in the treatment of children with inflammatory bowel disease.

During a 6-year period, we treated 21 patients with azathioprine, 2 mg/kg/day, as an adjunct to their customary regimen. Nine patients had ulcerative colitis and 12 patients had Crohn disease; the patients' ages ranged from 3 to 17 years. The median duration of disease before the start of azathioprine therapy was 2 years, and median follow-up was 2 years. Sixteen patients seemed to respond to azathioprine therapy: six patients in each disease group had complete responses and four patients (one with ulcerative colitis and three with Crohn disease) had partial responses. Two patients with ulcerative colitis and three patients with Crohn disease did not respond. The median time until patients responded was less than 3 months for patients with ulcerative colitis and 4 months for those with Crohn disease. Reduction of corticosteroid dose was possible for all patients who responded to azathioprine therapy. Only minimal side effects were attributable to the drug. We conclude that azathioprine is an effective adjunctive agent for the treatment of inflammatory bowel disease in childhood, but because questions remain regarding its long-term safety, its use should be reserved for children with refractory disease or severe and unacceptable side effects of corticosteroids.

Administration, Oral↗
PubMed1988

The CGTCA sequence motif is essential for biological activity of the vasoactive intestinal peptide gene cAMP-regulated enhancer.

cAMP-regulated transcription of the human vasoactive intestinal peptide gene is dependent upon a 17-base-pair DNA element located 70 base pairs upstream from the transcriptional initiation site. This element is similar to sequences in other genes known to be regulated by cAMP and to sequences in several viral enhancers. We have demonstrated that the vasoactive intestinal peptide regulatory element is an enhancer that depends upon the integrity of two CGTCA sequence motifs for biological activity. Mutations in either of the CGTCA motifs diminish the ability of the element to respond to cAMP. Enhancers containing the CGTCA motif from the somatostatin and adenovirus genes compete for binding of nuclear proteins from C6 glioma and PC12 cells to the vasoactive intestinal peptide enhancer, suggesting that CGTCA-containing enhancers interact with similar transacting factors.

Base Sequence↗
PubMed1982

An evaluation of two antigen nonspecific assays for circulating immune complexes using a model system.

Two methods of detecting circulating immune complexes (CIC) in serum, the Protein A-glass fiber filter assay (PA-GFF) and the polyethylene glycol (PEG) insolubilization assay, were compared using a model system of BSA/anti-BSA. Supernatants of the quantitative precipitin curve from extreme antigen excess through antibody excess were tested by both methods. The PEG assay detected soluble CIC in all areas where expected by comparison with the Farr Test; the filter assay did not, although it did detect aggregated globulins quantitatively. The PEG assay detected high levels of CIC in serum of rheumatoid arthritis patients and low levels in normal human serum. Postprandial samples and plasma with heparin or EDTA caused unpredictable increases in results in the PEG assay so that for best results the PEG assay should be run on serum from fasting individuals. PEG insolubilized complexes were analyzed and found to contain expected serum components. A slight modification of the original method was made and tested using two other model complex systems and known CIC-containing sera. The PEG turbidity assay it fast, easy, reproducible, and suitable for quantitating and isolating immune complexes in serum.

Animals↗