Cbl-mediated ubiquitinylation is required for lysosomal sorting of epidermal growth factor receptor but is dispensable for endocytosis

Ligand-induced down-regulation controls the signaling potency of the epidermal growth factor receptor (EGFR/ErbB1). Overexpression studies have identified Cbl-mediated ubiquitinylation of EGFR as a mechanism of ligand-induced EGFR down-regulation. However, the role of endogenous Cbl in EGFR down-regulation and the precise step in the endocytic pathway regulated by Cbl remain unclear. Using Cbl-/- mouse embryonic fibroblast cell lines, we demonstrate that endogenous Cbl is essential for ligand-induced ubiquitinylation and efficient degradation of EGFR. Further analyses using Chinese hamster ovary cells with a temperature-sensitive defect in ubiquitinylation confirm a crucial role of the ubiquitin machinery in Cbl-mediated EGFR degradation. However, internalization into early endosomes did not require Cbl function or an intact ubiquitin pathway. Confocal immunolocalization studies indicated that Cbl-dependent ubiquitinylation plays a critical role at the early endosome to late endosome/lysosome sorting step of EGFR down-regulation. These findings establish Cbl as the major endogenous ubiquitin ligase responsible for EGFR degradation, and show that the critical role of Cbl-mediated ubiquitinylation is at the level of endosomal sorting, rather than at the level of internalization.     

Quantification of hepatic carbohydrate metabolism in conscious mice using serial blood and urine spots

In vivo studies of hepatic carbohydrate metabolism in (genetically modified) conscious mice are hampered by limitations of blood and urine sample sizes. We developed and validated methods to quantify stable isotope dilution and incorporation in small blood and urine samples spotted onto filter paper. Blood glucose and urinary paracetamol-glucuronic acid were extracted from filter paper spots reproducibly and with high yield. Fractional isotopomer distributions of glucose and paracetamol-glucuronic acid when extracted from filter paper spots were almost identical to those isolated from the original body fluids. Rates of infusion of labeled compounds could be adjusted without perturbing hepatic glucose metabolism. This approach was used in mice to find the optimal metabolic condition for the study of hepatic carbohydrate metabolism. In fed mice, no isotopic steady state was observed during a 6-h label-infusion experiment. In 9-h-fasted mice, isotopic steady state was reached after 3 h of label infusion and important parameters in hepatic glucose metabolism could be calculated. The rate of de novo glucose-6-phosphate synthesis was 143 +/- 17 micromol kg(-1) min(-1) and partitioning to plasma glucose was 79.0 +/- 5.2%. In 24-h-fasted mice, abrupt changes were noticed in whole body and in hepatic glucose metabolism at the end of the experiment.     

Extraction and reconstitution of calponin and consequent contractile ability in permeabilized smooth muscle fibers

We demonstrate reduction and restoration of contractile ability in response to protein extraction and reconstitution in Triton X-100/glycerol-permeabilized smooth muscle fibers. Through significant reduction in the content of caldesmon (CaD), calponin (CaP), and the 20-kDa regulatory light chain (RLC) of myosin, but not other contractile proteins in "chemically skinned" fibers, we substantially reduced the contractile ability of these fibers, as measured by their ability to generate isometric force and to hydrolyze ATP by actomyosin Mg2+ ATPase. When the protein-depleted fibers were then reconstituted (either with a mixture of purified protein standards of CaD, CaP, and myosin RLC or with a protein extract from the demembranized muscle fibers containing CaD, CaP, and myosin RLC plus several low-molecular-mass proteins), all proteins used for reincorporation returned nearly to control levels, as did isometric force generation and rate of ATP hydrolysis. The fact that the low-molecular-mass proteins do not affect contractility in this model system indicates that our methods for reversible modulation of the content of CaP and CaD may provide a valuable tool for studying the thin-filament-based regulation of contractility.     

Typing and susceptibility to penicillin of Neisseria meningitidis isolated from patients in Cuba (1993-1999)

The susceptibility to penicillin of 111 Neisseria meningitidis strains was assessed by the agar-dilution procedure and serosubtypes were determined by a whole-cell enzyme-linked immunoassay using monoclonal antibodies reagents. Thirty-five isolates showed reduced sensitivity to penicillin (MIC > or = 0.1 mg/l and < or = 1 mg/l) and no resistant strains were detected. The most common phenotype was B:4:P1.15 (77.5%) and a rising trend of non-typeable and non-subtypeable strains was detected. The increase in levels of minimal inhibitory concentrations of meningococci to penicillin gives cause for concern and the increase in non-typeable and non-subtypeable isolation demand the use of molecular biology techniques for their typing.     

Identification of a novel microtubule-associated protein that regulates microtubule organization and cytokinesis by using a GFP-screening strategy

Microtubules play critical roles in a variety of cell processes, including mitosis, organelle transport, adhesion and migration, and the maintenance of cell polarity. Microtubule-associated proteins (MAPs) regulate the dynamic organization and stability of microtubules, often through either cell-specific or cell division stage-specific interactions. To identify novel cytoskeletal-associated proteins and peptides that regulate microtubules and other cytoskeletal and adhesive structures, we have developed a GFP cDNA screening strategy based on identifying gene products that localize to these structures. Using this approach, we have identified a novel MAP, GLFND, that shows homology to the Opitz syndrome gene product [6], localizes to a subpopulation of microtubules that are acetylated, and protects microtubules from depolymerization with nocodazole. Expression of an N-terminal deletion binds microtubules but alters their organization. During the cell cycle, GLFND dissociates from microtubules at the beginning of mitosis and then reassociates at cytokinesis. Furthermore, ectopic expression of GLFND inhibits cell division and cytokinesis in CHO cells. These observations make GLFND unique among MAPs characterized thus far.     

CD1 molecules efficiently present antigen in immature dendritic cells and traffic independently of MHC class II during dendritic cell maturation

Upon exposure to Ag and inflammatory stimuli, dendritic cells (DCs) undergo a series of dynamic cellular events, referred to as DC maturation, that involve facilitated peptide Ag loading onto MHC class II molecules and their subsequent transport to the cell surface. Besides MHC molecules, human DCs prominently express molecules of the CD1 family (CD1a, -b, -c, and -d) and mediate CD1-dependent presentation of lipid and glycolipid Ags to T cells, but the impact of DC maturation upon CD1 trafficking and Ag presentation is unknown. Using monocyte-derived immature DCs and those stimulated with TNF-alpha for maturation, we observed that none of the CD1 isoforms underwent changes in intracellular trafficking that mimicked MHC class II molecules during DC maturation. In contrast to the striking increase in surface expression of MHC class II on mature DCs, the surface expression of CD1 molecules was either increased only slightly (for CD1b and CD1c) or decreased (for CD1a). In addition, unlike MHC class II, DC maturation-associated transport from lysosomes to the plasma membrane was not readily detected for CD1b despite the fact that both molecules were prominently expressed in the same MIIC lysosomal compartments before maturation. Consistent with this, DCs efficiently presented CD1b-restricted lipid Ags to specific T cells similarly in immature and mature DCs. Thus, DC maturation-independent pathways for lipid Ag presentation by CD1 may play a crucial role in host defense, even before DCs are able to induce maximum activation of peptide Ag-specific T cells.     

Molecular phylogenetic analysis of the avian malarial parasite Plasmodium (Novyella) juxtanucleare

Plasmodium (Novyella) juxtanucleare is a widely distributed parasite that primarily infects chickens (Gallus gallus domesticus). All species of Novyella are characterized by very small schizonts, which in the case of P. juxtanucleare are always found juxtaposed to the erythrocyte nucleus, hence its name. Nearly complete small-subunit ribosomal RNA sequences have been obtained from 2 isolates of this species, and comparisons with other Plasmodium species have been made. Phylogenetic analysis reveals that this parasite is closely related to other avian-infecting Plasmodium species and that molecular relationships among the avian-infecting plasmodia do not correspond to their morphology-based subgeneric classifications.     

Interferon-regulated pathways that control hepatitis B virus replication in transgenic mice

We previously showed that the intrahepatic induction of cytokines such as alpha/beta interferon (IFN-alpha/beta) and gamma interferon (IFN-gamma) inhibits hepatitis B virus (HBV) replication noncytopathically in the livers of transgenic mice. The intracellular pathway(s) responsible for this effect is still poorly understood. To identify interferon (IFN)-inducible intracellular genes that could play a role in our system, we crossed HBV transgenic mice with mice deficient in IFN regulatory factor 1 (IRF-1), the double-stranded RNA-activated protein kinase (PKR), or RNase L (RNase L) (IRF-1(-/-), PKR(-/-), or RNase L(-/-) mice, respectively), three well-characterized IFN-inducible genes that mediate antiviral activity. We showed that unmanipulated IRF-1(-/-) or PKR(-/-) transgenic mice replicate HBV in the liver at slightly higher levels than the respective controls, suggesting that both IRF-1 and PKR individually appear to mediate signals that modulate HBV replication under basal conditions. These same animals were responsive to the antiviral effects of the IFN-alpha/beta inducer poly(I-C) or recombinant murine IFN-gamma, suggesting that under these conditions, either the IRF-1 or the PKR genes can mediate the antiviral activity of the IFNs or other IFN-inducible genes mediate the antiviral effects. Finally, RNase L(-/-) transgenic mice were undistinguishable from controls under basal conditions and after poly(I-C) or IFN-gamma administration, suggesting that RNase L does not modulate HBV replication in this model.     

Costs of phenotypic plasticity

Phenotypically plastic organisms display alternative phenotypes in different environments. It is widely appreciated that possessing alternative phenotypes can affect fitness. However, some investigators have suggested that simply carrying the ability to be plastic could also affect fitness. Evolutionary models suggest that high costs of plasticity could constrain the evolution of optimal phenotypes. However, costs (and limits) of plasticity are primarily hypothetical. Little empirical evidence exists to show that increased plasticity leads to reduced growth and development, leads to increased developmental instability, or limits the ability of organisms to produce more extreme phenotypes. I used half-sib families of larval wood frogs (Rana sylvatica) reared in outdoor mesocosms to examine how tadpoles altered behavioral, morphological, and life-historical traits in response to larval dragonfly predators (Anax longipes). The predators induced lower activity and the development of relatively large tails and small bodies in wood frogs. As a result, wood frogs experienced reduced growth and development. I then examined whether tadpole sibships with higher plasticity experienced fitness costs (above and beyond the costs of expressing a particular phenotype) and whether they were limited in producing extreme phenotypes. Fitness effects of plasticity were widespread. Depending on the trait examined and the environment experienced, increased plasticity had either positive effects, negative effects, or no effects on tadpole mass, development, and survivorship. I found no relationship between increased plasticity and greater developmental instability. There was also no evidence that sibships with increased plasticity produced less extreme phenotypes; the most extreme trait states were always produced by the most plastic genotypes. This work suggests that costs of plasticity may be pervasive in nature and may substantially impact the evolution of optimal phenotypes in organisms that live in heterogeneous environments.