Pituitary tumor-derived fibroblast growth factor receptor 4 isoform disrupts neural cell-adhesion molecule/N-cadherin signaling to diminish cell adhesiveness: a mechanism underlying pituitary neoplasia

We previously identified pituitary tumor-derived fibroblast growth factor receptor 4 (ptd-FGFR4), an alternatively transcribed N-terminally truncated cytoplasmic receptor isoform. Unlike wild-type FGFR4, ptd-FGFR4 facilitates cell transformation and results in pituitary tumor formation in transgenic mice. To investigate differences in the tumorigenic properties of FGFR4 and ptd-FGFR4, we examined their abilities to modulate cell adhesiveness. Introduction of ptd-FGFR4 into GH4 pituitary cells or NIH 3T3 fibroblasts resulted in significant reduction in cell adhesion to a collagen IV matrix compared with FGFR4- or empty vector-transfected cells. This adhesive difference was evident in the absence or presence of FGF stimulation. Furthermore, treatment with beta1-integrin neutralizing antibody markedly reduced adhesiveness in FGFR4-transfected cells but had little effect on the depressed adhesiveness of ptd-FGFR4-transfected cells. Unlike wild-type FGFR4, ptd-FGFR4 does not associate with neural cell-adhesion molecule (NCAM). Cells expressing FGFR4 demonstrate membranous N-cadherin with a noninvasive growth pattern identical to control GH4 cells when injected into immunodeficient mice. In contrast, ptd-FGFR4-expressing cells develop invasive tumors in vivo with marked loss of N-cadherin that localizes to the cytoplasm. Consistent with these changes, beta-catenin expression was diminished and its interaction with N-cadherin was disrupted in the presence of ptd-FGFR4, but both were intact in the presence of wild-type FGFR4. These data highlight the importance of membrane-anchored FGFR4 in assembling a multiprotein FGFR4 complex with NCAM and N-cadherin playing pivotal functions in maintaining normal cell adhesion. Disruption of distinct NCAM/N-cadherin proadhesive complexes by a tumor-derived FGFR4 isoform provides a novel mechanism beyond ligand independence that explains the pathobiology of proliferative and infiltrative but nonmetastatic neoplasms.     

Stereospecific synthesis of "para-hydroxymexiletine" and sodium channel blocking activity evaluation

Both enantiomers of "para-hydroxymexiletine" (PHM), one of the main metabolites of mexiletine, were synthesized and fully characterized. Properties of (R)- and (S)-PHM, in terms of blocking potency and stereoselectivity on frog skeletal muscle Na(+) channels, were evaluated. The presence of a hydroxy group on the aryloxy moiety in the 4-position, as in PHM, reduced potency with respect to mexiletine in reducing I(Na max). However, PHM showed clear use-dependent behavior similar to that of mexiletine and, in contrast with what is observed with the parent compound, maintained its stereoselectivity during the use-dependent block. Chirality 16:72-78, 2004.     

Isolation of nuclei from label-retaining cells and measurement of their turnover rates in rat colon

We describe here a new technique for isolating nuclei from long-term label-retaining cells (LRCs), a subpopulation enriched with stem cells from colon, and for measuring their proliferation rates in vivo. A double-label approach was developed, combining the use of bromodeoxyuridine (BrdU) and ²H₂O. Male Fisher 344 rats were administered BrdU in drinking water continuously for 2–8 wk. BrdU was then discontinued (BrdU washout), and animals (n = 33) were switched to ²H₂O in drinking water and killed after 2, 4, and 8 wk. Nuclei from BrdU-positive cells (LRCs) were collected by flow cytometry. The percentages of LRCs were 7 and 3.8% after 4 and 8 wk of BrdU washout, respectively. Turnover rates of LRCs were measured on the basis of deuterium incorporation from ²H₂O into DNA of LRC nuclei, as determined by mass spectrometry. The proliferation rate of the LRCs collected was 0.33–0.90% per day (half-life of 77–210 days). Significant contamination from other potentially long-lived colon cells was excluded. In conclusion, this double-labeling method allows both physical isolation of nuclei from colon epithelial LRCs and measurement of their in vivo proliferation rates. Use of this approach may allow better understanding of mechanisms by which agents induce or protect against colon carcinogenesis. carcinogenesis; deuterated water; long-term label-retaining cells; stable isotopes     

Lactate-utilizing bacteria, isolated from human feces, that produce butyrate as a major fermentation product

The microbial community of the human colon contains many bacteria that produce lactic acid, but lactate is normally detected only at low concentrations (<5 mM) in feces from healthy individuals. It is not clear, however, which bacteria are mainly responsible for lactate utilization in the human colon. Here, bacteria able to utilize lactate and produce butyrate were identified among isolates obtained from 10(-8) dilutions of fecal samples from five different subjects. Out of nine such strains identified, four were found to be related to Eubacterium hallii and two to Anaerostipes caccae, while the remaining three represent a new species within clostridial cluster XIVa based on their 16S rRNA sequences. Significant ability to utilize lactate was not detected in the butyrate-producing species Roseburia intestinalis, Eubacterium rectale, or Faecalibacterium prausnitzii. Whereas E. hallii and A. caccae strains used both D- and L-lactate, the remaining strains used only the d form. Addition of glucose to batch cultures prevented lactate utilization until the glucose became exhausted. However, when two E. hallii strains and one A. caccae strain were grown in separate cocultures with a starch-utilizing Bifidobacterium adolescentis isolate, with starch as the carbohydrate energy source, the L-lactate produced by B. adolescentis became undetectable and butyrate was formed. Such cross-feeding may help to explain the reported butyrogenic effect of certain dietary substrates, including resistant starch. The abundance of E. hallii in particular in the colonic ecosystem suggests that these bacteria play important roles in preventing lactate accumulation.     

Cloning and functional characterization of the human GLUT7 isoform SLC2A7 from the small intestine

Facilitated glucose transporters (GLUTs) mediate transport of sugars across cell membranes by using the chemical gradient of sugars as the driving force. Improved cloning techniques and database analyses have expanded this family of proteins to a total of 14 putative members. In this work a novel hexose transporter isoform, GLUT7, has been cloned from a human intestinal cDNA library by using a PCR-based strategy (GenBank accession no. AY571960). The encoded protein is comprised of 524 amino acid residues and shares 68% similarity and 53% identity with GLUT5, its most closely related isoform. When GLUT7 was expressed in Xenopus oocytes, it showed high-affinity transport for glucose (K(m) = 0.3 mM) and fructose (IC(50) = 0.060 mM). Galactose, 2-deoxy-d-glucose, and xylose were not transported. Uptake of 100 microM d-glucose was not inhibited by 200 microM phloretin or 100 microM cytochalasin B. Northern blotting indicated that the mRNA for GLUT7 is present in the human small intestine, colon, testis, and prostate. Western blotting and immunohistochemistry of rat tissues with an antibody raised against the predicted COOH-terminal sequence confirmed expression of the protein in the small intestine and indicated that the transporter is predominantly expressed in the enterocytes' brush-border membrane. The unusual substrate specificity and close sequence identity with GLUT5 suggest that GLUT7 represents an intermediate between class II GLUTs and the class I member GLUT2. Comparison between these proteins may provide key information as to the structural determinants for the recognition of fructose as a substrate.