Cbl-3-deficient mice exhibit normal epithelial development

Cbl family proteins are evolutionarily conserved ubiquitin ligases that negatively regulate signaling from tyrosine kinase-coupled receptors. The mammalian cbl family consists of c-Cbl, Cbl-b, and the recently cloned Cbl-3 (also known as Cbl-c). In this study, we describe the detailed expression pattern of murine Cbl-3 and report the generation and characterization of Cbl-3-deficient mice. Cbl-3 exhibits an expression pattern distinct from those of c-Cbl and Cbl-b, with high levels of Cbl-3 expression in epithelial cells of the gastrointestinal tract and epidermis, as well as the respiratory, urinary, and reproductive systems. Cbl-3 expression was not detected in nonepithelial cells, but within epithelial tissues, the levels of Cbl-3 expression varied from undetectable in the alveoli of the lungs to very strong in the cecum and colon. Despite this restricted expression pattern, Cbl-3-deficient mice were viable, healthy, and fertile and displayed no histological abnormalities up to 18 months of age. Proliferation of epithelial cells in the epidermises and gastrointestinal tracts was unaffected by the loss of Cbl-3. Moreover, Cbl-3 was not required for attenuation of epidermal growth factor-stimulated Erk activation in primary keratinocytes. Thus, Cbl-3 is dispensable for normal epithelial development and function.     

Neutrophil myeloperoxidase chlorinates and nitrates soy isoflavones and enhances their antioxidant properties

Soy isoflavones and other polyphenolics have a number of potentially important beneficial effects on the pro-oxidant aspects of chronic inflammation. The impact of inflammatory cell-specific metabolism of polyphenolics, which can include halogenation and nitration, on the properties of these compounds has not been examined. Using either human neutrophils or differentiated human leukemia cells (HL-60) stimulated with phorbol ester to elicit a respiratory burst, the hypothesis that local generation of reactive oxygen and nitrogen species may metabolize and modify the biological properties of the soy isoflavones was examined. Coincubation of the stimulated cells with genistein or daidzein had no effect on the respiratory burst. Medium from stimulated cells in the presence of the isoflavones and NO(2)(-) increased the inhibition of copper-induced LDL oxidation. Mass spectrometry analysis of this medium revealed that monochlorinated, dichlorinated, and nitrated isoflavones, formed through a myeloperoxidase-dependent mechanism, were present. The consumption of genistein in the presence of cells was both extensive and rapid with > 95% of the genistein converted to either the chlorinated or nitrated metabolites within 30 min. Chemically synthesized 3'-chlorogenistein and 3'-chlorodaidzein increased the inhibition of LDL oxidation by approximately 4-fold and 2-fold over genistein and daidzein, respectively. These results lead to the hypothesis that inflammatory cell-specific metabolism of polyphenolics can modify the properties of these compounds at the local site of inflammation.     

Externally accumulated flavonoids in three Mediterranean Ononis species

The Mediterranean Ononis species, O. fruticosa, O. natrix subsp. ramosissima and O. tridentata, have been analyzed for their exudate flavonoids. More than 20 flavonoid aglycones were identified, some of which are rather rare natural compounds. One of them, namely hypolaetin-8,3',4'-trimethyl ether, had been found only once before. The results are presented in a table along with literature data, and the chemotaxonomic impact of the flavonoid patterns is discussed.     

Retinoic acid signaling is essential for pancreas development and promotes endocrine at the expense of exocrine cell differentiation in Xenopus

How and when the vertebrate endoderm is first subdivided into discrete progenitor cell populations that will give rise to the different major organs, including pancreas and liver, are only poorly understood. We have used Xenopus laevis as a model system to characterize these events, since it is particularly suited to study the early embryonic patterning in vertebrates. Our experimental results support the notion that retinoic acid (RA) functions as an essential endodermal patterning signal in Xenopus and that it acts as early as during gastrulation. As a result of RA treatment, the expression of Sonic Hedgehog (Shh), a known inhibitor of pancreas development in other vertebrate systems, is negatively regulated in the dorsal prepancreatic endoderm. Furthermore, RA is found to promote endocrine at the expense of exocrine differentiation in the dorsal pancreas, correlating with a specific inhibition of Notch signaling activities in this territory. Conversely, RA enhances exocrine marker gene expression in the ventral pancreas.     

Experimental indication for the existence of multiple Trp rotamers in von Willebrand Factor A3 domain

The first step in both normal haemostasis and arterial thrombosis is the interaction between collagen, von Willebrand factor (vWF), and glycoprotein Ib. The A3 domain of vWF forms the principal binding site for collagen type I and type III. Inhibition of the vWF-collagen interaction by an anti-human vWF monoclonal antibody (MoAb) 82D6A3 can be a potential way to prevent arterial thrombosis. Identification of the epitope of MoAb 82D6A3 showed recently that the consensus sequence SPWR obtained by phage display could adopt the conformation of the discontinuous epitope. Modelling showed that Trp982 in the vWF had to obtain a more solvent accessible conformation. We performed a detailed fluorescence study of Trp982 in the vWF A3. Using the method described by Hellings et al. (Biophys J 2003;85:1894-1902), we were able to identify two different low-energy Trp982 rotamers and to link them with their experimentally derived fluorescence lifetimes. Fluorescence anisotropy showed no interconversion in the nanosecond timescale between the two different rotameric states. With these experiments, we gather strong indications for the existence of an exposed rotamer conformation and a rotamer that corresponds to the one observed in the X-ray structure. These results strongly support the modeling work (Vanhoorelbeke et al., J Biol Chem 2003;278:37815-37821).     

Paracrine actions of the stomach-derived leptin

Leptin, a 16 kilodalton protein-encoded by the ob gene, is involved in the regulation of food intake, body composition, and energy expenditure through a central feedback mechanism. Initially thought to be adipocyte-specific, the ob gene, as well as the leptin receptor, has been found in a variety of other tissues. Relevant to this review, the leptin gene and its receptor have been identified in the stomach, intestine, liver, and pancreas. Recent data also suggest that gut leptin may act locally within the gastrointestinal tract to influence intestinal functions such as nutrient absorption and may have a physiopathological implication. This review emphasises the concept that leptin may be a new gastrointestinal hormone.     

A human immunodeficiency virus type 1-infected individual with low viral load harbors a virus variant that exhibits an in vitro RNA dimerization defect

We investigated the in vitro RNA dimerization properties of the untranslated leader RNA derived from human immunodeficiency virus type 1 variants circulating in an individual with a low viral load and slow disease progression. The leader sequences of these viruses contain highly unusual polymorphisms within the dimerization initiation site (DIS): an insert that abolishes dimerization and a compensatory substitution. The dimerization of leader RNA from late stages of infection is further improved by additional mutations outside the DIS motif that facilitate a secondary structure switch from a dimerization-incompetent to a dimerization-competent RNA conformation.     

Flow cytometric and immunoblot assays for cell surface ADP-ribosylation using a monoclonal antibody specific for ethenoadenosine

NAD-dependent ADP-ribosylation is one of the posttranslational protein modifications. On mammalian cells, glycosylphosphatidylinositol-anchored cell surface ADP-ribosyltransferases (ARTs) ADP-ribosylate other cell surface proteins and thereby affect important cellular functions. Here we describe convenient flow-cytometric and immunoblot assays for monitoring ADP-ribosylation of cell surface proteins on living cells by exploiting the capacity of ARTs to utilize etheno-NAD as substrate. Etheno-ADP-ribosylation of cell surface proteins can be detected by flow cytometry with 1G4, a monoclonal antibody specific for ethenoadenosine. Labeling of cells with 1G4 is dependent on the expression of cell surface ARTs and occurs only after incubation of ART-expressing cells with etheno-NAD and not with etheno-ADP-ribose. Dose-response analyses show efficient 1G4 staining of ART-expressing cells at micromolar etheno-NAD concentrations. Half-maximal staining is obtained with 1-2 micro M etheno-NAD, saturation is reached at 5-20 micro M etheno-NAD. Immunoblot analyses confirm that ART-expressing cells incorporate ethenoadenosine covalently (i.e., SDS resistant) into several cell surface proteins. The flow-cytometric 1G4 staining assay can be used to identify subpopulations of cells expressing cell surface ART activity and to select ART(hi) cell variants. The immunoblot 1G4 staining assay can also be used to identify etheno-ADP-ribosylated target proteins. These new assays hold promise for many interesting applications in biochemistry and cell biology.