Cell-CAM105 (also named C-CAM) is a cell surface glycoprotein involved in intercellular adhesion of rat hepatocytes. It has four extracellular immunoglobulin (Ig) domains, a transmembrane domain and a cytoplasmic domain and therefore is a member of the Ig supergene family. We have characterized multiple cDNAs of the C-CAM genes in rat intestine. Sequence analyses showed that rat intestine contained not only the previously reported L-form and S-form C-CAMs (renamed C-CAM1 and C-CAM2 respectively) but also a new isoform, C-CAM3. The C-CAM3 transcript codes for a polypeptide with a truncated C-terminus that lacks 65 amino acids from the previously reported C-CAM1 cytoplasmic domain. Unlike C-CAM1, C-CAM3 did not mediate cell adhesion when expressed in insect cells using the baculoviral expression system. Thus the extra 65 amino acids in the cytoplasmic domain of C-CAM1 are important for adhesion phenotype when expressed in insect cells. Although C-CAM1 and C-CAM2 are encoded by different genes, sequence analysis suggests that C-CAM3 is probably derived from alternative splicing of the C-CAM1 gene. To examine this possibility, we have determined the exon organization of the C-CAM1 gene. C-CAM3 differed from C-CAM1 by the presence of a single unspliced intron which contained a stop codon immediately after the regular splice junction. As a result, translation of C-CAM3 terminates at the point where C-CAM1 and C-CAM3 sequences diverge. To investigate the expression of C-CAM1, C-CAM2 and C-CAM3 in different tissues, we used an RNAase-protection assay to simultaneously assess the levels of expression of these transcripts. Using total RNA prepared from various tissues, we showed that expression of C-CAM3 was tissue-specific, and the C-CAM3 transcript accounted for about 25% of the transcripts derived from the C-CAM1 gene. However, further analysis revealed that C-CAM3 transcript was not present in cytosolic RNA, rather it was enriched in nuclear RNA prepared from hepatocytes. Although C-CAM3 cDNA contains the polyadenylation signal and is polyadenylated, these results indicate that C-CAM3 is probably an incomplete spliced product of C-CAM1 gene.
Bortezomib (Velcade®) is a proteasome inhibitor that has been approved for the treatment of multiple myeloma and mantle cell lymphoma. It has been shown to inhibit the expression of cell adhesion molecules, co-stimulatory molecules, and NFĸB activation, to deplete alloreactive T lymphocytes, and to decrease Th1 cytokine production. The anti-inflammatory effects of bortezomib were further investigated in this current set of studies. Systemic treatment with bortezomib was efficacious in the thioglycolate-induced MCP-1 production model, and the dinitrofluorobenzene-induced delayed-type hypersensitivity model. Psoriasis is an autoimmune disease that affects about 2% of the world population. Many treatments have been reported with varying degrees of efficacy. A topical bortezomib formulation was developed to minimize systemic exposure. Its tolerability was investigated in a topical imiquimod (IMQ)-induced psoriasis model. Daily application of IMQ on mouse skin induced inflamed scaly skin lesions resembling plaque-type psoriasis. Fatality was observed in the 1-mg/ml dose group. At 0.1 and 0.01 mg/ml, bortezomib potentiated IMQ-induced erythema, scaling, skin thickening, and caused necrotic lesions. Lower doses had no effect on the clinical observations. Histologically, bortezomib dose-dependently increased parakeratosis, hyperkeratosis, acanthosis, and inflammatory cell infiltration. This study demonstrated that topical bortezomib is not suitable for the treatment of psoriasis.
C-CAMs are epithelial cell-adhesion molecules of the immunoglobulin supergene family with sequences highly homologous to carcinoembryonic antigen (CEA). C-CAMs and their human homologues, biliary glycoproteins, are unique among the CEA-family proteins in that they have cytoplasmic domains. Furthermore, alternative splicing generates C-CAM isoforms with different cytoplasmic domains, suggesting that the cytoplasmic domains of C-CAM may play important roles in regulating the function or functions of C-CAM. By using both sense and antisense approaches, we have shown that C-CAM1 is a tumour suppressor in prostate carcinogenesis. This observation raises the possibility that the cytoplasmic domain of C-CAM1 may be involved in signal transduction or interaction with cytoskeletal elements to elicit the tumour suppressor function. The cytoplasmic domain of C-CAM1 contains several potential phosphorylation sites, including putative consensus sequences for cyclic AMP-dependent kinase and tyrosine kinase. One of the potential tyrosine phosphorylation sites is located within the antigen-receptor homology (ARH) domain. The ARH domain of the membrane-bound IgM molecule is necessary for signal transduction in B-cells. These structural features suggest that the cytoplasmic domain of C-CAM1 may be important for signal transduction. To test this possibility, we generated several site-directed C-CAM1 mutants and tested their ability to support adhesion and their abilities to be phosphorylated in vivo. Results from these studies revealed that Tyr-488 is phosphorylated in vivo. However, replacing this tyrosine with phenylalanine did not significantly compromise its adhesion function. Similarly, Ser and Thr residues are phosphorylated in vivo, but deletion of the potential cyclic AMP-dependent kinase site did not significantly reduce the adhesion function. These results suggest that the kinase phosphorylation sites in the cytoplasmic domain of C-CAM1 are not required for the adhesion function. However, these phosphorylation sites are probably involved in the regulation of C-CAM-mediated signal transduction. Thus, there are probably distinct structural requirements for the adhesion and the signal transduction functions of C-CAM. Incidentally, a C-CAM1 deletion mutant containing a 10-amino-acid cytoplasmic domain was able to support adhesion activity. This is in contrast to our previous finding that a C-CAM isoform, C-CAM3, with a 6-amino-acid cytoplasmic domain could not support cell adhesion. This result indicates that the extra four amino acids, which are absent in C-CAM3 and contain a potential Ser/Thr phosphorylation site, are important for the adhesion function.
Our data indicated that etanercept is not a suitable treatment for 5-FU-induced mucositis. Despite decreased apoptosis in the gut, cyclosporine did not affect the severity of the diarrhea or survival. Therefore, we concluded that cyclosporine treatment was only effective in mediating the short-term apoptotic events in the intestines but has no long-term effect on the animals' survival and diarrhea.
Background: Doxazosin is an α1-adrenergic receptor antagonist for the treatment of high blood pressure and benign prostatic hyperplasia. Peripheral α-adrenergic receptors have been implicated in inflammation. Aim: To examine the anti-inflammatory effects of doxazosin in rodent models of inflammation. Method: The anti-inflammatory properties of doxazosin were investigated in 4 models. In all studies, drug treatment was administered 15 min prior to challenge. In the lipopolysaccharide (LPS)-induced systemic inflammation model, LPS was injected systemically at 0.25 mg/kg. At 90 min after challenge, blood samples were collected for analysis. In the LPS-induced pulmonary inflammation model, LPS was instilled intranasally. Four hours after challenge, the lungs were harvested for monocyte chemoattractant protein-1 (MCP-1) analysis. In a delayed-type hypersensitivity model, the mice were injected intravenously with sheep red blood cells, and rechallenged in the left footpad 7 days later. Drug treatment was given on day 6 and 7 just prior to the rechallenge. The thickness of hind footpads was measured at 15 min after rechallenge. In the thioglycollate-induced peritoneal monocyte infiltration model, mice were challenged with 3% thioglycollate, and 2 h later peritoneal lavage fluid was collected for MCP-1 analysis. Results: In animals challenged systemically and intranasally with LPS, doxazosin inhibited TNF-α and MCP-1 production, respectively. In the delayed-type hypersensitivity model, footpad swelling was inhibited by doxazosin. Doxazosin decreased the level of MCP-1 release in the peritoneal cavity of thioglycollate-stimulated animals, though this effect was not statistically significant. Conclusion: This is the first set of studies that reports the novel anti-inflammatory effects of doxazosin.
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