Endocrine cells of the human gastrointestinal tract have no proliferative capacity

Summary There is compelling evidence that the epithelial cell lineages of the gastrointestinal tract are derived from a common stem cell precursor, but the details of the subsequent cellular hierarchies remain uncertain. In this context, it is important to know the arrangement of cell proliferation that gives rise to the final cell populations. In rodents, a number of studies have been performed examining the possible proliferative capacity of endocrine cells, but a wide range of technical problems makes interpretation of these data difficult. Continuous labelling studies suggest that there is potential for proliferation in endocrine cells but flash labelling studies have not been conclusive. In man there are no data on this issue. We have taken advantage of the ability to perform double immunostaining for operational markers of proliferation (Ki67 antigen) and endocrine cell phenotype (chromogranin expression). We demonstrate that there are no double-labelled cells in the normal stomach, small intestine or colon of fetal, neonatal or adult humans. Moreover, no double-labelled cells are found in pathological states associated with endocrine cell hyperplasia (gastritis, ulcerative colitis). These data indicate that the normal endocrine cells of the human gut have no proliferative capacity and that, in this cell lineage, population expansion precedes differentiation.     

Reovirus infection and tissue injury in the mouse central nervous system are associated with apoptosis.

Reovirus serotype 3 strains infect neurons within specific regions of the neonatal mouse brain and produce a lethal meningoencephalitis. Viral replication and pathology colocalize and have a predilection for the cortex, hippocampus, and thalamus. We have shown previously that infection of cultured fibroblasts and epithelial cells with reovirus type 3 Dearing (T3D) and other type 3 reovirus strains results in apoptotic cell death, suggesting that apoptosis is a mechanism of cell death in vivo. We now report that T3D induces apoptosis in infected mouse brain tissue. To determine whether reovirus induces apoptosis in neural tissues, newborn mice were inoculated intracerebrally with T3D, and at various times after inoculation, brain tissue was assayed for viral antigen by immunostaining and apoptosis was identified by DNA oligonucleosomal laddering and in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. Cells were also stained with cresyl violet to detect morphological changes characteristic of apoptosis, including chromatin condensation and cell shrinkage. DNA laddering was detected in T3D- but not in mock-infected brain tissue. Apoptotic cells were restricted to the same regions of the brain in which infected cells and tissue damage were observed. These findings suggest that virus-induced apoptosis is a mechanism of cell death, tissue injury, and mortality in reovirus-infected mice. The correlation between apoptosis and pathogenesis in vivo identifies apoptosis as a potential target for molecular and pharmacological strategies designed to curtail or prevent diseases resulting from induction of this cell death pathway.     

Reovirus-induced apoptosis of MDCK cells is not linked to viral yield and is blocked by Bcl-2.

In this study, we investigated the relationship between reovirus-induced apoptosis and viral growth. Madin-Darby canine kidney (MDCK) epithelial cells infected with prototype reovirus strains type 1 Lang (T1L) or type 3 Dearing (T3D) were found to undergo apoptosis, and T3D induced apoptosis of MDCK cells to a substantially greater extent than T1L. By using T1L x T3D reassortant viruses, we found that differences in the capacities of these strains to induce apoptosis are determined by the viral S1 and M2 gene segments. These genes encode viral outer-capsid proteins that play important roles in viral entry into cells. T1L grew significantly better in MDCK cells than T3D, and these differences in growth segregated with the viral L1 and M1 gene segments. The L1 and M1 genes encode viral core proteins involved in viral RNA synthesis. Bcl-2 overexpression in MDCK cells inhibited reovirus-induced apoptosis but did not substantially affect reovirus growth. These findings indicate that differences in the capacities of reovirus strains to induce apoptosis and grow in MDCK cells are determined by different viral genes and that premature cell death by apoptosis does not limit reovirus growth in MDCK cells.     

Inhibition of cysteine proteinases and dipeptidyl peptidase I by egg-white cystatin.

The interactions between egg-white cystatin and the cysteine proteinases papain, human cathepsin B and bovine dipeptidyl peptidase I were studied. Cystatin was shown to be a competitive reversible inhibitor of cathepsin B (Ki 1.7 nM, k-1 about 2.3 X 10(-3) s-1). The inhibition of dipeptidyl peptidase I was shown to be reversible (Ki(app.) 0.22 nM, k-1 about 2.2 X 10(-3) s-1). Cystatin bound papain too tightly for Ki to be determined, but an upper limit of 5 pM was estimated. The association was a second-order process, with k+1 1.0 X 10(7) M-1 X s-1. Papain was shown to form equimolar complexes with cystatin. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of complexes formed between papain or cathepsin B and an excess of cystatin showed no peptide bond cleavage after incubation for 72 h. The reaction of the active-site thiol group of papain with 5,5'-dithiobis-(2-nitrobenzoic acid) at pH 8 and 2,2'-dithiobispyridine at pH 4 was blocked by complex-formation. Dipeptidyl peptidase I and papain were found to compete for binding to cystatin, contrary to a previous report. The two major isoelectric forms of cystatin were found to have similar specific inhibitory activities for papain, and similar affinities for papain, cathepsin B and dipeptidyl peptidase I. This, together with specific oxidation of the N-terminal serine residue with periodate, showed the N-terminal amino group of cystatin 1 to be unimportant for inhibition. General citraconylation of amino groups resulted in a large decrease in the affinity of cystatin for dipeptidyl peptidase I. It is concluded that the interaction of cystatin with cysteine proteinases has many characteristics similar to those of an inhibitor such as aprotinin with serine proteinases.     

Proteinase inhibitors of bovine nasal cartilage.

Extracts from bovine nasal cartilage with 1 M-guanidinium chloride were fractionated by ultrafiltration. Gel chromatography of the low-molecular-weight material resolved three distinct fractions with inhibitory activity against (a) collagenases (22000 mol.wt.), (b) thiol proteinases cathepsin B and papain (13000 mol.wt.), and (c) trypsin and other serine proteinases (7000 mol.wt.).