A Wnt kinase network alters nuclear localization of TCF-1 in colon cancer.

Constitutive activation of the Wnt/beta-catenin pathway has been implicated as the primary cause of colon cancer. However, the major transducers of Wnt signaling in the intestine, T-cell factor 1 (TCF-1) and TCF-4, have opposing functions. Knockout of TCF-4 suppresses growth and maintenance of crypt stem cells, whereas knockout of TCF-1 leads to adenomas. These phenotypes suggest that TCF-4 is Wnt-promoting, whereas TCF-1 acts like a tumor suppressor. Our study of TCF expression in human colon crypts reveals a mechanistic basis for this paradox. In normal colon cells, a dominant-negative isoform of TCF-1 (dnTCF-1) is expressed that is equally distributed between nuclear and cytoplasmic compartments. In colon cancer cells, TCF-1 is predominantly cytoplasmic. Localization is because of active nuclear export and is directed by an autocrine-acting Wnt ligand that requires Ca2+/calmodulin-dependent kinase II (CaMKII) activity for secretion and a downstream step in the export pathway. TCF-4 remains nuclear; its unopposed activity is accompanied by downregulation of dnTCF-1 and increased expression of full-length isoforms. Thus, the dnTCF-1 and TCF-4 balance is corrupted in cancer by two mechanisms, a Wnt/CaMKII kinase signal for nuclear export and decreased dnTCF-1 expression. We propose that dnTCF-1 provides homeostatic regulation of Wnt signaling and growth in normal colon, and the alterations in nuclear export and promoter usage contribute to aberrant Wnt activity in colon cancer.

Impaired apoptosis, extended duration of immune responses, and a lupus-like autoimmune disease in IEX-1-transgenic mice.

Susceptibility of activated T cells to apoptosis must be tightly regulated to ensure sufficient T cell progeny for an effective response, while allowing a rapid depletion of them at the end of the immune response. We show here that a previously isolated, NF-kappa B/rel target gene IEX-1 (Immediate Early response gene X-1) is highly expressed in T cells at early stages of activation, but declines with a prolonged period of activation time, coincident with an increased susceptibility of T cells to apoptosis during the late phases of an immune response. Transgenic expression of IEX-1 specifically in lymphocytes impaired apoptosis in activated T cells, extended a duration of an effector-phase of a specific immune response, and increased the accumulation of effector/memory-like T cells and the susceptibility to a lupus-like autoimmune disease. Our study demonstrated an antiapoptotic effect of IEX-1 on T cell apoptosis triggered by ligation of Fas and T cell receptor (TCR)/CD3 complex. The ability of extending life expectancy of T effectors, in line with a decrease in its expression following prolonged T cell activation, suggests a key role for IEX-1 in regulating T cell homeostasis during immune responses.

Crystal structure of the human ubiquitin conjugating enzyme complex, hMms2-hUbc13.

The ubiquitin conjugating enzyme complex Mms2-Ubc13 plays a key role in post-replicative DNA repair in yeast and the NF-kappaB signal transduction pathway in humans. This complex assembles novel polyubiquitin chains onto yet uncharacterized protein targets. Here we report the crystal structure of a complex between hMms2 (Uev1) and hUbc13 at 1.85 A resolution and a structure of free hMms2 at 1.9 A resolution. These structures reveal that the hMms2 monomer undergoes a localized conformational change upon interaction with hUbc13. The nature of the interface provides a physical basis for the preference of Mms2 for Ubc13 as a partner over a variety of other structurally similar ubiquitin-conjugating enzymes. The structure of the hMms2-hUbc13 complex provides the conceptual foundation for understanding the mechanism of Lys 63 multiubiquitin chain assembly and for its interactions with the RING finger proteins Rad5 and Traf6.

Phenotypic consequences of lung-specific inducible expression of FGF-3.

Members of the fibroblast growth factor (FGF) family play a critical role in embryonic lung development and adult lung physiology. The in vivo investigation of the role FGFs play in the adult lung has been hampered because the constitutive pulmonary expression of these factors often has deleterious effects and frequently results in neonatal lethality. To circumvent these shortcomings, we expressed FGF-3 in the lungs under the control of the progesterone antagonist-responsive binary transgenic system. Four binary transgenic lines were obtained that showed ligand-dependent induction of FGF-3 with induced levels of FGF-3 expression dependent on the levels of expression of the GLp65 regulator as well as the dose of the progesterone antagonist, RU486, administered. FGF-3 expression in the adult mouse lung resulted in two phenotypes depending on the levels of induction of FGF-3. Low levels of FGF-3 expression resulted in massive free alveolar macrophage infiltration. High levels of FGF-3 expression resulted in diffuse alveolar type II cell hyperplasia. Both phenotypes were reversible after the withdrawal of RU486. This system will be a valuable means of investigating the diverse roles of FGFs in the adult lung.

Inside or outside: detecting the cellular location of bacterial pathogens.

Salmonella are intracellular pathogens that infect and multiply inside macrophages. Although Salmonella are some of the best-studied pathogens, it is difficult to determine quickly and reliably whether the bacteria are intracellular or extracellular. We have developed a novel method using differential fluorescence of two fluorescent proteins to determine the cellular location of pathogenic bacteria in macrophage infection assays. Using the differential expression of two unique fluorescent proteins that are expressed under specific conditions, we have developed a real-time assay for macrophage infections. The critical advantages of this system are that it does not alter the bacterial surface, it is not toxic to either the bacteria or the host cell, and it may be used in real-time quantitative assays. This assay can be readily applied to any other model pathogenic systems such as Listeria, Mycobacteria, and Legionella in which intracellular gene expression has been characterized.

Unilocular retroperitoneal cyst of mesothelial origin presenting as a renal mass.

We report the first 2 cases, to our knowledge, of retroperitoneal cysts with features of mesothelial differentiation that clinically mimic renal masses. The first lesion occurred in a 71-year-old man who presented with flank pain. Ultrasound and magnetic resonance imaging studies showed a unilocular cystic structure arising from the upper pole of the left kidney. The second lesion was in a 44-year-old woman who presented with left flank pain. Imaging studies revealed an 8-cm hemorrhagic cyst at the lower pole of the left kidney. Histologic examination of the nephrectomy specimens in each case revealed a unilocular cyst with intracystic and pericystic hemorrhage. In each case, the cyst was lined by a single layer of cells with ample eosinophilic cytoplasm and benign nuclear features without mucinous or müllerian differentiation. Histochemical staining showed Alcian blue positivity on the cell surface, which was sensitive to hyaluronidase digestion. Intracytoplasmic mucin, however, was not detected. Immunostaining showed that the cyst lining cells were positive for keratin, vimentin, HBME-1, WT1, and thrombomodulin but negative for carcinoembryonic antigen, B72.3, Leu-M1, and BerEP4. The first case was positive for calretinin, whereas the second was negative. These findings support the mesothelial nature of the cysts.

Telemedicine in vascular surgery: feasibility of digital imaging for remote management of wounds.

PURPOSE: Telemedicine coupled with digital photography could potentially improve the quality of outpatient wound care and decrease medical cost by allowing home care nurses to electronically transmit images of patients' wounds to treating surgeons. To determine the feasibility of this technology, we compared bedside wound examination by onsite surgeons with viewing digital images of wounds by remote surgeons. METHODS: Over 6 weeks, 38 wounds in 24 inpatients were photographed with a Kodak DC50 digital camera (resolution 756 x 504 pixels/in2). Agreements regarding wound description (edema, erythema, cellulitis, necrosis, gangrene, ischemia, and granulation) and wound management (presence of healing problems, need for emergent evaluation, need for antibiotics, and need for hospitalization) were calculated among onsite surgeons and between onsite and remote surgeons. Sensitivity and specificity of remote wound diagnosis compared with bedside examination were calculated. Potential correlates of agreement, level of surgical training, certainty of diagnosis, and wound type were evaluated by multivariate analysis. RESULTS: Agreement between onsite and remote surgeons (66% to 95% for wound description and 64% to 95% for wound management) matched agreement among onsite surgeons (64% to 85% for wound description and 63% to 91% for wound management). Moreover, when onsite agreement was low (i.e., 64% for erythema) agreement between onsite and remote surgeons was similarly low (i.e., 66% for erythema). Sensitivity of remote diagnosis ranged from 78% (gangrene) to 98% (presence of wound healing problem), whereas specificity ranged from 27% (erythema) to 100% (ischemia). Agreement was influenced by wound type (p < 0.01) but not by certainty of diagnosis (p > 0.01) or level of surgical training (p > 0.01). CONCLUSIONS: Wound evaluation on the basis of viewing digital images is comparable with standard wound examination and renders similar diagnoses and treatment in the majority of cases. Digital imaging for remote wound management is feasible and holds significant promise for improving outpatient vascular wound care.

beta-Catenin associates with the actin-bundling protein fascin in a noncadherin complex.

Catenins were first characterized as linking the cytoplasmic domains of cadherin cell-cell adhesion molecules to the cortical actin cytoskeleton. In addition to their essential role in modulating cadherin adhesivity, catenins have more recently been indicated to participate in cell and developmental signaling pathways. beta-Catenin, for example, associates directly with at least two receptor tyrosine kinases and transduces developmental signals within the Wnt pathway. Catenins also complex with the tumor suppressor protein adenomatous polyposis coli (APC), which appears to have a role in regulating cell proliferation. We have used the yeast two-hybrid method to reveal that fascin, a bundler of actin filaments, binds to beta-catenin's central Armadillo repeat domain. Western blotting of immunoprecipitates from cell line and mouse and rat brain extracts indicate that this interaction exists in vivo. Fascin and beta-catenin's association was further substantiated in vitro using purified proteins isolated from recombinant bacterial and baculoviral sources. Immunoprecipitation analysis indicates that fascin additionally binds to plakoglobin, which is highly homologous to beta-catenin but not to p120cas, a newly described catenin which contains a more divergent Armadillo-repeat domain. Immunoprecipitation, in vitro competition, and domain-mapping experiments demonstrate that fascin and E-cadherin utilize a similar binding site within beta-catenin, such that they form mutually exclusive complexes with beta-catenin. Immunofluorescence microscopy reveals that fascin and beta-catenin colocalize at cell-cell borders and dynamic cell leading edges of epithelial and endothelial cells. In addition to cell-cell borders, cadherins were unexpectedly observed to colocalize with fascin and beta-catenin at cell leading edges. It is conceivable that beta-catenin participates in modulating cytoskeletal dynamics in association with the microfilament-bundling protein fascin, perhaps in a coordinate manner with its functions in cadherin and APC complexes.

Identification of major and minor chaperone proteins involved in the export of 987P fimbriae.

The 987P fimbriae of Escherichia coli consist mainly of the major subunit, FasA, and two minor subunits, FasF and FasG. In addition to the previously characterized outer membrane or usher protein FasD, the FasB, FasC, and FasE proteins are required for fimbriation. To better understand the roles of these minor proteins, their genes were sequenced and the predicted polypeptides were shown to be most similar to periplasmic chaperone proteins of fimbrial systems. Western blot (immunoblot) analysis and immunoprecipitation of various fas mutants with specific antibody probes identified both the subcellular localizations and associations of these minor components. FasB was shown to be a periplasmic chaperone for the major fimbrial subunit, FasA. A novel periplasmic chaperone, FasC, which stabilizes and specifically interacts with the adhesin, FasG, was identified. FasE, a chaperone-like protein, is also located in the periplasm and is required for optimal export of FasG and possibly other subunits. The use of different chaperone proteins for various 987P subunits is a novel observation for fimbrial biogenesis in bacteria. Whether other fimbrial systems use a similar tactic remains to be discovered.

Surface denaturation of proteins: the thermal inactivation of beta-galactosidase (Escherichia coli) on wall-liquid surfaces.

Irreversible inactivation of dilute beta-galactosidase (Escherichia coli) at relatively low temperatures was found to occur as a result of interactions of beta-galactosidase with wall-liquid surfaces. The rate of inactivation was directly proportional to the wall-liquid surface area, but independent of the air-liquid surface area, and the rate was also dependent on the wall composition. A small portion of the beta-galactosidase molecules was found to bind strongly to the surfaces of vessels in which the beta-galactosidase was stored. Bovine serum albumin eliminated the inactivation and it also eliminated the binding of beta-galactosidase to the wall. On the other hand, EDTA eliminated the inactivation, but it did not decrease the amount of beta-galactosidase bound. The addition of some transition metals increased the rate of inactivation. Protection of beta-galactosidase from surface inactivation by EDTA is not, therefore, a result of decreased binding of the enzyme to the walls of the vessels, but is probably a result of the ability of EDTA to scavenge certain trace metal ions present in solution, which are needed for the inactivation. The content of protein in the solution did not change as a result of the inactivation and, thus, the inactive enzyme does not accumulate at the surface. Since beta-galactosidase is often stored for long periods of time and since it is used to decrease the lactose content of milk for lactose intolerant individuals, this study may have practical significance. The presence of metal chelators and extraneous proteins should improve the stability of the enzyme, especially for processes that are carried out at elevated temperatures.