Control of EGF receptor signaling by clathrin-mediated endocytosis.

Epidermal growth factor receptor (EGFR) signaling was analyzed in mammalian cells conditionally defective for receptor-mediated endocytosis. EGF-dependent cell proliferation was enhanced in endocytosis-defective cells. However, early EGF-dependent signaling events were not uniformly up-regulated. A subset of signal transducers required the normal endocytic trafficking of EGFR for full activation. Thus, endocytic trafficking of activated EGFR plays a critical role not only in attenuating EGFR signaling but also in establishing and controlling specific signaling pathways.

An oocytic membrane receptor for biotin-binding protein.

The chicken oocyte accumulates a biotin-binding protein (BBP) in the yolk that is distinct from the avidin in the 'egg white'. An identical BBP to that of the yolk is also present in the circulation of the laying hen. We report the first evidence for the existence of a BBP receptor in the oocyte vitelline membrane. Reduction of the 100 kDa receptor results in loss of BBP-binding activity; this suggests that a disulfide bonded region of the receptor is necessary for maintaining BBP-binding activity. We show further that the levels of serum BBP are strictly dependent on the presence of estrogen. As expected, BBP is not detected in significant quantities in rooster serum. Thus, these results suggest that circulatory BBP, like other estrogen-dependent components of serum, has a cognate binding activity on the oocyte membrane that may mediate its endocytosis.

Identification of a circulatory and oocytic avian apolipoprotein D.

Apolipoprotein D (apo D) is an unusual apolipoprotein with respect to structure and sites of synthesis. It has been identified in the circulatory system of certain mammals, but its physiological role remains poorly understood. In this report, it is shown that apo D is not exclusively a mammalian apolipoprotein, and evidence is presented which suggests a novel function for this protein during oogenesis in the chicken. The avian apo D which we identify has the same molecular mass (29 kDa) as the human protein and also associates preferentially with the plasma lipoprotein fraction. In addition to the 29 kDa avian apo D species, an immunoreactive 24 kDa protein is observed in chicken serum. The chicken apo D (along with the 24 kDa species) is also demonstrated to be present in the yolk of the rapidly growing chicken oocyte, a cell with high endocytic activity. Clathrin-coated vesicles from chicken oocytes, which we have previously shown to contain specific lipoproteins along with their oocytic receptors (Bujo et al., 1994: EMBO J 13:5165-5175), also contain chicken apo D. Thus, apo D represents a novel candidate for plasma-to-oocyte transport of lipids and/or their mobilization during embryogenesis in oviparous species.

Retinoids: transport, metabolism, and mechanisms of action.

The retinoids comprise a family of polyisoprenoid lipids that includes vitamin A (retinol) and structurally related compounds. The biological activity of retinoids can be modified, for example, by changes in the molecules' state of oxidation and cis/trans isomerization. Their activity is also dependent on the levels of specific types of retinoid-binding proteins which exist in extracellular, cytosolic and nuclear compartments. The role of retinoids in gene expression represents an important biological function for this family of molecules. Retinoid-dependent modulation of gene expression is critical for normal cell and tissue function in mature as well as developing animals. Despite significant advances in the understanding of retinoid biological activity, important questions remain concerning aspects of retinoid metabolism, cellular uptake, intracellular trafficking and regulation of gene transcription. The purpose of this review is to present these topics as a compendium of retinoid endocrinology.

Retinol in avian oogenesis: molecular properties of the carrier protein.

Normal embryo development in oviparous (egg-laying) species requires the coordinated targeting to growing oocytes of nutrients and regulatory molecules such as retinol, the precursor of active retinoids. Serum retinol-binding protein (RBP) is the major carrier protein for retinol in the circulatory system of vertebrates. In oviparous animals, RBP is thought to function in the delivery of retinol to yolk, in analogy to other important nutrients and vitamins known to accumulate within the oocyte. Here, immunoelectron microscopy revealed that RBP indeed accumulates in yolk, in particular in the electron-lucent phase of yolk organelles known to harbor other serum-derived yolk proteins and their receptors. To gain understanding of the RBP-mediated serum-to-yolk transport of retinol, we have characterized the chicken carrier protein at the molecular level. The essential function of RBPs is emphasized by the first known avian RBP structure, which confirms that these vitamin carriers are among the most highly conserved serum proteins known. Interestingly, by analysis of RBP hepatic RNA and serum protein levels, we identified a unique property of chicken RBP relative to other known RBPs and yolk precursors, i.e., the absence of estrogen induction. One cause of the observed reduction in RBP RNA is an estrogen-dependent decrease of RBP gene transcription. Furthermore, Northern blot analysis of tissues of the hen demonstrated a lack of RBP synthesis by the oocyte or other ovarian cells, confirming the exogenous (hepatic) origin of yolk RBP. These results provide strong evidence that chicken RBP is an essential serum-to-yolk vitamin carrier with certain properties different from those of other such transporters.

Chicken yolk contains bona fide high density lipoprotein particles.

Lipoproteins, the major nutrient source for developing embryos in egg-laying species, are thought to be transported from the circulation of the hen to the yolk of growing oocytes. In order to fully understand the contribution of the different lipoprotein species to oocyte growth, yolk formation, and embryo development, we have started to elucidate the relationships between the high density lipoproteins (HDL) in serum with the hitherto uncharacterized yolk HDL fraction. Immunoblotting with antibodies against apolipoprotein (apo) A-I, the major protein moiety of circulating HDL, revealed, for the first time, significant amounts of this protein in yolk. Importantly, yolk apoA-I was an integral component of bona fide lipoprotein particles: i) the apoA-I-containing particles could be purified by ultracentrifugal flotation and immunoaffinity chromatography on immobilized anti-apoA-I IgG; ii) the particles resembled serum HDL in ultrastructural, chemical, and biochemical aspects; and iii) in particular, these particles contained another major apolipoprotein, apo II. To date, apo II has been assumed to be unique to the very low density lipoprotein (VLDL) and HDL fractions of laying hen serum. Its residence on yolk HDL particles, together with the other results, strongly implies that yolk HDL, at least to a large part, is derived from serum. This implication is supported by the presence of apoA-I in oocytic coated vesicles. However, an oocyte plasma membrane receptor for the transport of HDL could not be identified; furthermore, immunoelectron microscopy demonstrated that yolk HDL particles do not colocalize with VLDL, known to be endocytosed via a specific receptor. Thus, these studies have revealed that HDL particles are taken up into the oocyte from the serum of the laying hen, and are deposited into the yolk by a mechanism distinct from that involved in the uptake of other yolk lipoproteins.

Transport of serum transthyretin into chicken oocytes. A receptor-mediated mechanism.

Transthyretin (TTR) is involved in the transport of thyroid hormones and, due to its interaction with serum retinol-binding protein, also of vitamin A. The importance of both ligands in vertebrate embryonic development has prompted us to investigate the molecular details of TTR transport function in a powerful germ cell system, the rapidly growing chicken oocytes. Yolk TTR is derived from the circulatory system, since biotinylated TTR was recovered by immunoaffinity chromatography of yolk obtained from a hen previously infused with in vitro biotinylated chicken serum proteins. In concordance with the intraoocytic localization in an endosomal compartment, ligand blotting and chemical cross-linking experiments revealed the presence of a approximately 115-kDa TTR-binding oocyte membrane protein. This putative TTR receptor was not detected in chicken ovarian granulosa cells or embryonic fibroblasts and was different from the previously described oocyte-specific receptor for two estrogen-induced chicken serum lipoproteins, vitellogenin and very low density lipoprotein (Barber, D. L., Sanders, E. J., Aebersold, R., and Schneider, W. J. (1991) J. Biol. Chem. 266, 18761-18770). Furthermore, in contrast to the serum levels of the yolk precursor lipoproteins, those of TTR were not significantly changed by estrogen; thus, TTR represents a newly defined, estrogen-independent class of yolk precursor proteins. These data strongly suggest that oocytic TTR is derived from the circulation, where it is a constitutive component, and deposited into yolk as a result of endocytosis mediated by a specific receptor.

Transport and uptake of retinol during chicken oocyte growth.

Most, if not all, components found in the yolk of a chicken egg are extracted from the plasma compartment during the rapid growth phase of the oocyte. Uptake of the major yolk constituents, very-low-density lipoprotein and vitellogenin, has been shown to be mediated by a specific receptor in the plasma membrane of the oocyte (Barber, D.L., Aebersold, R., Sanders, E.J. and Schneider, W.J. (1991) J. Biol. Chem. 266, 18761-18770). In the current study, we sought biochemical evidence for the uptake into oocytes of a minor but biologically very important component, the vitamin retinol. For transport in serum, retinol is bound to retinol-binding protein (RBP), which in turn forms a complex with transthyretin (TTR). In order to gain insight into the biochemical details of transport of the vitamin, we have identified, purified and characterized RBP, TTR, and RBP-TTR complexes from chicken serum and yolk. The results demonstrate that both serum and yolk contain the tertiary retinol-RBP-TTR complexes as well as free RBP and TTR. Western blots of yolk collected from oocytes at different stages of growth show that both RBP and TTR, but not albumin, are more abundant at early stages relative to total yolk protein. In addition, we find both RBP and TTR in endocytic clathrin-coated vesicles of the oocyte. Our results support the hypothesis that retinol, which must be imported by the oocyte for proper embryonic development, is internalized by the chicken oocyte bound to its serum protein-transport complex.

One-step chromatographic method for the purification of avian serotransferrin.

Existing purification procedures for serum transferrins involve multistep chromatographic separations and require several days to complete. In addition, they have not been tested for purification of transferrins directly from the blood of egg-laying animals, where large amounts of circulatory lipoproteins can interfere with standard chromatographic separations. We have developed a procedure for purifying transferrin in one step directly from the serum of ovulating chickens. The method, which is based on hydrophobic interaction chromatography, gives a yield of about 12 mg (80%) of purified serotransferrin from 3 ml of serum and can be completed in a few hours.

[Asbestos and bronchogenic carcinoma: a search for fibers in the pulmonary tissue of 3 patients with bronchogenic carcinoma]. / Asbesto e carcinoma broncogênico: pesquisa de fibras em tecido pulmonar de três pacientes portadores de carcinoma broncogênico.

Based on the highly suspicious radiological findings of exposure to asbestos (case 1) or on a positive occupational history (cases 2 and 3), the authors looked for the presence of fibers in blocks of lung tissue removed in autopsy or surgical biopsies of three cases of bronchogenic carcinoma. The blocks were submitted to sodium hypochloride digestion followed by fiber identification in phase contrast light microscopy. The authors were able to demonstrate the presence of fibres in the three cases. The likelyhood of those carcinomas being caused by exposure to asbestos is very high, as two out of the three cases showed pulmonary fibrosis (cases 1 and 2) and the other case showed typical parietal pleural plaques at thoracotomy.