A delocalizable cationic headgroup together with an oligo-oxyethylene spacer in gemini cationic lipids improves their biological activity as vectors of plasmid DNA

Lipoplex nano-aggregates constituted of plasmid DNA (pDNA) pEGFP-C3 and mixed cationic liposomes, consisting of several percentages of a gemini cationic lipid (GCL) of the 1,2-bis(hexadecyl imidazolium) oxyethylene series, referred to as (C(16)Im)(2)(C2O)(n), with oxyethylene spacers (n = 1, 2 or 3) between the imidazolium cationic groups and the DOPE zwitterionic helper lipid, have been characterized by various biophysical and biological approaches carried out at several GCL compositions (alpha), and either the mass or the effective charge ratio of the lipoplex. The electrochemical study by zeta-potential confirms that the three GCLs yield a 10% lower effective charge than the nominal one, while compacted pDNA yields only a 25% effective negative charge. The SAXS study reveals, irrespective of the spacer length (n) and effective charge ratio (rho(eff)), the presence of two lamellar structures, i.e., one (L-alpha,L-main) in the whole GCL composition and another (L-alpha,L-DOPE,L-rich) with higher periodicity values that coexists with the previous one at low GCL composition (alpha = 0.2). The cryo-TEM analysis shows two types of multilamellar structures consisting of cationic lipidic bilayers with pDNA sandwiched between them: a cluster-type (C-type) at low alpha = 0.2 and a fingerprint-type (FP-type) at alpha >= 0.5, both with similar interlamellar spacing (d) in agreement with the L-alpha,L-main structure determined by SAXS. Transfection efficacies (TEs) of each lipid mixture were determined in four different cell lines (HEK293T, HeLa, Caco-2 and A549) at several alpha and rho(eff) values in the absence and presence of serum (FBS). The optimized formulations (alpha = 0.2 and rho(eff) = 2.0) substantially transfect cells much better than a commercial transfection reagent, Lipofectamine 2000 and previously studied efficient lipoplexes containing other cationic head groups or spacers both in the absence and presence of serum. The activity of optimized formulations may be attributed to the combination of several factors, such as: (a) the fusogenic character of DOPE which results in higher fluidity of the lipoplexes at alpha = 0.2, (b) the coexistence of two lamellar structures at alpha = 0.2 that synergizes the TE of these lipid vectors, and mainly (c) the higher biocompatibility of the GCLs reported in this work due to the presence of two imidazolium cationic groups together with an oligo-oxyethylene spacer. The length of the spacer in the GCL seems to have less impact, although (C(16)Im)(2)(C2O)(n)/DOPEpDNA lipoplexes with n = 1 and 3 show higher gene transfection than n = 2. All the optimum formulations reported herein are all highly efficient with negligible levels of toxicity, and thus, may be considered as very promising gene vectors for in vivo applications.

Expression of Bioactive Callithrix jacchus Follicle-Stimulating Hormone in Pichia pastoris

Callithrix jacchus (common marmoset) is a New World primate monkey, used as an animal model in biomedical research. Marmoset-specific follicle-stimulating hormone (FSH) preparation is required to improve superovulation protocols and to develop homologous FSH monitoring assays in these monkeys. In this study, we document the large-scale expression of recombinant marmoset FSH in methylotropic yeast, Pichia pastoris. The recombinant preparation was found to be immunologically active in Western blotting and radioimmunoassay. The preparation displayed receptor binding ability in radioreceptor assay. Based on the receptor binding ability, the yield of fermentation was estimated to be 7.2 mg/L. FSH-induced cAMP assay and estradiol assay revealed that the recombinant hormone is able to induce signal transduction. Both immunological and in vitro biological activity of marmoset FSH was found to be comparable to purified human pituitary FSH, which served as reference hormone for these assays. Thus, the study suggests that a Pichia expression system can be used for large-scale expression of bioactive recombinant marmoset FSH.

Evaluation of bioactive sphingolipids in 4-HPR-resistant leukemia cells

Background -- N-(4-hydroxyphenyl)retinamide (4-HPR, fenretinide) is a synthetic retinoid with potent pro-apoptotic activity against several types of cancer, but little is known regarding mechanisms leading to chemoresistance. Ceramide and, more recently, other sphingolipid species (e.g., dihydroceramide and dihydrosphingosine) have been implicated in 4-HPR-mediated tumor cell death. Because sphingolipid metabolism has been reported to be altered in drug-resistant tumor cells, we studied the implication of sphingolipids in acquired resistance to 4-HPR based on an acute lymphoblastic leukemia model. Methods -- CCRF-CEM cell lines resistant to 4-HPR were obtained by gradual selection. Endogenous sphingolipid profiles and in situ enzymatic activities were determined by LC/MS, and resistance to 4-HPR or to alternative treatments was measured using the XTT viability assay and annexin V-FITC/propidium iodide labeling. Results -- No major crossresistance was observed against other antitumoral compounds (i.e. paclitaxel, cisplatin, doxorubicin hydrochloride) or agents (i.e. ultra violet C, hydrogen peroxide) also described as sphingolipid modulators. CCRF-CEM cell lines resistant to 4-HPR exhibited a distinctive endogenous sphingolipid profile that correlated with inhibition of dihydroceramide desaturase. Cells maintained acquired resistance to 4-HPR after the removal of 4-HPR though the sphingolipid profile returned to control levels. On the other hand, combined treatment with sphingosine kinase inhibitors (unnatural (dihydro)sphingosines ((dh)Sph)) and glucosylceramide synthase inhibitor (PPMP) in the presence or absence of 4-HPR increased cellular (dh)Sph (but not ceramide) levels and were highly toxic for both parental and resistant cells. Conclusions -- In the leukemia model, acquired resistance to 4-HPR is selective and persists in the absence of sphingolipid profile alteration. Therapeutically, the data demonstrate that alternative sphingolipid-modulating antitumoral strategies are suitable for both 4-HPR-resistant and sensitive leukemia cells. Thus, whereas sphingolipids may not be critical for maintaining resistance to 4-HPR, manipulation of cytotoxic sphingolipids should be considered a viable approach for overcoming resistance.

Delivery of small nucleic acids by conjugation to carbohydrates and lipids as novel research and therapeutic tools

399 p. : il., graf.

Geochemistry of lipids in lacustrine sediments

An explanation of the basic premises of the subject and its terminology is given. The article then outlines aspects of research on the organic material found in lacustrine sediments, and the analytical methods involved.

Solid-state NMR study of bioactive binary borosilicate glasses

A series of binary borosilicate glasses prepared by the sol-gel method are shown to be bioactive. Tetraethyl orthosilicate (TEOS) and trimethylborate (TMB) in acidic medium are used to prepare xB(2)O(3)center dot(1-x)SiO2 glass systems for x = 0.045-0.167. The formation of a layer of apatite-like mineral on the glass surface becomes apparent after soaking in simulated body fluid for 48 h. We have measured the B-11-B-11 homonuclear second moments of the borosilicate glasses and inferred that no macroscopic phase separation occurred in our glasses. The B-11 chemical shift data also show that the formation of clustered boroxol rings is negligible in our glass system. Although the bioactivity of our borosilicate glasses is less than that of CaO-SiO2 sol-gel glasses, these simple binary systems could be taken as reference glass systems for the search of new bioactive borosilicate glasses. (C) 2008 Elsevier Ltd. All rights reserved.

Physico-chemical interactions between compartment-forming lipids and other prebiotically relevant biomolecules

Lipids are essential constituents of contemporary living cells, serving as structural molecules that are necessary to form membranous compartments. Amphiphilic lipid-like molecules may also have contributed to prebiotic chemical evolution by promoting the synthesis, aggregation and cooperative encapsulation of other biomolecules. The resulting compartments would allow systems of molecules to be maintained that represent microscopic experiments in a natural version of combinatorial chemistry. Here we address these possibilities and describe recent results related to interactions between amphiphiles and other biomolecules during early evolution toward the first living cells.

Shrimp disease management using bioactive marine secondary metabolites: an eco-friendly approach

Vibriosis caused by opportunistic and secondary bacterial pathogens is still a serious disease problem in aquaculture of the black tiger shrimp Penaeus monodon. Attempts were made for controlling shrimp bacterial disease using Marine Secondary Metabolites (MSMs). Findings indicated that the MSMs of seaweed Ulva fasciata and Dendrilla nigra are effective for controlling shrimp bacterial pathogens.

Studies on fish lipids. Pt. 2. Fatty acid composition of lipids of oil sardine (Sardinella longiceps)

Gas-liquid chromatography has been employed for the qualitative and quantitative analysis of the component fatty acids in lipids of oil sardine (Sardinella longiceps). Phospholipids and triglycerides of the lipids were previously separated by column chromatography before they were converted into the methyl esters of the fatty acids. The predominant acids present in the depot fat of the fish have been found to be C14:0=8.13%, C16:0=27.9%, C18:0=3.8%, C18:1=15.4%., C20:5=10.6% and C22:6=8.8%. Apart from the above acids the distribution of minor acids belonging to Cl8, C20 and C22 groups have also been worked out. The separated phospholipid fraction contained more than 70% polyunsaturated acids of which the important constituents were docosahexaenoic (C22:6=28%) and eicosapentaenoic (C20:5=10.6%). A marked reduction was found in the amounts of polyunsaturated acids in triglycerides, their total amount registering about 20%. This fraction recorded about 48% of C16 acids of which palmitic and palmitoleic acids amounted to 25.8% and 19.1% respectively. Occurrence of odd numbered fatty acids C15 and C17 has also been noted in the phospholipid and composite samples of the fish.