Biochemical characterization of the uptake and release of [3H]dopamine by dopaminergic hypothalamic neurons: a developmental study using serum-free medium cultures

The development of the biochemical properties of mouse hypothalamic dopaminergic neurons has been analyzed in vivo and in cultures of cell taken on the 16th day of gestation and grown in serum-free medium for up to 3 weeks. In the course of in vivo development, the dopamine (DA) content remains low during fetal life (10% of the adult value), beginning to increase on the 19th fetal day. In contrast, the specific accumulation of [3H]DA increased markedly during the last days of gestation from 20% of the adult value on the 16th fetal day to 70-80% of the adult value on Postnatal Day 3. Hypothalamic DA neurons in culture accumulate endogenous DA although at a lower level than in vivo. They take up [3H]DA by an active transport system which is specific for DA, and which shows time, temperature, and sodium dependency (Km = 1 microM). HPLC analysis showed that the newly taken up [3H]DA was not metabolized in the short run under the conditions used. It was stored in a form that could be released when neurons were depolarized in a high K+ (60 mM) medium. The K+-evoked [3H]DA release was found to be strictly dependent on extracellular Ca2+. Moreover the release of [3H]DA was also stimulated by veratridine in a Ca2+-dependent manner. Similar data have been obtained with the release of endogenous dopamine. No specific uptake and no K+-evoked dopamine release occurred in 2-day-old cultures. The specific [3H]DA uptake and the K+-evoked release appeared in 5-day-old cultures and increased with time in culture at least until Day 15. We examined the effects on [3H]DA release of polyunsaturated fatty acid, triiodothyronine, and corticosterone, all of which have been shown to play an important role in synaptogenesis in culture. These components, either separately or together, did not modify the percentage of the basal or the stimulated [3H]DA release. These results showed that hypothalamic DA neurons grown in serum-free medium progressively acquired the functional properties of adult DA neurons as concerns DA synthesis, DA uptake, and release. From a development point of view, this study suggests that the capacity to specifically take up [3H]DA and to respond to high K+ concentration is not expressed at early stages of neuronal development.(ABSTRACT TRUNCATED AT 400 WORDS)     

Subcellular localization of secretogranin II and synaptophysin by immunoelectron microscopy in differentiated hypothalamic neurons in culture

Secretogranin II (SgII), a tyrosine-sulfated secretory protein, is a widespread component of endocrine and neuronal cells. In the present study we used mouse hypothalamic neurons differentiated in culture and studied the subcellular localization of SgII by two methods, i.e., by the use of immunoperoxidase or immunogold electron microscopy. By immunoperoxidase labeling, SgII was mainly detected in the matrix of large dense-core vesicles (LDCVs). In addition, usually in nerve terminals containing LDCVs, peroxidase reaction product was also found in association with the membrane of small synaptic vesicles (SSVs). By immunogold labeling, SgII was detected only in the matrix of LDCVs. We also compared the localization of SgII and synaptophysin (SY), an integral membrane protein of SSVs, by double labeling, using a combination of pre-embedding immunogold and -peroxidase techniques for SgII and SY, respectively. In perikarya, SgII-positive LDCVs were observed in the vicinity of the Golgi complex and scattered in the cytoplasm. In contrast, SY labeling was restricted to electron-translucent vesicles and tubular membranes in the Golgi area. Moreover, membrane structures positive for both SgII and SY were not found either in the Golgi zone or in other regions of the cytoplasm. In synaptic boutons, immunolabeling of LDCVs and SSVs with anti-SgII and anti-SY, respectively, was mutually exclusive. In summary, within the limitation of the methods used, our data are consistent with the notion that SgII and SY are segregated from each other on exit from the trans-Golgi network, than follow two distinct membrane traffic pathways, and that the presence of SgII on the membrane of some SSVs is due to endocytosis.     

omega-(Imidazol-4-yl)alkane-1-sulfonamides: a new series of potent histamine H(3) receptor antagonists.

omega-(1H-Imidazol-4-yl)alkane-1-sulfonamides were prepared and found to be potent histamine H(3) receptor antagonists. High receptor affinity and a low difference in the data between the bioassays were achieved with 5-(1H-imidazol-4-yl)pentane-1-sulfonic acid 4-chlorobenzylamide (16). Good in vitro profiles were also obtained for 2-hydroxysulfonamide and vinylsulfonamide analogues. This complements and completes the existing set of imidazole-based sulfonamides and sulfamides.     

Comparison of Color and Body Condition Between Early and Late Breeding King Penguins

Early breeding is associated with greater reproductive success in many species. In king penguins, Aptenodytes patagonicus , laying extends for 6 mo. Early breeders may fledge a single chick at best, but late breeders virtually never fledge a chick. For early and late breeders, we compared colored ornaments known to be important in mate choice: yellow–orange feathers of the breast and auricular areas, and an ultraviolet and yellow–orange beak spot. Our purpose was to discern differences between males and females in this highly sexually monomorphic species, as well as to discern whether colored ornaments are more important for the more successful early breeders (aspects of color were hue, chroma, and brightness). For this, we weighed and measured 130 penguins. Early males had greater reflectance of ultraviolet color from the beak spot than did early females and late breeders of both sexes, and the early males were heavier and in better condition than late breeding males or females. Late breeding females were the yellowest in breast hue, a trait that has been linked to immunocompetence. Within pairs, males and females were significantly correlated in body mass, but only early in the breeding season. We concluded that early in the breeding season when reproductive success was greatest, potential mates were not only more similar in body mass, but also that females may have chosen males that had brighter beak spots and were in better body condition.     

Assessment of the allelochemical activity of Ostreopsis cf. ovata and the ovatoxins towards competitive benthic microalgae

Aquatic Ecology - Recurrent blooms of the toxic dinoflagellate Ostreopsis cf. ovata are frequently reported in the Northwestern Mediterranean Sea. The impact of these proliferations on other...     

The alternative translated MDMXp60 isoform regulates MDM2 activity

Isoforms derived from alternative splicing, mRNA translation initiation or promoter usage extend the functional repertoire of the p53, p63 and p73 genes family and of their regulators MDM2 and MDMX. Here we show cap-independent translation of an N-terminal truncated isoform of hMDMX, hMDMX^p60, which is initiated at the 7th AUG codon downstream of the initiation site for full length hMDMX^FL at position +384. hMDMX^p60 lacks the p53 binding motif but retains the RING domain and interacts with hMDM2 and hMDMX^FL. hMDMX^p60 shows higher affinity for hMDM2, as compared to hMDMX^FL. In vitro data reveal a positive cooperative interaction between hMDMX^p60 and hMDM2 and in cellulo data show that low levels of hMDMX^p60 promote degradation of hMDM2 whereas higher levels stabilize hMDM2 and prevent hMDM2-mediated degradation of hMDMX^FL. These results describe a novel alternatively translated hMDMX isoform that exhibits unique regulatory activity toward hMDM2 autoubiquitination. The data illustrate how the N-terminus of hMDMX regulates its C-terminal RING domain and the hMDM2 activity.     

MDM2 protein-mediated ubiquitination of numb protein: identification of a second physiological substrate of MDM2 that employs a dual-site docking mechanism

The E3 ubiquitin ligase, MDM2, uses a dual-site mechanism to ubiquitinate and degrade the tumor suppressor protein p53, involving interactions with the N-terminal hydrophobic pocket and the acidic domain of MDM2. The results presented here demonstrate that MDM2 also uses this same dual-site mechanism to bind to the cell fate determinant NUMB with both the N-terminal hydrophobic pocket and the acidic domain of MDM2 also involved in forming the interaction with NUMB. Furthermore, the acidic domain interactions are crucial for MDM2-mediated ubiquitination of NUMB. Contrary to p53, where two separate domains form the interface with MDM2, only one region within the phosphotyrosine binding domain of NUMB (amino acids 113-148) mediates binding to both these regions of MDM2. By binding to both domains on MDM2, NUMB disrupts the MDM2-p53 complex and MDM2-catalyzed ubiquitination of p53. Therefore, we have identified the mechanism NUMB uses to regulate the steady-state levels of the p53 in cells. By targeting the acidic domain of MDM2 using acid domain-binding ligands we can overcome MDM2-mediated ubiquitination and degradation of NUMB impacting on the stabilization of p53 in cells. Furthermore, delivery of MDM2 acid domain-binding ligands to cancer cells promotes p53-dependent growth arrest and the induction of apoptosis. This highlights the dual-site mechanism of MDM2 on another physiological substrate and identifies the acid domain as well as N terminus as a potential target for small molecules that inhibit MDM2.     

Genome sequence of the biocontrol strain Pseudomonas fluorescens F113

Pseudomonas fluorescens F113 is a plant growth-promoting rhizobacterium (PGPR) that has biocontrol activity against fungal plant pathogens and is a model for rhizosphere colonization. Here, we present its complete genome sequence, which shows that besides a core genome very similar to those of other strains sequenced within this species, F113 possesses a wide array of genes encoding specialized functions for thriving in the rhizosphere and interacting with eukaryotic organisms.