Fkbp8: novel isoforms, genomic organization, and characterization of a forebrain promoter in transgenic mice

The immunophilin homolog FKBP8 has been implicated in the regulation of apoptosis. Here we show that the 38-kDa form of FKBP8 (FKBP38) derives from a truncated ORF. The extended FKBP8 ORFs are 46 and 44 kDa in mouse and 45 kDa in human. Although the genomic organization of mouse and human FKBP8 is evolutionarily conserved, additional first exons are encoded by the murine locus. A 4.4-kb murine Fkbp8 gene fragment, containing a GC-rich potential promoter, directed expression of a LacZ reporter gene to forebrain neurons in transgenic mice. Expression of the transgene was observed in CA1 pyramidal neurons of the hippocampus in transgenic mice from three lines. One transgenic founder mouse exhibited widespread forebrain expression of the LacZ transgene that resembles the pattern for the endogenous Fkbp8 gene. Thus promoter/enhancer elements for forebrain expression are located around the first exons of the mouse Fkbp8 gene.     

Hepatocyte growth factor induces MDCK cell morphogenesis without causing loss of tight junction functional integrity

Hepatocyte growth factor (HGF) induces mitogenesis, motogenesis, and tubulogenesis of cultured Madin-Darby canine kidney (MDCK) epithelial cells. We report that in addition to these effects HGF stimulates morphogenesis of tight, polarized MDCK cell monolayers into pseudostratified layers without loss of tight junction (TJ) functional integrity. We tested TJ functional integrity during formation of pseudostratified layers. In response to HGF, the TJ marker ZO-1 remained in morphologically complete rings and functional barriers to paracellular diffusion of ruthenium red were maintained in pseudostratified layers. Transepithelial resistance (TER) increased transiently two- to threefold during the morphogenetic transition from monolayers to pseudostratified layers and then declined to baseline levels once pseudostratified layers were formed. In MDCK cells expressing the trk/met chimera, both HGF and NGF at concentrations of 2.5 ng/ml induced scattering. However, 2.5 ng/ml HGF did not affect TER. The peak effect of HGF on TER was at a concentration of 100 ng/ml. In contrast, NGF at concentrations as high as 25 µg/ml had no effect on TER or pseudostratified layer morphogenesis of trk/met-expressing cultures. These results suggest that altered presentation of the stimulus, such as through HGF interaction with low-affinity sites, may change the downstream signaling response. In addition, our results demonstrate that HGF stimulates pseudostratified layer morphogenesis while inducing an increase in TER and maintaining the overall tightness of the epithelial layer. Stimulation of epithelial cell movements by HGF without loss of functional TJs may be important for maintaining epithelial integrity during morphogenetic events such as formation of pseudostratified epithelia, organ regeneration, and tissue repair. c-met protooncogene; transepithelial resistance; Madin-Darby canine kidney cell     

Detection of intracellular iron by its regulatory effect

Intracellular iron regulates gene expression by inhibiting the interaction of iron regulatory proteins (IRPs) with RNA motifs called iron-responsive elements (IREs). To assay this interaction in living cells we have developed two fluorescent IRE-based reporters that rapidly, reversibly, and specifically respond to changes in cellular iron status as well as signaling that modifies IRP activity. The reporters were also sufficiently sensitive to distinguish apo- from holotransferrin in the medium, to detect the effect of modifiers of the transferrin pathway such as HFE, and to detect the donation or chelation of iron by siderophores bound to the lipocalin neutrophil gelatinase-associated lipocalin (Ngal). In addition, alternative configurations of the IRE motif either enhanced or repressed fluorescence, permitting a ratio analysis of the iron-dependent response. These characteristics make it possible to visualize iron-IRP-IRE interactions in vivo. iron regulatory proteins; iron-responsive element; labile iron pool; transferrin; HFE; neutrophil gelatinase-associated lipocalin; siderophore     

Enzymatic mechanisms for catalysis of enolization: ketosteroid isomerase

Breaking a carbon-hydrogen bond adjacent to a carbonyl is a slow step in a large number of chemical reactions. However, many enzymes are capable of catalyzing this reaction with great efficiency. One of the most proficient of these enzymes is 3-oxo-Delta5-steroid isomerase (KSI), which catalyzes the isomerization of a wide variety of 3-oxo-Delta5-steroids to their Delta4-conjugated isomers. In this review, the mechanism of KSI is discussed, with particular emphasis on energetic considerations. Both experimental and theoretical approaches are considered to explain the mechanistic details of the reaction.     

IL-10 modulates intestinal damage and epithelial cell apoptosis in T cell-mediated enteropathy

In vivo T cell activation by anti-CD3 monoclonal antibody (mAb) results in intestinal damage characterized by loss of villi and epithelial cell apoptosis. The role of the increased interleukin (IL)-10 released during this process is not clear. We assessed the effects of IL-10 on T cell-induced mucosal damage in vivo using IL-10-deficient C57BL/6 [IL-10 knockout (KO)] mice. IL-10 KO and wild-type C57BL/6 mice were injected with anti-CD3 mAb and observed for diarrhea. Changes in serum cytokine levels were measured by ELISA. Histological changes and epithelial cell apoptosis were analyzed on hematoxylin- and eosin-stained tissue sections. Fas expression on intestinal epithelial cells was assessed by flow cytometry analysis of freshly isolated intestinal epithelial cells. Anti-CD3-treated IL-10 KO mice developed more severe diarrhea, a greater loss of intestinal villi, and an increase in the numbers of apoptotic cells in the crypt epithelium. This difference in IL-10 KO mice was associated with an increase in serum tumor necrosis factor-alpha and interferon-gamma levels and with an increase in Fas expression on fresh, isolated, small intestinal epithelial cells. In addition, the enhanced intestinal tissue damage induced by anti-CD3 in IL-10 KO mice was significantly diminished by treatment with recombinant murine IL-10. Therefore, the lack of IL-10 allowed for an increased T cell-induced intestinal tissue damage, and this was associated with an increase in T cell cytokine release and an increase in epithelial cell Fas expression.     

FlyGEM, a full transcriptome array platform for the Drosophila community

We have constructed a DNA microarray to monitor expression of predicted genes in Drosophila. By using homotypic hybridizations, we show that the array performs reproducibly, that dye effects are minimal, and that array results agree with systematic northern blotting. The array gene list has been extensively annotated and linked-out to other databases. Incyte and the NIH have made the platform available to the community via academic microarray facilities selected by an NIH committee.     

The Protein Information Resource

The Protein Information Resource (PIR) is an integrated public resource of protein informatics that supports genomic and proteomic research and scientific discovery. PIR maintains the Protein Sequence Database (PSD), an annotated protein database containing over 283 000 sequences covering the entire taxonomic range. Family classification is used for sensitive identification, consistent annotation, and detection of annotation errors. The superfamily curation defines signature domain architecture and categorizes memberships to improve automated classification. To increase the amount of experimental annotation, the PIR has developed a bibliography system for literature searching, mapping, and user submission, and has conducted retrospective attribution of citations for experimental features. PIR also maintains NREF, a non-redundant reference database, and iProClass, an integrated database of protein family, function, and structure information. PIR-NREF provides a timely and comprehensive collection of protein sequences, currently consisting of more than 1 000 000 entries from PIR-PSD, SWISS-PROT, TrEMBL, RefSeq, GenPept, and PDB. The PIR web site (http://pir.georgetown.edu) connects data analysis tools to underlying databases for information retrieval and knowledge discovery, with functionalities for interactive queries, combinations of sequence and text searches, and sorting and visual exploration of search results. The FTP site provides free download for PSD and NREF biweekly releases and auxiliary databases and files.