Use of trypsin for rapid and efficient purification of murine sarcoma and leukemia virus

A method is described for purification of MSV-MuLV from culture supernatant of chronically infected 78A₁ rat embryo cell line. This method involves direct polyethylene glycol-NaCl precipitation of the low speed supernatant of culture fluid followed by digestion of the pellet with trypsin. This procedure efficiently disrupts large aggregates which normally entrap most of the virus. Highly purified virus can be obtained in very good yield by a combination of sedimentation velocity and isopycnic centrifugation : yields up to 100 A₂₈₀ units (17 mg of protein) of purified virus per liter of culture fluid can be observed. This procedure appears well suited for large scale isolation of virion associated enzymatic activities.     

Androgen on the estrogen receptor I — Binding and in vivo nuclear translocation

In the immature rat uterus, high concentrations of androgens competed specifically with estradiol on the estrogen receptor (RE). This competition was stereospecific for C₁₉ steroids bearing a 17β and/or 3 hydroxyl group. Very low affinity ligands, such as testosterone, could not compete with estradiol at equilibrium but decreased the association rate of estradiol on its receptor. High doses (> 0.4mg) of 5 α aihydrotestosterone provoked $\text{in vivo}$ as $\text{in vitro}$ the nuclear translocation of RE. The nuclear receptor thus formed displayed the same 5.2 S sedimentation constant as that induced by estradiol. We conclude that the weak affinity binding of androgens to the estrogen receptor is sufficient to induce its nuclear translocation $\text{in vivo}$ provided androgen concentration is high enough in uterus to occupy the estradiol binding site. Conversely, progesterone which does not bind RE could not provoke its nuclear translocation.     

Ostracodes devoniens du Sud-Ouest de l'ile Ellesmere (archipel arctique-canadien): Première partie

On south western Ellesmere Island, near the head of Eids Fiord, the Eids Formation and thelower member of the Blue Fiord Formation, to which an Emsian age may be assigned on conodont evidence, yield large and diversified ostracode faunas. Over ninety species and subspecies are distinguished, including the fauna described by I. P. Tolmachoff (1926) from the «Series Db in the Goose Fiord area near the southern shore of the island. The present report, the first one of a series of four, deals with the families Beyrichiidae, Hollinidae, Pribylitidae, Rozhdestvenskayitidae and Youngiellidae. Twenty eight taxa, among them three new genera, eleven new species and one new subspecies, are described.     

Effect of apolipoprotein A-I lipidation on the formation and function of pre-beta and alpha-migrating LpA-I particles

A unique class of lipid-poor high-density lipoprotein, pre-beta1 HDL, has been identified and shown to have distinct functional characteristics associated with intravascular cholesterol transport. In this study we have characterized the structure/function properties of poorly lipidated HDL particles and the factors that mediate their conversion into multimolecular lipoprotein particles. Studies were undertaken with homogeneous recombinant HDL particles (LpA-I) containing apolipoprotein (apo) A-I and various amounts of palmitoyloleoylphosphatidylcholine (PC) and cholesterol. Complexation of apoA-I with small amounts of PC and cholesterol results in the formation of discrete lipoprotein structures that have a hydrated diameter of about 6 nm but contain only one molecule of apoA-I (Lp1A-I). While the molecular charge and alpha-helix content of apoA-I are unaffected by lipidation, the thermodynamic stability of the protein is reduced significantly (from 2.4 to 0.9 kcal/mol of apoA-I). Evaluation of apoA-I conformation by competitive radioimmunoassay with monoclonal antibodies shows that addition of small amounts of PC and cholesterol to apoA-I significantly increases the immunoreactivity of a number of domains over the entire molecule. Increasing the ratio of PC:apoA-I to 10:1 in the Lp1A-I complex is associated with increases in the alpha-helix content and stability of apoA-I. However, incorporation of 10-15 mol of PC destabilizes the Lp1A-I complex and promotes the formation of more thermodynamically stable (1.8 kcal/mol of apoA-I) bimolecular structures (Lp2A-I) that are approximately 8 nm in diameter. The formation of an Lp2A-I particle is associated with an increased immunoreactivity of most of the epitopes studied, with the exception of one central domain (residues 98-121), which becomes significantly less exposed. This structural change parallels a significant increase in the net negative charge on the complex. Characterization of the ability of these lipoproteins to act as substrates for lecithin:cholesterol acyltransferase (LCAT) shows that unstable Lp1A-I complexes stimulate a higher rate of cholesterol esterification by LCAT than the small but more stable Lp2A-I particles (Vmax values are 5.8 and 0.3 nmol of free cholesterol esterified/h, respectively). The ability of LCAT to interact with lipid-poor apoA-I suggests that LCAT does not need to bind to the lipid interface on an HDL particle but that LCAT may directly interact with apoA-I. The data suggests that lipid-poor HDL particles may be metabolically reactive particles because they are thermodynamically unstable.     

Fine-scale structure and cross-taxon congruence of bird and beetle assemblages in an old-growth boreal forest mosaic

Aim Nestedness occurs when species present in depauperate sites are subsets of those found in species‐rich sites. The degree of congruence of site nestedness among different assemblages can inform commonalities of mechanisms structuring the assemblages. Well‐nested assemblages may still contain idiosyncratic species and sites that notably depart from the typical assemblage pattern. Idiosyncrasy can arise from multiple processes, including interspecific interactions and habitat preferences, which entail different consequences for species co‐occurrences. We investigate the influence of fine‐scale habitat variation on nestedness and idiosyncrasy patterns of beetle and bird assemblages. We examine community‐level and pairwise species co‐occurrence patterns, and highlight the potential influence of interspecific interactions for assemblage structure. Location Côte‐Nord region of Québec, Canada. Methods We sampled occurrences of ground‐dwelling beetles, flying beetles and birds at sites within old‐growth boreal forest. We examined the nestedness and idiosyncrasy of sites and sought relationships to habitat attributes. We analysed non‐random species co‐occurrence patterns at pairwise and community levels, using null model analysis and five ‘association’ indices. Results All three assemblages were significantly nested. There was limited congruence only between birds and flying beetles whose nestedness was related to canopy openness. For ground‐dwelling beetles, nestedness was related to high stand heterogeneity and sapling density, whereas site idiosyncrasy was inversely related to structural heterogeneity. For birds, site idiosyncrasy increased with canopy cover, and most idiosyncratic species were closed‐canopy specialists. In all assemblages, species idiosyncrasy was positively correlated with the frequency of negative pairwise associations. Species co‐occurrence patterns were non‐random, and for flying beetles and birds positive species pairwise associations dominated. Community‐level co‐occurrence summaries may not, however, always reflect these patterns. Main conclusions Nestedness patterns of different assemblages may not correlate, even when sampled at common locations, because of different responses to local habitat attributes. We found idiosyncrasy patterns indicating opposing habitat preferences, consistent with antagonistic interactions among species within assemblages. Analysis of such patterns can thus suggest the mechanisms generating assemblage structures, with implications for biodiversity conservation.     

Coencapsulation of irinotecan and floxuridine into low cholesterol-containing liposomes that coordinate drug release in vivo

A liposomal delivery system that coordinates the release of irinotecan and floxuridine in vivo has been developed. The encapsulation of floxuridine was achieved through passive entrapment while irinotecan was actively loaded using a novel copper gluconate/triethanolamine based procedure. Coordinating the release rates of both drugs was achieved by altering the cholesterol content of distearoylphosphatidylcholine (DSPC)/distearoylphosphatidylglycerol (DSPG) based formulations. The liposomal retention of floxuridine in plasma after intravenous injection was dramatically improved by decreasing the cholesterol content of the formulation below 20 mol%. In the case of irinotecan, the opposite trend was observed where increasing cholesterol content enhanced drug retention. Liposomes composed of DSPC/DSPG/Chol (7:2:1, mole ratio) containing co-encapsulated irinotecan and floxuridine at a 1:1 molar ratio exhibited matched leakage rates for the two agents so that the 1:1 ratio was maintained after intravenous administration to mice. The encapsulation of irinotecan was optimal when copper gluconate/triethanolamine (pH 7.4) was used as the intraliposomal buffer. The efficiency of irinotecan loading was approximately 80% with a starting drug to lipid molar ratio of 0.1/1. Leakage of floxuridine from the liposomes during irinotecan loading at 50 degrees C complicated the ability to readily achieve the target 1:1 irinotecan/floxuridine ratio inside the formulation. As a result, a procedure for the simultaneous encapsulation of irinotecan and floxuridine was developed. This co-encapsulation method has the advantage over sequential loading in that extrusion can be performed in the absence of chemotherapeutic agents and the drug/drug ratios in the final formulation can be more precisely controlled.     

Membrane Rafts Are Involved in Intracellular Miconazole Accumulation in Yeast Cells

Azoles inhibit ergosterol biosynthesis, resulting in ergosterol depletion and accumulation of toxic 14α-methylated sterols in membranes of susceptible yeast. We demonstrated previously that miconazole induces actin cytoskeleton stabilization in Saccharomyces cerevisiae prior to induction of reactive oxygen species, pointing to an ancillary mode of action. Using a genome-wide agar-based screening, we demonstrate in this study that S. cerevisiae mutants affected in sphingolipid and ergosterol biosynthesis, namely ipt1, sur1, skn1, and erg3 deletion mutants, are miconazole-resistant, suggesting an involvement of membrane rafts in its mode of action. This is supported by the antagonizing effect of membrane raft-disturbing compounds on miconazole antifungal activity as well as on miconazole-induced actin cytoskeleton stabilization and reactive oxygen species accumulation. These antagonizing effects point to a primary role for membrane rafts in miconazole antifungal activity. We further show that this primary role of membrane rafts in miconazole action consists of mediating intracellular accumulation of miconazole in yeast cells.     

Cloning and characterization of the durable tomato mosaic virus resistance gene Tm-22 from Lycopersicon esculentum

In tomato, infections by tomato mosaic virus are controlled by durable Tm-2² resistance. In order to gain insight into the processes underlying disease resistance and its durability, we cloned and analysed the Tm-2² resistance gene and the susceptible allele, tm-2. The Tm-2² gene was isolated by transposon tagging using a screen in which plants with a destroyed Tm-2² gene survive. The Tm-2² locus consists of a single gene that encodes an 861 amino acid polypeptide, which belongs to the CC-NBS-LRR class of resistance proteins. The putative tm-2 allele was cloned from susceptible tomato lines via PCR with primers based on the Tm-2² sequence. Interestingly, the tm-2 gene has an open reading frame that is comparable to the Tm-2² allele. Between the tm-2 and the Tm-2² polypeptide 38 amino acid differences are present of which 26 are located in the second half of the LRR-domain. Susceptible tomato plants, which were transformed with the Tm-2² gene, displayed resistance against ToMV infection. In addition, virus specificity, displayed by the Tm-2² resistance was conserved in these transgenic lines. To explain the durability of this resistance, it is proposed that the Tm-2²-encoded resistance is aimed at the Achilles' heel of the virus.