Interactions of phospholipid vesicles with rat hepatocytes in vitro influence of vesicle-incorporated glycolipids

We examined the interaction of glycolipid-containing phospholipid vesicles with rat hepatocytes in vitro. Incorporation of either $\text{N-}\text{lignoceroyldihydrolactocerebroside}$ or the monosialoganglioside, G_M1, enhanced liposomal lipid uptake 4–5-fold as judged by the uptake of radioactive phosphatidylcholine as a vesicle marker. Cerebroside enhanced phospholipid uptake only when incorporated into dimyristoyl, but not into egg phosphatidylcholine vesicles. The lack of cerebroside effect in egg phosphatidylcholine-containing vesicles appeared to be due to a limited exposure of the carbohydrate part of the glycolipid as suggested by the reduced agglutinability of those vesicles by Ricinus communis agglutinin.In contrast to the results with radioactive phosphatidylcholine, we observed only a 20% increase in vesicle-cell association as a result of glycolipid incorporation, when a trace amount of [¹⁴C]cholesteryloleate served as a marker of the liposomal lipids or when using the fluorescent dye, carboxyfluorescein, as a marker of the aqueous space of the vesicles. By the same token, intracellular delivery of vesicle-contents was only slightly enhanced (approx. 10%).The discrepancy between the association with the cells of phosphatidylcholine on the one hand and cholesteryoleate or entrapped marker on the other suggests different mechanisms of uptake for these markers. Our results are compatible with the notion that the main effect of incorporation of glycolipids into the vesicles is the enhancement of exchange or transfer of phospholipid molecules between vesicles and cells. Incubation of the cells with galactose or lactose, prior to addition of vesicles, suggests that this enhanced phospholipid exchange or transfer involves specific recognition of the terminal galactose residues of the glycolipid vesicles by a receptor present on the plasma membranes of hepatocytes.     

Interaction of phospholipid vesicles with rat hepatocytes in primary monolayer culture

We studied the interaction of positively and negatively charged unilamellar and multilamellar phospholipid vesicles (liposomes) with rat-liver parenchymal cells in primary monolayer culture. Radioactive liposomal phosphatidylcholine was taken up more rapidly and to a larger extent from unilamellar than from multilamellar vesicles. No significant difference in uptake characteristics was observed between vesicles of different charge. The presence of serum greatly reduced uptake of liposomal phosphatidylcholine of both unilamellar and multilamellar vesicles. This serum effect was independent of surface charge of the vesicles. When cells were allowed to take up radioactive liposomal phospholipid and then incubated further in absence of vesicles, part of the radioactivity associated with the cells was released into the medium, most of it as water soluble degradation products. When cells were preincubated with vesicles containing horseradish peroxidase and then, after removal of the vesicles, further incubated, peroxidase activity could be demonstrated in the culture medium, part of it only after addition of Triton X-100. These observations were taken to indicate that part of the phospholipid taken up the cells represented vesicles binding to the cell surface rather than having been internalized. Vesicle-entrapped [125I]albumin was taken up by the cells and rapidly hydrolyzed as indicated by the appearance of radioactivity soluble in trichloroacetic acid within minutes after starting the incubation. No uptake of free albumin could be demonstrated. The kinetics of albumin uptake and release of trichloroacetic acid-soluble radioactivity from the cells suggest that, initially, liposomes are internalized predominantly by endocytosis, while during prolonged incubation fusion of the liposomal membrane with the plasma membrane gradually contributes more substantially to the overall uptake process. The significance of these findings is emphasized with special reference to the use of liposomes as intravenous carriers of enzymes or drugs.     

Reproducibility and complications in gene searches: linkage on chromosome 6, heterogeneity, association, and maternal inheritance in juvenile myoclonic epilepsy

Evidence for genetic influences in epilepsy is strong, but reports identifying specific chromosomal origins of those influences conflict. One early study reported that human leukocyte antigen (HLA) markers were genetically linked to juvenile myoclonic epilepsy (JME); this was confirmed in a later study. Other reports did not find linkage to HLA markers. One found evidence of linkage to markers on chromosome 15, another to markers on chromosome 6, centromeric to HLA. We identified families through a patient with JME and genotyped markers throughout chromosome 6. Linkage analysis assuming equal male-female recombination probabilities showed evidence for linkage (LOD score 2.5), but at a high recombination fraction (theta), suggesting heterogeneity. When linkage analysis was redone to allow independent male-female thetas, the LOD score was significantly higher (4.2) at a male-female theta of.5,.01. Although the overall pattern of LOD scores with respect to male-female theta could not be explained solely by heterogeneity, the presence of heterogeneity and predominantly maternal inheritance of JME might explain it. By analyzing loci between HLA-DP and HLA-DR and stratifying the families on the basis of evidence for or against linkage, we were able to show evidence of heterogeneity within JME and to propose a marker associated with the linked form. These data also suggest that JME may be predominantly maternally inherited and that the HLA-linked form is more likely to occur in families of European origin.     

Hypoglycaemia-insulin test: discordant growth hormone and cortisol response in paediatric patients regarding recovery from hypoglycaemia with or without oral glucose solution

BACKGROUND: Hypoglycaemia-insulin test (HIT) is the 'gold standard' for the diagnosis of adrenal-pituitary-hypothalamic axis disorders. Controversy exists on the convenience of recovery from an insulin-induced hypoglycaemia since this test is not risk-free. OBJECTIVE: To ascertain whether recovery from insulin-induced hypoglycaemia with an oral glucose solution produces a different response of growth hormone (GH) and cortisol at different times of the study compared with spontaneous recovery from hypoglycaemia. PATIENTS AND METHODS: Prospective study of 100 children and adolescents with growth delay who underwent an HIT. Patients were consecutively assigned to two groups of 50. In one group recovery from hypoglycaemia occurred spontaneously and in the other recovery was achieved with an oral glucose solution (20 g of glucose) when glycaemia was under 30 mg/dl. The two groups did not differ in age, sex, pubertal status, weight, height and IGF-I levels. RESULTS: The response of GH at 30, 60, 90 and 120 min and cortisol at 10, 60, 90 and 120 min was lower and statistically significant in patients with recovery from hypoglycaemia with oral glucose solution. GH deficiency was diagnosed more frequently in patients recovered with glucose solutions (94%) compared to those with spontaneous recovery (68%). CONCLUSIONS: Oral glucose solution administration when glycaemia was under 30 mg/dl in HIT produced a lower GH and cortisol response to insulin stimulus and a greater frequency of GH deficit diagnosis.     

Expression of mutated cationic trypsinogen reduces cellular viability in AR4-2J cells

Mutations in the human cationic trypsinogen are associated with hereditary pancreatitis. The cDNA coding for human cationic trypsinogen was subcloned into the expression vector pcDNA3. The mutations R122H, N29I, A16V, D22G, and K23R were introduced by site directed mutagenesis. We constructed an expression vector coding for active trypsin by subcloning the cDNA of trypsin lacking the coding region for the trypsin activating peptide behind an appropriate signal peptide. Expression of protein was verified by Western blot and measurement of enzymatic activity. AR4-2J cells were transiently transfected with the different expression vectors and cell viability and intracellular caspase-3 activity were quantified. In contrast to wild-type trypsinogen, expression of active trypsin and mutated trypsinogens reduced cell viability of AR4-2J cells. Expression of trypsin and R122H trypsinogen induced caspase-3 activity. Acinar cells might react to intracellular trypsin activity by triggering apoptosis.     

Cloning of IP15, a pancreatitis-induced gene whose expression inhibits cell growth

We describe the cloning and expression of the mouse gene interferon-inducible-protein 15 (IP15), whose activation is related to the acute phase of experimental pancreatitis. Analysis of its structure indicates that it encodes a putative transmembrane protein of 137 amino acids. This gene contains a predicted IFN-stimulable-response element. In vivo studies showed that IP15 is strongly activated in pancreas early during caerulein-induced pancreatitis. In situ hybridization of IP15 mRNA showed that its expression is restricted to acinar cells. IP15 was also induced in pancreas under systemic-lipopolysaccharide treatment and in intestine under Salmonella infection. In vitro studies using NIH3T3 fibroblasts showed that IP15 is induced by IFN-alpha. Growth rate was significantly lower in cells transfected with pcDNA4/IP15 plasmid. In addition, cells expressing IP15 showed less capacity to develop colonies after antibiotic selection. In conclusion, we identified a new interferon-inducible gene that is activated early in pancreas with pancreatitis and whose expression inhibits cell growth.     

Female reproductive biology of the sand smelt in brackish lagoons of southern France

The breeding season of Atherina boyeri living in the brackish lagoons of Mauguio, Pérols and Méjean (Hérault, southern France) was very protracted (February-September). The peak of the reproductive effort occurred in April, May and June. Larger fishes started spawning earlier, and stopped later, than smaller ones. Batch fecundity varied from 4 to 447 oocytes and was positively related to female length and weight. Monthly mean fecundity increased at the beginning of the breeding season and decreased between June and July. Individual mean oocyte diameter ranged from 1.34 to 1.94 mm and was not related to fish length. Monthly mean oocyte size declined throughout the breeding season as water temperature rose. There was no trade-off between number and egg size. The strategy of these sand smelts seemed well adapted to ensure spawning success without endangering their survival. The highest reproductive effort period occurred in such a way that environmental conditions were suitable to the best survival and growth of hatched larvae. When breeding ceased, fishes, particularly the youngest ones, had enough time to improve their condition before overwintering.     

Historical colonization of the Mediterranean Sea by Atlantic fishes: do biological traits matter?

Since its opening 5.33 million years ago, the Gibraltar Strait has always contributed to the Mediterranean fauna and flora. Despite the increasing importance of the phenomenon, ecological determinants underlying colonization success of Atlantic fishes in the Mediterranean Sea have been poorly investigated. Here we reconstruct the recent historical colonization of the whole Mediterranean Sea by Atlantic fishes and we aim to determine where Atlantic fishes preferentially establish and whether some biological traits and ecological factors can be correlated to the colonization success (climate match, position in the water column, maximum body length, propagules, confamilial resistance, depth). A database on Atlantic fish species records from 1810 to 2006 was built and the colonization rate of each introduced species was estimated. Analysis of Variance, Chi squared test and logistic regression were used to investigate the relationships between ecological variables and colonization success. In addition, an index of asymmetry was used to analyse the relative colonization on the two sides of the Mediterranean Sea. Overall 48.33% of Atlantic species introduced in the Mediterranean Sea succeeded in colonizing eastwards. We found that habitat depth of Atlantic species is significantly related to their colonization success due to the obstacle of the shallow depth of the Gibraltar Strait (300 m). It also appears that despite the cyclonic general water circulation on the North Western basin, the northern side is more colonized than the southern one: 70.40% of the studied species colonize the northern side, while only 29.62% colonize the southern one. Two hypotheses may explain this trend: the bottom-up process that enhances the colonization success of Atlantic fishes along the Spanish coast of Alboran Sea owning to its high productivity and the intensiveness of the scientific explorations along the northern Mediterranean side. We conclude that crossing the Gibraltar Strait does not guarantee the colonization success and that species life-history and functional traits are poor predictors. Instead we suggest that environmental factors may determine favourable locations for invasive species installation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Handling editor: J. Cambray.     

A PH domain within OCRL bridges clathrin‐mediated membrane trafficking to phosphoinositide metabolism

OCRL, whose mutations are responsible for Lowe syndrome and Dent disease, and INPP5B are two similar proteins comprising a central inositol 5‐phosphatase domain followed by an ASH and a RhoGAP‐like domain. Their divergent NH2‐terminal portions remain uncharacterized. We show that the NH2‐terminal region of OCRL, but not of INPP5B, binds clathrin heavy chain. OCRL, which in contrast to INPP5B visits late stage endocytic clathrin‐coated pits, was earlier shown to contain another binding site for clathrin in its COOH‐terminal region. NMR structure determination further reveals that despite their primary sequence dissimilarity, the NH2‐terminal portions of both OCRL and INPP5B contain a PH domain. The novel clathrin‐binding site in OCRL maps to an unusual clathrin‐box motif located in a loop of the PH domain, whose mutations reduce recruitment efficiency of OCRL to coated pits. These findings suggest an evolutionary pressure for a specialized function of OCRL in bridging phosphoinositide metabolism to clathrin‐dependent membrane trafficking.