Mice Lacking Platelet-Derived Growth Factor D Display a Mild Vascular Phenotype

Platelet-derived growth factor D (PDGF-D) is the most recently discovered member of the PDGF family. PDGF-D signals through PDGF receptor β, but its biological role remains largely unknown. In contrast to other members of the PDGF family of growth factors, which have been extensively investigated using different knockout approaches in mice, PDGF-D has until now not been characterized by gene inactivation in mice. Here, we present the phenotype of a constitutive Pdgfd knockout mouse model (Pdgfd^-/-), carrying a LacZ reporter used to visualize Pdgfd promoter activity. Inactivation of the Pdgfd gene resulted in a mild phenotype in C57BL/6 mice, and the offspring was viable, fertile and generally in good health. We show that Pdgfd reporter gene activity was consistently localized to vascular structures in both postnatal and adult tissues. The expression was predominantly arterial, often localizing to vascular bifurcations. Endothelial cells appeared to be the dominating source for Pdgfd, but reporter gene activity was occasionally also found in subpopulations of mural cells. Tissue-specific analyses of vascular structures revealed that NG2-expressing pericytes of the cardiac vasculature were disorganized in Pdgfd^-/- mice. Furthermore, Pdgfd^-/- mice also had a slightly elevated blood pressure. In summary, the vascular expression pattern together with morphological changes in NG2-expressing cells, and the increase in blood pressure, support a function for PDGF-D in regulating systemic arterial blood pressure, and suggests a role in maintaining vascular homeostasis.     

Sar1 GTPase Activity Is Regulated by Membrane Curvature

The majority of biosynthetic secretory proteins initiate their journey through the endomembrane system from specific subdomains of the endoplasmic reticulum. At these locations, coated transport carriers are generated, with the Sar1 GTPase playing a critical role in membrane bending, recruitment of coat components, and nascent vesicle formation. How these events are appropriately coordinated remains poorly understood. Here, we demonstrate that Sar1 acts as the curvature-sensing component of the COPII coat complex and highlight the ability of Sar1 to bind more avidly to membranes of high curvature. Additionally, using an atomic force microscopy-based approach, we further show that the intrinsic GTPase activity of Sar1 is necessary for remodeling lipid bilayers. Consistent with this idea, Sar1-mediated membrane remodeling is dramatically accelerated in the presence of its guanine nucleotide-activating protein (GAP), Sec23-Sec24, and blocked upon addition of guanosine-5′-[(β,γ)-imido]triphosphate, a poorly hydrolysable analog of GTP. Our results also indicate that Sar1 GTPase activity is stimulated by membranes that exhibit elevated curvature, potentially enabling Sar1 membrane scission activity to be spatially restricted to highly bent membranes that are characteristic of a bud neck. Taken together, our data support a stepwise model in which the amino-terminal amphipathic helix of GTP-bound Sar1 stably penetrates the endoplasmic reticulum membrane, promoting local membrane deformation. As membrane bending increases, Sar1 membrane binding is elevated, ultimately culminating in GTP hydrolysis, which may destabilize the bilayer sufficiently to facilitate membrane fission.     

Identification of plant volatiles activating single receptor neurons in the pine weevil (Hylobius abietis)

Naturally produced plant volatiles, eliciting responses of single olfactory receptor neurons in the pine weevil, have been identified by gas chromatography linked with mass spectrometry. The receptor neurons (n = 72) were classified in 30 types, according to the compound which elicited the strongest response in each neuron, 20 of which compounds were identified. Most potent for 14 types of neurons (n = 50) were monoterpenes, including bicyclic (e.g. α-pinene, camphor and myrtenal) for 8 types (n = 32), monocyclic (limonene, carvone, α-terpinene) for 3 types (n = 12) and acyclic (e.g. β-myrcene and linalool) for 3 types (n = 6). Other compounds eliciting strongest responses of a neuron were five sesquiterpenes, including α-copaene and a farnesene-isomer, and an anethole type which has no biosynthetic relationship with terpenes. Within one type, receptor neurons with quite selective responses to the most potent compound as well as neurons with additional responses to several, structurally similar compounds were found, indicating that the neurons may have the same functional types of membrane receptors, but different sensitivities. Response spectra of neurons within the bicyclic-, mono-cyclic and acyclic types showed more overlapping than across the neuron types. Minimal overlapping response spectra was found between monoterpene and sesquiterpene neurons. The results suggest that this structure-activity relationship is significant for encoding plant odour information in the pipe weevil. Accepted: 6 January 1997     

Zn2+ binding to human calbindin D(28k) and the role of histidine residues

We have studied the binding of Zn2+ to the hexa EF-hand protein, calbindin D(28k)-a strong Ca2+-binder involved in apoptosis regulation-which is highly expressed in brain tissue. By use of radioblots, isothermal titration calorimetry, and competition with a fluorescent Zn2+ chelator, we find that calbindin D(28k) binds Zn2+ to three rather strong sites with dissociation constants in the low micromolar range. Furthermore, we conclude based on spectroscopic investigations that the Zn2+-bound state is structurally distinct from the Ca2+-bound state and that the two forms are incompatible, yielding negative allosteric interaction between the zinc- and calcium-binding events. ANS titrations reveal a change in hydrophobicity upon binding Zn2+. The binding of Zn2+ is compatible with the ability of calbindin to activate myo-inositol monophosphatase, one of the known targets of calbindin. Through site-directed mutagenesis, we address the role of cysteine and histidine residues in the binding of Zn2+. Mutation of all five cysteines into serines has no effect on Zn2+-binding affinity or stoichiometry. However, mutating histidine 80 into a glutamine reduces the binding affinity of the strongest Zn2+ site, indicating that this residue is involved in coordinating the Zn2+ ion in this site. Mutating histidines 5, 22, or 114 has significantly smaller effects on Zn2+-binding affinity.     

Somatic embryogenesis of Pinus rigida?×?P. taeda and the relationship between the initiation of embryogenic tissue and zygotic embryo development

The pitch-loblolly pine hybrid (Pinus rigida × P. taeda) has useful characteristics of the parents, but its exploitation is hindered by restrictions of conventional breeding and propagation methods. This study was undertaken to establish an effective in vitro system for propagating pitch-loblolly hybrid pine through somatic embryogenesis and to unravel the relationship between the efficiency of embryogenic tissue initiation and zygotic embryo development. To initiate embryogenic tissue, megagametophytes of developing seeds were used as explants. Seeds were collected weekly, examined, and tested during June and July 2004. The medium and seed collection date were the most important factors for the successful somatic embryogenesis of P. rigida × P. taeda. Five embryogenic lines were obtained using a modified P. taeda basal medium, and the highest initiation rate was 0.55%, for seeds collected in 2 weeks, between July 3 and 16. Histological observation revealed that zygotic embryos of those seeds were mostly at the proembryonic stage or in transition to precotyledonary stages. For the successful maturation of somatic embryos, abscisic acid and gellan gum were needed in the medium. The results show that, although further tests and development are required, somatic embryogenesis could provide a viable option for propagating P. rigida × P. taeda hybrids.     

Trikaryon formation and nuclear selection in pairings between heterokaryons and homokaryons of the root rot pathogen Heterobasidion parviporum

Pairings between heterokaryons and homokaryons of Agaricomycete fungi (he-ho pairings) can lead to either heterokaryotization of the homokaryon or displacement of the homokaryotic nucleus through migration of nuclei from the heterokaryon into the homokaryon. In species of Agaricomycetes with multinucleate cells (>2 nuclei per cell), he-ho pairings could result in the stable or transient formation of a hypha with three genetically different nuclei (trikaryons). In this study, he-ho pairings were conducted using the multinucleate Agaricomycete Heterobasidion parviporum to determine whether trikaryons can be formed in the laboratory and whether nuclear genotype affects migration and heterokaryon formation. Nuclei were tracked by genotyping the heterokaryotic mycelium using nucleus-specific microsatellite markers. The data indicated that certain nuclear combinations were favored, and that nuclei from some strains had a higher rate of migration. A high percentage of trikaryons (19 %) displaying three microsatellite alleles per locus were identified among subcultures of the he-ho pairings. Using hyphal tip and conidial isolation, we verified that nuclei of three different mating types can inhabit the same mycelium, and one of the trikaryotic strains was judged to be semi-stable over multiple sub-culturing steps, with some hyphal tips that retained three alleles and others that reduced to two alleles per locus. These results demonstrate that nuclear competition and selection are possible outcomes of heterokaryon-homokaryon interactions in H. parviporum and confirm that ratios of component nuclei in heterokaryons are not strictly 1:1. The high rate of trikaryon formation in this study suggests that fungi with multinucleate cells may have the potential for greater genetic diversity and recombination relative to dikaryotic fungi.     

YcfDRM is a thermophilic oxygen-dependent ribosomal protein uL16 oxygenase

Extremophiles - YcfD from Escherichia coli is a homologue of the human ribosomal oxygenases NO66 and MINA53, which catalyse histidyl-hydroxylation of the 60S subunit and affect cellular...     

TFG clusters COPII‐coated transport carriers and promotes early secretory pathway organization

In mammalian cells, cargo‐laden secretory vesicles leave the endoplasmic reticulum (ER) en route to ER‐Golgi intermediate compartments (ERGIC) in a manner dependent on the COPII coat complex. We report here that COPII‐coated transport carriers traverse a submicron, TFG (Trk‐fused gene)‐enriched zone at the ER/ERGIC interface. The architecture of TFG complexes as determined by three‐dimensional electron microscopy reveals the formation of flexible, octameric cup‐like structures, which are able to self‐associate to generate larger polymers in vitro. In cells, loss of TFG function dramatically slows protein export from the ER and results in the accumulation of COPII‐coated carriers throughout the cytoplasm. Additionally, the tight association between ER and ERGIC membranes is lost in the absence of TFG. We propose that TFG functions at the ER/ERGIC interface to locally concentrate COPII‐coated transport carriers and link exit sites on the ER to ERGIC membranes. Our findings provide a new mechanism by which COPII‐coated carriers are retained near their site of formation to facilitate rapid fusion with neighboring ERGIC membranes upon uncoating, thereby promoting interorganellar cargo transport.     

Symposium De toekomst van de informele zorg

Due to the reform of long term care in 2015, there is growing concern about whether groups at risk receive the care they need. People in need of care have to rely more on help from their social network. The increased need for informal care requires resilience and organizational skills of families, but also of volunteers, professionals and employers. What does this mean for the provision of informal care in the next decennia? The symposium ‘The future of informal care’, organized on January 26 2017 by the National Institute for Social Research and the Institute for Societal Resilience of the Vrije Universiteit, addressed possible answers to this question. In her inaugural speech Alice de Boer discussed social inequality as possible determinant and outcome of informal care. Some conclusions:Until 2050 the absolute number of 75-plus doubled to about 3 million persons, but the number of informal caregivers will decrease. In addition to the importance of social and economic resources (the ‘have & have-nots’), the ability to arrange care (the ‘can & can-nots’) gains importance.Almost half of the older employers provides informal care just before retirement. Flexibility in working hours and work location facilitates combining work and care, but about half of the employers indicates that partial retirement and working at home are no options.Informal caregivers and professionals often provide care from comparable perspectives and identities. Addressing similarities rather than differences improves their chances for collaboration.The number of adult children providing household care to older parents increased between 2002 and 2014. This suggests an increase in family solidarity, but current reform policies may increase the gender inequality in caregiving families.Spouses and children remain primary caregivers in the future, preferably supported by many different types of caregivers. Not everybody has the capabilities to organize and direct such a large care network.Providing informal care increases the risk for overburden and absence at work or education. Informal caregivers at risk remain, also in the future, women, spouses, migrants, and younger carers.     

Comparing the epidermal growth factor interaction with four different cell lines: intriguing effects imply strong dependency of cellular context

The interaction of the epidermal growth factor (EGF) with its receptor (EGFR) is known to be complex, and the common over-expression of EGF receptor family members in a multitude of tumors makes it important to decipher this interaction and the following signaling pathways. We have investigated the affinity and kinetics of ¹²⁵I-EGF binding to EGFR in four human tumor cell lines, each using four culturing conditions, in real time by use of LigandTracer®.Highly repeatable and precise measurements show that the overall apparent affinity of the ¹²⁵I-EGF – EGFR interaction is greatly dependent on cell line at normal culturing conditions, ranging from K_D≈200 pM on SKBR3 cells to K_D≈8 nM on A431 cells. The ¹²⁵I-EGF – EGFR binding curves (irrespective of cell line) have strong signs of multiple simultaneous interactions. Furthermore, for the cell lines A431 and SKOV3, gefitinib treatment increases the ¹²⁵I-EGF - EGFR affinity, in particular when the cells are starved. The ¹²⁵I-EGF - EGFR interaction on cell line U343 is sensitive to starvation while as on SKBR3 it is insensitive to gefitinib and starvation.The intriguing pattern of the binding characteristics proves that the cellular context is important when deciphering how EGF interacts with EGFR. From a general perspective, care is advisable when generalizing ligand-receptor interaction results across multiple cell-lines.