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Biomedical subjects

G D Wu

Publications and source records attributed to G D Wu.

At least 73 records · Page 4Linked to original sources
PubMed1992

Isolation and characterization of the human sucrase-isomaltase gene and demonstration of intestine-specific transcriptional elements.

The molecular mechanisms that regulate intestine-specific gene expression and the transition from proliferating, undifferentiated crypt cells to nonproliferating, differentiated villus cells are unknown. Sucrase-isomaltase is an apical membrane disaccharidase that is found exclusively in enterocytes of adult intestine and is expressed in a complex pattern along the intestinal crypt-villus axis. To investigate the regulation of sucrase-isomaltase, we have cloned and sequenced 3.6 kilobases of the 5'-flanking region of the human sucrase-isomaltase gene. The transcriptional start site was mapped in human small intestine and in a colonic adenocarcinoma cell line (Caco-2) using an anchored polymerase chain reaction, primer extension, and RNase protection assays. The 5'-flanking DNA of the gene was linked to either chloramphenicol acetyltransferase or luciferase reporter genes and used for transfection into Caco-2, HeLa, and HepG2 cells. This analysis demonstrated that intestine-specific transcription of the sucrase-isomaltase gene involves both proximal and distal regulatory elements. Use of sucrase-isomaltase as a model gene will allow investigation of the mechanisms that regulate transcription of enterocyte-specific genes, developmental gene expression in the small intestine and colon, and the process of differentiation as epithelial cells migrate from intestinal crypts onto the villus in adult intestine.

Adenocarcinoma↗
PubMed1992

The prolongation of concordant hamster-to-rat cardiac xenografts by brequinar sodium.

Brequinar sodium (BQR) prevents cell proliferation by virtue of its inhibition of de novo pyrimidine biosynthesis. The immunosuppressive activity of BQR is highly effective in prolonging heart, liver, and kidney allograft survival in the rat. In these experiments, we have tested the ability of BQR to prevent the rejection of concordant cardiac xenografts. LEW inbred rats transplanted with heterotopic hamster hearts were treated orally with brequinar sodium as a single agent. The survival of the cardiac xenografts was significantly prolonged with a variety of treatment regimens. The most effective treatment was the daily oral administration of BQR at 3 mg/kg. At this level, the median graft survival was approximately 25 days. Four animals had hamster heart xenografts that functioned for more than 90 days. The prolonged survival of the xenografts was associated with relatively constant plasma drug levels of approximately 1 to 3 micrograms/ml and a marked suppression of IgM production. At rejection, there was a significant rise in IgM levels compared with those of recipients with stable xenografts. In vitro MLR responses were effectively inhibited by BQR, with an IC50 of 0.08 microgram/ml. The results of these experiments demonstrate that BQR is a new immunosuppressive agent that is highly effective as a single agent in prolonging the survival of hamster-to-rat cardiac xenografts. The prolonged xenograft survival is associated with effective suppression of rat antihamster antibody production, suggesting that brequinar sodium may be an important addition to multidrug immunosuppressive regimes designed to prevent B and T lymphocyte-mediated immune responses.

Animals↗
PubMed1992

Novel DNA-binding proteins regulate intestine-specific transcription of the sucrase-isomaltase gene.

Sucrase-isomaltase (SI) is an enterocyte-specific gene which exhibits a complex pattern of expression during intestinal development and in the adult intestinal mucosa. In the studies described in this report, we demonstrate that enterocyte-specific transcription of the SI gene is regulated by an evolutionarily conserved promoter that extends approximately 180 bp upstream of the transcription start site. DNase I footprint analysis allowed the identification of three nuclear protein-binding sites within the SI promoter (SIF1, SIF2, and SIF3 [SI footprint]), each of which acted as a positive regulatory element for transcription in intestinal cell lines. SIF1 was shown to bind nuclear protein complexes present in primary mouse small intestinal cell and in an intestinal cell line (Caco-2). However, SIF1-binding proteins were absent in a variety of other epithelial and nonepithelial cells. In vitro mutagenesis experiments demonstrated that the SIF1 site is required for high-level promoter activity in intestinal cells. The SIF3 element formed prominent binding complexes with intestinal and liver nuclear extracts, whereas nuclear proteins from other epithelial and nonepithelial cells formed weaker complexes of different mobilities. The SIF2 element bound nuclear proteins in a pattern similar to that of SIF3, and cross-competition studies suggested that SIF2 and SIF3 may bind the same nuclear proteins. Taken together, these data have allowed the identification of novel DNA-binding proteins that play an important role in regulating intestine-specific transcription of the SI gene.

Animals↗
PubMed1992

Sucrase-isomaltase gene expression along crypt-villus axis of human small intestine is regulated at level of mRNA abundance.

The mucosal lining of the small intestine is a complex epithelium that is continually renewed by division of a stem cell population located in intestinal crypts, migration of daughter cells along the villus, and, finally, extrusion of senescent cells into the lumen. The majority of cells in both crypt and villus cell compartments are enterocytes that acquire differentiated functions as they migrate out of the crypt. Sucrase-isomaltase (SI) is an enterocyte-specific, brush-border enzyme that has little activity in crypt cells and maximal activity in low and mid villus cells. The mechanism by which enterocytes acquire SI enzymatic activity as they move from crypt to villus is controversial. In this study we examined the distribution of SI mRNA along the crypt-villus axis of human small intestine using isolated epithelial cells and in situ hybridization. A complementary DNA to the 5' portion of the human SI mRNA was amplified and cloned using the polymerase chain reaction. Hybridization analysis of RNA extracted from human intestinal epithelial cells showed that the cloned cDNA recognized a single 6.5-kb mRNA. In situ hybridization of duodenal biopsy specimens was performed using a single-stranded RNA probe derived from this cDNA. This analysis showed that there was little SI mRNA in crypt cells and appearance of mRNA in enterocytes located at the crypt-villus junction. The mRNA levels were maximal in lower and mid villus cells with decreased levels noted in villus tip cells. These results are identical to those previously described in rat intestine and suggest that expression of the SI gene as enterocytes emerge from intestinal crypts is regulated primarily at the level of mRNA accumulation. Study of SI gene regulation may provide a useful model to investigate the mechanisms that regulate enterocyte-specific gene expression and intestinal differentiation.

Amino Acid Sequence↗
PubMed1990

Cardiac transplantation in the rat. II. Alteration of the severity of donor graft arteriosclerosis by modulation of the host immune response.

Cardiac transplantation between inbred rat strains that differ for weak histocompatibility antigens is associated with the development of arteriosclerosis in the arteries of the donor graft myocardium. The lesions are seen in donor/recipient pairs that differ for both MHC and non-MHC histocompatibility antigens that apparently stimulate a low-level, chronic rejection of the donor heart graft. The arteriosclerosis associated with this chronic rejection consists of a diffuse, concentric proliferation of the intima and pathologically resembles the lesions observed in the coronary arteries of long-term human cardiac graft recipients. We have recently examined the influence of positive and negative manipulation of the host immune response on the development of the graft arteriosclerosis. Our results demonstrate that delayed harvest of the cardiac grafts or immunization with donor skin grafts or splenic lymphocytes increases the sensitivity of the recipient to the donor heart grafts--and, under conditions that allow for the long-term survival of the graft--increases the severity of the arteriosclerotic lesions. Alternatively, suppression of the host immune responses with cyclosporine or FK506, substantially reduces the arteriosclerotic changes. These results suggest that control of accelerated graft arteriosclerosis in long-term human cardiac recipient may require more careful and effective immunosuppression of the allograft reaction.

Animals↗
PubMed1989

A comparison of routine light microscopy, immunohistochemistry, and in situ hybridization for the detection of cytomegalovirus in gastrointestinal biopsies.

Eighty (80) paraffin-embedded intestinal biopsies from 35 AIDS patients were evaluated for the presence of cytomegalovirus by use of biotinylated DNA probe, immunohistochemical assay, and routine hematoxylin and eosin (H&E) staining. Cytomegalovirus was detected in 13 biopsies (16.3%) from five patients (14.2%). The distribution of positive biopsies was one in seven esophageal biopsies, three in 19 small bowel biopsies, six in 27 colonic biopsies, and three in 17 rectal biopsies. Immunoelectron microscopy was employed to confirm the presence of CMA in four of the positive in situ hybridization cases. In situ hybridization for CMV DNA was more sensitive than immunostaining, and both proved superior to routine H&E staining in terms of sensitivity.

Acquired Immunodeficiency Syndrome↗
PubMed1987

A versatile and simplified non-random strategy for nucleotide sequencing.

We describe a versatile and simplified non-random strategy for nucleotide sequencing. This procedure avoids exposure of the vector DNA to endo- or exonucleases during construction of the subfragment library. The advantages resulting from this strategy are: (1) minimal manipulation in construction of the subfragment library, (2) no need for compatible restriction sites, (3) no insert size limitation, (4) can be used with both chemical and enzymatic sequencing methods. Hence, the procedure provides simplicity, universality and versatility for non-random nucleotide sequencing. We demonstrate the usefulness of this procedure by obtaining the nucleotide sequence of a goat 3.7-kb EcoRI fragment, which is 5' of the epsilon globin gene.

Animals↗