Jan Kornelis Dijkstra

EFFICIENCY OF CYTOPLASMIC DELIVERY BY PH-SENSITIVE LIPOSOMES TO CELLS IN CULTURE

The intracellular processing of pH-sensitive liposomes composed of cholesterylhemisuccinate (CHEMS) and dioleoylphosphatidylethanolamine (DOPE) by eukaryotic cell lines has been compared to non-pH-sensitive liposomes made of CHEMS and dioleoylphosphatidylcholine (DOPC). The pH-sensitive liposomes can deliver encapsulated fluorescent molecules [calcein, fluoresceinated dextran, fluoresceinated polypeptide, and diphtheria toxin A chain (DTA)] into the cytoplasm. Cytoplasmic delivery can be blocked in the presence of ammonium chloride or EDTA, indicating that the process requires a low-pH environment and the presence of divalent cations. Inhibition of cellular protein synthesis by DTA delivery from the pH-sensitive liposome is orders of magnitude greater than from the non-pH-sensitive liposome composition. The delivery of DTA into the cytoplasm by pH-sensitive liposomes is at least 0.01% of cell-associated liposomal DTA. There is no significant difference in the degradation rate of bovine serum albumin (BSA) or the rate of acidification of pH-sensitive dye, 8-hydroxy-l,3,6-pyrene-trisulfonate (HPTS), when delivered to cells in pH-sensitive and non-pH-sensitive liposomes. Thus the efficiency of cytoplasmic delivery is less than 10% of the cell-associated liposome contents, which is the smallest difference that can be detected by these two assays. Based upon the various assays used to measure liposome content disposition in the cell, we conclude that the efficiency of cytoplasmic delivery by the CHEMS/DOPE liposomes is greater than 0.01% and less than 10% of the cell-associated liposomal contents.

Beyond the Class Norm: Bullying Behavior of Popular Adolescents and its Relation to Peer Acceptance and Rejection

This study examined to what extent bullying behavior of popular adolescents is responsible for whether bullying is more or less likely to be accepted or rejected by peers (popularity-norm effect) rather than the behavior of all peers (class norm). Specifically, the mean level of bullying by the whole class (class norm) was split into behavior of popular adolescents (popularity-norm) and behavior of non-popular adolescents (non-popularity-norm), and examined in its interaction with individual bullying on peer acceptance and peer rejection. The data stem from a peer-nominations subsample of TRAILS, a large population-based sample of adolescent boys and girls (N = 3312). The findings of multilevel regression analyses demonstrated that the negative impact of individual bullying on peer acceptance and the positive impact on peer rejection were particularly weakened by bullying by popular adolescents. These results place the class-norm effects found in previous person-group dissimilarity studies in a different light, suggesting that particularly bullying by popular adolescents is related to the social status attached to bullying.

THE INVOLVEMENT OF PARENCHYMAL, KUPFFER AND ENDOTHELIAL LIVER-CELLS IN THE HEPATIC-UPTAKE OF INTRAVENOUSLY INJECTED LIPOSOMES - EFFECTS OF LANTHANUM AND GADOLINIUM SALTS

125I-labeled albumin or poly(vinyl pyrrolidone) encapsulated in intermediate size multilamellar or unilamellar liposomes with 30-40% of cholesterol were injected intravenously into rats. In other experiments liposomes containing phosphatidyl[Me-14C]choline was injected. 1 h after injection parenchymal or non-parenchymal cells were isolated. Non-parenchymal cells were separated by elutriation centrifugation into a Kupffer cell fraction and an endothelial cell fraction. From the measurements of radioactivities in the various cell fractions it was concluded that the liposomes are almost exclusively taken up by the Kupffer cells. Endothelial cells did not contribute at all and hepatocytes only to a very low extent to total hepatic uptake of the 125I-labels. Of the 14C-label, which orginates from the phosphatidylcholine moiety of the liposomes, much larger proportions were recovered in the hepatocytes. A time-dependence study suggested that besides the involvement of phosphatidylcholine exchange between liposomes and high density lipoprotein, a process of intercellular transfer of lipid label from Kupffer cells to the hepatocytes may be involved in this phenomenon. Lanthanum or gadolinium salts, which effectively block Kupffer cell activity, failed to accomplish an increase in the fraction of liposomal material recovered in the parenchymal cells. This is compatible with the notion that liposomes of the type used in these experiments have no, or at most very limited, access to the liver parenchyma following their intravenous administration to rats.

Same-Gender and Cross-Gender Peer Acceptance and Peer Rejection and Their Relation to Bullying and Helping Among Preadolescents: Comparing Predictions From Gender-Homophily and Goal-Framing Approaches

The relation between bullying and helping and same-gender and cross-gender peer acceptance and peer rejection was examined in a sample of preadolescents aged 11 and 12 years (N=1,065). The authors tested predictions from a gender-homophily approach vs. predictions from a goal-framing approach in which acceptance and rejection are seen as being generated by approach and avoidance goals, respectively. For preadolescents, both approaches predicted a central role for gender, but the gender-homophily approach predicted symmetrical effects for acceptance and rejection, whereas the goal-framing approach predicted strong asymmetries. The data supported the goal-framing approach. The most important findings were that for preadolescents, acceptance is much more frequent and much more gendered than rejection; the absolute impact of helping on acceptance is much larger than that of bullying (and vice versa for rejection); for acceptance, there is a prototypicality effect (i.e., boys accept bullying girls better than nonbullying girls, and girls accept helping boys better than nonhelping boys); and for acceptance, there is a cross-gender ignorance effect (i.e., boys ignore helping in girls, and girls ignore bullying in boys).

Influence of liposome charge on the association of liposomes with Kupffer cells in vitro. Effects of divalent cations and competition with latex particles.

We studied the interaction of large unilamellar liposomes carrying different surface charges with rat Kupffer cells in maintenance culture. In addition to 14C-labeled phosphatidylcholine, all liposome preparations contained either 3H-labeled inulin or 125I-labeled bovine serum albumin as a non-degradable or a degradable aqueous space marker, respectively. With vesicles carrying no net charge, intracellular processing of internalized liposomes caused nearly complete release of protein label into the medium in acid-soluble form, while phospholipid label was predominantly retained by the cells, only about one third being released. The presence of the lysosomotropic agent, ammonia, inhibited the release of both labels from the cells. At 4 degrees C, the association and degradation of the vesicles were strongly reduced. These results are very similar to what we reported on negatively charged liposomes (Dijkstra, J., Van Galen, W.J.M., Hulstaert, C.E., Kalicharan, D., Roerdink, F.H. and Scherphof, G.L. (1984) Exp. Cell Res. 150, 161-176). The interaction of both types of vesicles apparently proceeds by adsorption to the cell surface followed by virtually complete internalization by endocytosis. Similar experiments with positively charged vesicles indicated that only about half of the liposomes were taken up by the endocytic route, the other half remaining adsorbed to the cell-surface. Attachment of all types of liposomes to the cells was strongly dependent on the presence of divalent cations; Ca2+ appeared to be required for optimal binding. Neutral liposomes only slightly competed with the uptake of negatively charged vesicles, both at 4 degrees and 37 degrees C, whereas negatively charged small unilamellar vesicles and negatively charged latex beads were found to compete very effectively with the large negatively charged liposomes. Neutral vesicles competed effectively for uptake with positively charged ones. These results suggest that neutral and positively charged liposomes are largely bound by the same cell-surface binding sites, while negatively charged vesicles attach mainly to other binding sites.

Popularity and Adolescent Friendship Networks: Selection and Influence Dynamics

This study examined the dynamics of popularity in adolescent friendship networks across 3 years in middle school. Longitudinal social network modeling was used to identify selection and influence in the similarity of popularity among friends. It was argued that lower status adolescents strive to enhance their status through befriending higher status adolescents, whereas higher status adolescents strive to maintain their status by keeping lower status adolescents at a distance. The results largely supported these expectations. Selection partially accounted for similarity in popularity among friends; adolescents preferred to affiliate with similar-status or higher status peers, reinforcing the attractiveness of popular adolescents and explaining stability of popularity at the individual level. Influence processes also accounted for similarity in popularity over time, showing that peers increase in popularity and become more similar to their friends. The results showed how selection and influence processes account for popularity dynamics in adolescent networks over time.

PREPARATION, PROPERTIES, AND APPLICATIONS OF RECONSTITUTED INFLUENZA-VIRUS ENVELOPES (VIROSOMES)

Publisher Summary This chapter discusses the preparation, properties, and applications of reconstituted influenza virus envelopes (virosomes). Enveloped viruses utilize a membrane fusion strategy to introduce their genome into the cytoplasm of the host cell. This infectious entry, in principle, can occur through direct fusion of the viral envelope with the cell plasma membrane, or it may occur from within endosomes after cellular uptake of intact virions through receptor-mediated endocytosis. In the latter route of entry, the target membrane for fusion of the viral envelope is the limiting membrane of the endosomal cell compartment. The expression of the fusion activity of the virus is strictly dependent on a mildly acidic pH, precluding direct fusion of the viral envelope with the plasma membrane. After endocytic uptake, the virus encounters an acidic environment in the lumen of the endosomes, activating its fusion capacity. In addition, the membrane fusion activity of influenza virus is mediated solely by the major envelope glycoprotein, hemagglutinin (HA). HA represents the best-characterized membrane fusion protein.

Effects of ammonium chloride and chloroquine on endocytic uptake of liposomes by Kupffer cells in vitro

In this study we investigated the interaction of liposomes with rat Kupffer cells in maintenance culture by using the lysosomotropic amines ammonium chloride and chloroquine as inhibitors of intralysosomal degradation. The liposomes (large unilamellar vesicles) contained either the metabolically inert 3H-labeled inulin or the degradable 125I-labeled bovine serum albumin. In control incubations, the cells released nearly all accumulated protein label and about 30% of the lipid label when they were incubated in the absence of liposomes, after an initial uptake period of 1 h in the presence of liposomes. This release of label was, for the greater part, suppressed in the presence of ammonia or chloroquine. When the inhibitors were present during the initial uptake period, a several-fold increase in the amount of protein label accumulating in the cells and a smaller, but still marked, increase in lipid label accumulation were observed. The effect of ammonia when present during uptake was readily reversible in contrast to that of chloroquine. Experiments with encapsulated inulin revealed that both lysosomotropic agents also affected the uptake process per se to some extent, probably as a result of impaired membrane/receptor recycling. Labeled liposomes adsorbed to the cells at 4 degrees C were effectively internalized and processed intracellulary after shifting the temperature to 37 degrees C, even when a 500-fold excess of unlabeled liposomes was present in the medium during the 37 degrees C incubation. The observed effects of ammonia and chloroquine indicate that, after uptake, the liposomes are degraded within lysosomes, thus confirming our previous conclusion that endocytosis is the major uptake mechanism at 37 degrees C. From the temperature-change experiments we conclude that, at 4 degrees C, the liposomes are bound with high affinity to the cells, remaining firmly attached to the cell-surface structures which initiate their internalization when the temperature is raised to 37 degrees C.

Same-Gender and Cross-Gender Likeability: Associations with Popularity and Status Enhancement--The TRAILS Study.

This study examined the associations of popularity, substance use, athletic abilities, physical attractiveness, and physical and relational aggression with likeability by same-gender and cross-gender peers among early adolescents (N = 3,312; M age = 13.60, with 92.7% of the participants in the 12-14 age range). Data collection consisted of peer nominations in 172 classrooms of 34 secondary schools. Taking a goal-framing perspective, it was argued that key to understanding the association between popularity and likeability is the distinction between features that help to achieve popularity and features that help to maintain popularity. In support of our hypotheses, popularity and substance use, athletic abilities, and physical attractiveness (characteristics that help to become popular) contributed significantly to likeability, whereas physical and relational aggression (characteristics that help to maintain popularity) negatively predicted likeability. These specific nature of these effects depended on the reference group (same-gender vs. other-gender peers) and were further moderated by popularity.

EFFECT OF LIPOPROTEIN-FREE PLASMA ON THE INTERACTION OF HUMAN-PLASMA HIGH-DENSITY LIPOPROTEIN WITH EGG-YOLK PHOSPHATIDYLCHOLINE LIPOSOMES

Liposomes consisting of 14C-labeled egg yolk phosphatidylcholine were incubated with whole human plasma or plasma subfractions. The transfer of liposomal phospholipid to plasma high density lipoprotein was determined by gel filtration. Whole plasma degraded the liposomes considerably faster than isolated high density lipoprotein. The phospholipid-transferring activity of whole plasma could be recovered in an equivalent mixture of isolated high density lipoprotein and lipoprotein-free plasma. The transfer stimulating activity in lipoprotein-free plasma was not associated with albumin but with a component of higher molecular weight. Upon incubation of lipoprotein-free plasma with liposomes this component appeared to be adsorbed to the liposomes and could thus be separated from the bulk protein by gel filtration. This binding to liposomes is taken as an indication that the component acts by modifying the lipid-water interface thus facilitating the insertion of the lipoprotein into the liposomal bilayer.