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The concept that a general intracellular pool serves as the sole precursor of amino acids for protein biosynthesis has been vigorously debated in recent years. To help resolve this controversy, we followed the distribution of intraperitoneally administered [(3)H]valine in the tRNA and the extracellular and intracellular compartments of rat liver. The specific radioactivity of the valine released from isolated tRNA was 2-3 times higher than that of intracellular valine, suggesting that the intracellular pool cannot be the sole precursor of amino acids used for charging tRNA. In addition, the specific radioactivity of the tRNA was only half that of the extracellular valine. Therefore it is unlikely that the valyl-tRNA is charged exclusively with amino acids derived from the extracellular pool. A model is proposed which stipulates that both extracellular and intracellular amino acids contribute to a restricted compartment that funnels amino acids towards protein biosynthesis.

Intestinal cell kinase (ICK), originally cloned from the intestine and expressed in the intestinal crypt epithelium, is a highly conserved serine/threonine protein kinase that is similar to mitogen-activated protein kinases (MAPKs) in the catalytic domain and requires dual phosphorylation within a MAPK-like TDY motif for full activation. Despite these similarities to MAPKs, the biological functions of ICK remain unknown. In this study, we report that suppression of ICK expression in cultured intestinal epithelial cells by short hairpin RNA (shRNA) interference significantly impaired cellular proliferation and induced features of gene expression characteristic of colonic or enterocytic differentiation. Downregulation of ICK altered expression of cell cycle regulators (cyclin D1, c-Myc, and p21(Cip1/WAF1)) of G(1)-S transition, consistent with the G(1) cell cycle delay induced by ICK shRNA. ICK deficiency also led to a significant decrease in the expression and/or activity of p70 ribosomal protein S6 kinase (S6K1) and eukaryotic initiation factor 4E (eIF4E), concomitant with reduced expression of their upstream regulators, the mammalian target of rapamycin (mTOR) and the regulatory associated protein of mTOR (Raptor). Furthermore, ICK interacts with the mTOR/Raptor complex in vivo and phosphorylates Raptor in vitro. These results suggest that disrupting ICK function may downregulate protein translation of specific downstream targets of eIF4E and S6K1 such as cyclin D1 and c-Myc through the mTOR/Raptor signaling pathway. Taken together, our findings demonstrate an important role for ICK in proliferation and differentiation of intestinal epithelial cells.

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Antineutrophil cytoplasmic antibodies (ANCA) have been identified in the serum of 50-80% of ulcerative colitis (UC) patients. UC-associated ANCA yield a perinuclear staining pattern (pANCA) with alcohol-fixed neutrophils. More recently, pANCA have been detected in the serum of patients with primary sclerosing cholangitis (PSC) and other autoimmune liver diseases. Up to 70% of PSC patient sera and up to 92% of sera from patients with well-defined type 1 autoimmune hepatitis (type 1 AIH) were found to express pANCA. Such expression by patients with PSC and type 1 AIH raises questions concerning the relationship of these pANCA to each other and to that of UC. Differences and similarities in pANCA characteristics are found among the three diseases, suggesting the use of pANCA to define specific disease subgroups. Our recent finding that the UC-associated pANCA reactive antigen was localized within the nuclear domain prompted an examination of whether DNase treatment of neutrophils would alter antigenic recognition by the pANCA of UC, PSC, and type 1 AIH. While loss of antigenic recognition after DNase digestion of neutrophils was a dominant feature of the UC-associated pANCA, the majority of PSC and type 1 AIH pANCA recognized cytoplasmic constituents. These results further support the feasibility of defining and/or distinguishing disease subgroups based on the characterization of respective pANCA.