Expression In Intestinal Tissue Research Articles

CD44 EXPRESSION IN REJECTING RAT SMALL BOWEL ALLOGRAFTS1,2

CD44 is a widely expressed cell surface protein that recognizes multiple ligands and is involved in extra- and intercellular adhesion. The precise role of CD44 in immune interactions is currently unknown, but it is believed to be a homing receptor involved in lymphocyte trafficking and inflammatory responses. This study investigated CD44 expression in intestinal tissue after heterotopic rat small bowel transplantation and assessed the effect of transplantation on intestinal epithelial cell proliferation using an antibody to the nuclear activation Ag Ki67. Lamina propria and intestinal epithelial cell expression of CD44 was graded blindly by five observers, and villus epithelial cells were noted as being positive or negative for Ki67 staining. CD44 expression was high in the lamina propria of both allografted and isografted animals; however, there was no significant difference between the two groups. In contrast, the expression of CD44 on the villus epithelium was greater in allografted animals and progressed toward the villus tips as rejection developed, declining thereafter because of loss of villus integrity. Ki67 positivity was also greater in allografted animals but did not progress toward the villus tip. This is the first reported observation of CD44 expression on intestinal epithelium that is not restricted to the crypts. The findings indicate the involvement of CD44 in the rejection process and demonstrate changes in the proliferative profile of rejecting small intestinal epithelium. Further studies into adhesion molecules, such as CD44, may help to improve understanding of graft failure and promote the development of new therapeutic approaches for controlling and preventing graft rejection.

Cloning of the Rat Ecotropic Retroviral Receptor and Studies of Its Expression in Intestinal Tissues

A long-term goal of our laboratory is to establish a rat model to study the feasibility of using the intestinal tract as a site for somatic gene therapy. As a step toward that goal, the current study reports the cloning of the rat ecotropic retroviral receptor (EcoR) cDNA and the study of various aspects of its expression in the intestinal tissues. The cDNA for rat EcoR was cloned by screening a size-selected rat intestinal cDNA library with mouse EcoR cDNA. A clone of approximately 7 kb, designated MP10, was obtained. Partial sequencing of MP10 from the 5' end revealed a level of similarity of 92% compared with mouse EcoR. The presence of a 5' untranslated region and a 3' poly(A) tract, together with the overall size of the cDNA, suggest that is very close to being a full-length cDNA for this large transcript. Northern blots with MP10 showed an RNA of approximately 7.9 kb present along the entire length of the small intestine and somewhat less abundant in the colon. Developmental studies showed high levels of EcoR in fetal rat intestine, a decline in the early postnatal period, then a gradual rise to adulthood. Caco-2 cells were used to assess the expression of EcoR in proliferating compared with differentiated intestinal epithelial cells. EcoR mRNA was found to be very much more abundant in nondifferentiated cells and declined to low levels as the cells underwent spontaneous differentiation. These patterns of EcoR expression indicate that ecotropic retroviruses should be suitable vectors with which to attempt gene transfer into the intestinal epithelium. In addition, since the endogenous role of EcoR is as the y+ cationic amino acid transporter, these data have significance for understanding patterns of amino acid transport in the intestinal epithelium.

An in situ hybridization histochemistry method for the use of alkaline phosphatase-labeled oligonucleotide probes in small intestine.

We have developed a method of non-radioactive in situ hybridization histochemistry using alkaline phosphatase-labeled oligonucleotide probes to detect gene expression in the intestine. Because the intestine contains a large amount of endogenous alkaline phosphatase activity, mild acid pretreatment of the tissue sections was required to inactivate the alkaline phosphatase. Acid pre-treatment dramatically reduced the endogenous activity without affecting the efficiency of hybridization or the probe's ability to reveal a positive mRNA signal. Furthermore, the addition of polyvinyl alcohol to the substrate solution helped to keep the background staining low without adversely affecting the intensity of the signal. The current protocol allows rapid and sensitive detection of sites of gene expression in intestinal tissue.

Identification of a new type of mammalian myosin heavy chain by molecular cloning. Overlap of its mRNA with preprotachykinin B mRNA.

In this paper, we report the polypeptide sequence, the mRNA sequence, and gene organization of a new category of mammalian myosin heavy chain, termed myosin I heavy chain-like protein (MIHC), on the basis of sequence analyses of cDNA clones isolated from a bovine intestinal cDNA library and a genomic DNA clone containing the MIHC gene. The MIHC mRNA shares its 3' sequence with the 5' sequence of the preprotachykinin B mRNA encoding the precursor for the neuromedin K neuropeptide. An overlap of these functionally unrelated mRNAs results from overlapping transcription of the same strand of the genomic DNA sequence. The deduced amino acid sequence comprising the amino-terminal two-thirds of the 119-kDa MIHC polypeptide is highly homologous to that portion of conventional myosin heavy chains which forms the globular head domain. The homology is particularly strong in the regions of sequence thought to be involved in forming the nucleotide- and actin-binding sites. By contrast, the deduced sequence comprising the carboxyl-terminal one-third of MIHC shows no significant homology to that portion of conventional myosin heavy chains which forms the alpha-helical coiled-coil rod-like tail. In this regard, MIHC more closely resembles the monomolecular, non-filamentous, low molecular weight myosin I heavy chain from Acanthamoeba castellanii. Furthermore, the size of the MIHC polypeptide, together with its preferential expression in intestinal tissue, leads us to speculate that MIHC and the 110-kDa intestinal brush-border protein (Collins, J. H., and Borysenko, C. W. (1984) J. Biol. Chem. 259, 14128-14135), which binds actin and has myosin-like ATPase activity, are the same or closely related proteins.