Visualization of binding and receptor-mediated uptake of low density lipoproteins by human endothelial cells

Low density lipoproteins (LDL) were conjugated to colloidal gold for investigation of the ultrastructural aspects of binding and receptor-mediated internalization of LDL by cultured endothelial cells from the human umbilical artery and vein. The number of LDL receptors was increased by preincubation in lipoprotein-depleted serum. When the cells were incubated with LDL-gold particles for 2 h at 4 degrees C, the complexes were found in coated pits as well as in clusters attached to the plasma membrane. Small vesicles containing a few LDL-gold complexes appeared in the cytoplasm close to the plasma membrane when the cells were incubated with the conjugate for 5 min at 37 degrees C. After 15 min at 37 degrees C, larger vesicles with a pale matrix and membrane-orientated LDL-gold complexes were seen. After incubation for 30 min at 37 degrees C, colloidal gold particles were present in dense bodies. Quantification of the binding of LDL-gold complexes to the plasma membrane at 4 degrees C showed no differences between arterial and venous endothelial cells.     

Isoenzymes of vitamin-K-dependent carboxylase

Vitamin-K-dependent carboxylase was prepared from bovine liver, kidney, lung and testis and it was checked that these systems obeyed the laws of normal enzyme kinetics. Four carboxylatable substrates were obtained from different sources and the apparent Michaelis constants of the various carboxylases for these four substrates were measured. From the results thus obtained we concluded that carboxylase is a group name for a number of isoenzymes which are present in hepatic as well as in various non-hepatic tissues.     

Discovery of a gamma-carboxyglutamic acid-containing protein in human spermatozoa

Here we describe the identification of a gamma-carboxyglutamic acid-containing protein in human spermatozoa. After thermal decarboxylation the protein is a good substrate for vitamin K-dependent carboxylase from various origins. A quick purification procedure for the decarboxylated protein is presented and in a preliminary characterization we have established its Mr (28 000-30 000) and its amino acid composition.     

In vitro prothrombin synthesis from a purified precursor protein III. Preparation of an acid-soluble substrate for vitamin K-dependent carboxylase by limited proteolysis of bovine descarboxyprothrombin

Bovine descarboxyprothrombin and descarboxyfragment-1 can be used as substrates for rat and bovine vitamin K-dependent carboxylase. In both enzyme systems, however, these substrates have a high K_m (0.3–0.4 mM). A better substrate (K_m = 0.001–0.003 mM) was prepared from bovine descarboxyprothrombin by limited proteolysis with subtilisin Carlsberg. This substrate is called Fragment-Su and is composed of the amino acids 13–29 of descarboxyprothrombin.     

In vitro prothrombin synthesis from a purified precursor protein. II. Partial purification of bovine carboxylase

In this paper, we describe the isolation and partial purification of an enzyme system that converts bovine decarboxyfactor II (PIVKA-II) into prothrombin (factor II). It is shown that the increase in factor II activity occurs in parallel with 14CO2 incorporation into BaSO4 adsorbable proteins. The system is not strictly vitamin K-dependent because it is obtained from the livers of normal healthy cows. By preincubating the enzyme(s) with an excess of warfarin, an absolute vitamin K1-dependence can be obtained. The reaction is inhibited by its own product, factor II.     

Preliminary Report on the Distribution of <Emphasis Type="Italic">Callicebus oenanthe</Emphasis> on the Eastern Feet of the Andes

In 2007 we conducted a field study of almost 6 mo to determine the distribution of Callicebus oenanthe, formerly known as the Andean titi monkey. There previously has been no extensive study on the distribution and status by other fieldworkers. We visited a total of 96 localities within or around the presumed distribution of this rare primate species to determine the distribution of Callicebus oenanthe. We collected additional information on group size and threats to the species. Our expeditions revealed that the species is not endemic to the Alto Mayo Valley, as earlier authors suggested, but that its distribution extends into the Bajo Mayo and Huallaga Central. The study area is heavily deforested, and to date only one area was found where a viable population might live, although further research is needed to confirm this. The species lives in the southern part of its distribution in sympatry with another, undescribed species of Callicebus. We will continue the study to determine more precisely the distribution and conservation status of the Callicebus oenanthe, to determine if conservation measures are necessary for this species. This is the first activity of a long-term project for the conservation of Callicebus oenanthe initiated by La Vallée des Singes Primate park.     

Genetic differentiation in the urban habitat: the great tits (Parus major) of the parks of Barcelona city

The increase of urban areas has led to a fragmentation of habitats for many forest‐living species. Man‐made parks might be a solution, but they can also act as sinks that are unable to maintain themselves without immigration from natural areas. Alternatively, parks might act as true metapopulations with extinctions and colonizations. In both cases, we can expect genetic variation to be reduced in the parks compared to the natural habitat. A third alternative is that the parks have sufficient reproduction to maintain themselves. To test these hypotheses, we analysed the pattern of genetic variation in the great tit (Parus major) in 12 parks in central Barcelona, and in an adjacent forest population using microsatellites. Genetic variation was not lower in the parks compared to the forest population, but larger, and gene flow was higher from the town to the forest compared to vice versa. We found a significant genetic differentiation among the parks, with a structure that only partly reflected the geographic position of the parks. Relatedness among individuals within parks was higher than expected by chance, although we found no evidence of kin groups. Assignment tests suggest that some parks are acting as net donors of individuals to other parks. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99 , 9–19.     

Control of pH responsive peptide self-association during endocytosis is required for effective gene transfer

Cationic amphipathic histidine rich peptides demonstrate differential nucleic acid binding capabilities at neutral and acidic pH and adopt conformations at acidic pH that enable interaction with endosomal membranes, their subsequent disordering and facilitate entry of cargo to the cell cytosol. To better understand the relative contributions of each stage in the process and consequently the structural requirements of pH responsive peptides for optimal nucleic acid transfer, we used biophysical methods to dissect the series of events that occur during endosomal acidification. Far-UV circular dichroism was used to characterise the solution conformation of a series of peptides, containing either four or six histidine residues, designed to respond at differing pH while a novel application of near-UV circular dichroism was used to determine the binding affinities of the peptides for both DNA and siRNA. The peptide induced disordering of neutral and anionic membranes was investigated using (2)H solid-state NMR. While each of these parameters models key stages in the nucleic acid delivery process and all were affected by increasing the histidine content of the peptide, the effect of a more acidic pH response on peptide self-association was most notable and identified as the most important barrier to further enhancing nucleic acid delivery. Further, the results indicate that Coulombic interactions between the histidine residues modulate protonation and subsequent conformational transitions required for peptide mediated gene transfer activity and are an important factor to consider in future peptide design.