Amphipathic peptides and drug delivery

The discovery of cell-penetrating peptides as gene delivery systems and the interest in the mechanism by which these vectors cross the cell membrane have generated a large number of studies. Among the parameters involved in the translocation process, controversy has arisen about the role of the amphipathicity of the carriers in the interaction and reorganization of the cell membrane. In this review we have summarized the vectors with primary or secondary amphipathicity related to secondary structure. Some of the insights into the relationship between the aggregation state of the peptide at the concentrations used for internalization studies and its interaction with the cell membrane result from our contribution to the field with a new family of amphipathic proline-rich peptides.     

Conjugation of meningococcal lipooligosaccharides through their lipid A terminus conserves their inner epitopes and results in conjugate vaccines having improved immunological properties

The importance of conserved inner saccharide epitopes to the immune performance of meningococcal lipooligosaccharide-protein conjugate vaccines was demonstrated in the following experiments. Two different oligosaccharides were obtained by chemical degradations of the same L7 lipooligosaccharide, and both were linked terminally to tetanus toxoid. One was a truncated oligosaccharide in which the inner epitopes were incomplete and was obtained by mild acid hydrolysis of the L7 lipooligosaccharide. This oligosaccharide was conjugated by direct reductive amination through its newly exposed terminal Kdo residue. The second, a full-length oligosaccharide, was obtained by O-deacylation of the L7 lipooligosaccharide, with subsequent removal of phosphate substituents from its lipid A moiety using alkaline phosphatase. This permitted the full-length oligosaccharide to be conjugated directly to tetanus toxoid by reductive amination through its newly exposed terminal 2-N-acyl-2-deoxy-D-glucopyranose residue. Comparison of the immune performance of the two conjugates in mice revealed, that while both were able to induce significant levels of L7-lipooligosaccharide-specific IgG antibody, the conjugate made with the full-length saccharide was able to induce antibodies with increased bactericidal activity against homologous meningococci.     

Fungal chitin-glucan derivatives exert protective or damaging activity on plasmid DNA

Water-soluble derivatives of the chitin-glucan (Ch-G) complex isolated from the fungal mycelium of the industrial strain of Aspergillus niger have been previously shown to possess potent antimutagenic protective activity in vivo. Their direct action on DNA has not been yet evaluated. Using carboxymethylation, sulfoethylation and subsequent ultrasonic treatment, lower molecular weight water-soluble derivatives were obtained from the crude fungal Ch-G. The biological effects of the prepared compounds were evaluated in direct interaction on plasmid DNA in vitro. Monitoring of electrophoretic mobility of different conformers of plasmid DNA implied that carboxymethyl chitin-glucan (CM-Ch-G) induced single- and double-strand breaks into supercoiled DNA in a concentration-dependent manner. On the other hand, sulfoethyl chitin-glucan (SE-Ch-G) alone did not induce any DNA breaks in plasmid DNA. However, process of DNA damaging induced by free-radical oxidation initiated with Fe(2+) was inhibited, while the process of DNA breakage induced by H(2)O(2) was increased in the presence of SE-Ch-G.     

Dinucleosome DNA of human K562 cells: experimental and computational characterizations

Dinucleosome formation is the first step in the organization of the higher order chromatin structure. With the ultimate aim of elucidating the dinucleosome structure, we constructed a library of human dinucleosome DNA. The library consists of PCR-amplifiable DNA fragments obtained by treatment of nuclei of erythroid K562 cells with micrococcal nuclease followed by extraction of DNA and adaptor ligation to the blunt-ended DNA fragments. The library was then cloned using a plasmid vector and the sequences of the clones were determined. The dominating clones containing the Alu elements were removed. A total of 1002 clones, which comprised a dinucleosome database, contained 84 and 918 clones from the clones before and after removing Alu elements, respectively. Approximately 70% of the clones were between 300 and 400 bp in size and they were distributed to various locations of all chromosomes except the Y chromosome. The clones containing A(2)N(8)A(2)N(8)A(2) or T(2)N(8)T(2)N(8)T(2) sequences were classified into three types, Type I (N shape), Type II (V shape) and Type III (M shape) according to DNA curvature plots. The locations of experimentally determined curved DNA segments matched well with the calculated ones though the clones of Types I and III showed additional curved DNA segments as revealed by the curvature plots. The distributions of complementary dinucleotides in the nucleosome DNA, at the ends of the dinucleosome DNA clones, allowed us to predict the positions of the nucleosome dyad axis, and estimate the size of the nucleosome core DNA, 125nt. The distributions of AA and TT dinucleotides, as well as other RR and YY dinucleotides, showed a periodicity with an average period of 10.4 bases, close to the values observed before. Mapping of nucleosome positions in the dinucleosome database based on the observed periodicity revealed that the nucleosomes were separated by a linker of 7.5+ approximately 10 x n nt. This indicates that the nucleosome-nucleosome orientations are, typically, halfway between parallel and antiparallel. Also an important finding is that the distributions of AA/TT and other RR/YY dinucleotides, apparently, reflect both DNA curvature and DNA bendability, cooperatively contributing to the nucleosome formation.     

Antigenotoxic potential of glucomannan on four model test systems

Antimutagenic, anticlastogenic, and bioprotective effect of polysaccharide glucomannan (GM) isolated fromCandida utilis was evaluated in four model test systems. The antimutagenic effect of GM against 9-aminoacridine (9-AA)- and sodium azide (NaN₃)-induced mutagenicity was revealed in theSalmonella typhimurium strains TA97 and TA100, respectively. GM showed anticlastogenic effect against N-nitroso-N-methylurea (NMU) induced chromosome aberrations in theVicia sativa assay. The bioprotective effect of GM co-treated with methyl-methane-sulphonate (MMS) was also established inChlamydomonas reinhardtii repair deficient strainsuvs10 anduvs14. The statistically significant antimutagenic potential of GM was not proved against 4-nitro-quinoline-1-oxide (4-NQO)-induced mutagenicity inSaccharomyces cerevisiae D7 assay. It may be due to bioprotectivity of -mannan and -glucan, which are integral part ofS. cerevisiae cell walls. Due to the good water solubility, low molecular weight (30 kDa), antimutagenic/anticlastogenic, and bioprotective activity against chemical compounds differing in mode of action, GM appears to be a promising natural protective (antimutagenic) agent.     

The <Emphasis Type="Italic"> GATE </Emphasis>retrotransposon in<Emphasis Type="Italic"> Drosophila melanogaster</Emphasis>: mobility in heterochromatin and aspects of its expression in germline tissues

A full-length copy of the retrotransposon GATE was identified as an insertion in the tandemly repeated, heterochromatic, Stellate genes, which are expressed in the testis of Drosophila melanogaster. Sequencing of this heterochromatic GATE copy revealed that it is closely related to the BEL retrotransposon, a representative of the recently defined BEL-like group of LTR retrotransposons. This copy contains identical LTRs, indicating that the insertion is a recent event. By contrast, the euchromatic part of the D. melanogaster genome contains only profoundly damaged GATE copies or fragments of the transposon. The preferential localization of GATE sequences in heterochromatin was confirmed for the other species in the melanogaster subgroup. The level of GATE expression is dramatically increased in ovaries, but not in testes, of spn-E(1) homozygous flies. We speculate that spn-E is involved in the silencing of GATE via an RNA interference mechanism.     

PML-RARA-targeted DNA vaccine induces protective immunity in a mouse model of leukemia

Despite improved molecular characterization of malignancies and development of targeted therapies, acute leukemia is not curable and few patients survive more than 10 years after diagnosis. Recently, combinations of different therapeutic strategies (based on mechanisms of apoptosis, differentiation and cytotoxicity) have significantly increased survival. To further improve outcome, we studied the potential efficacy of boosting the patient's immune response using specific immunotherapy. In an animal model of acute promyelocytic leukemia, we developed a DNA-based vaccine by fusing the human promyelocytic leukemia-retinoic acid receptor-alpha (PML-RARA) oncogene to tetanus fragment C (FrC) sequences. We show for the first time that a DNA vaccine specifically targeted to an oncoprotein can have a pronounced effect on survival, both alone and when combined with all-trans retinoic acid (ATRA). The survival advantage is concomitant with time-dependent antibody production and an increase in interferon-gamma (IFN-gamma). We also show that ATRA therapy on its own triggers an immune response in this model. When DNA vaccination and conventional ATRA therapy are combined, they induce protective immune responses against leukemia progression in mice and may provide a new approach to improve clinical outcome in human leukemia.     

Protective effect of the yeast glucomannan against cyclophosphamide-induced mutagenicity.

Glucomannan (GM) isolated from Candida utilis with molecular weight 30 kDa was administered either intraperitoneally or orally prior to cyclophosphamide (CP) injection and its effect on the frequency of micronuclei was evaluated in polychromatic erythrocytes of mouse bone marrow. GM administration by either route decreased significantly (p<0.002) the clastogenic effect of CP. The protective effect was concentration-dependent, with a higher decrease achieved by 200 mg/kg than by 100 mg/kg b. wt. (body weight). The fact that GM was effective also at oral administration is indicative of the passage of GM molecules through the wall of the gastrointestinal tract. The important characteristics of GM isolated from C. utilis, such as good water solubility, relatively small molecular weight (30 kDa), and antimutagenic effect exerted also at oral administration, appear to be promising features for its prospective use as a natural protective agent.     

In vitro formation of resting spores by the insect pathogenic fungus Entomophaga maimaiga

Field-collected resting spores (azygospores) of the fungal pathogen of Lymantria dispar (gypsy moth), Entomophaga maimaiga, have been used to release this biological control agent in areas where this pathogen is not established. We have found that E. maimaiga can produce resting spores in vitro using Grace's insect tissue culture medium (95%) plus fetal bovine serum (5%). The majority of spores become mature between 7 and 21 days after cultures are initiated. Spore production varies by fungal isolate; of 38 isolates tested, 10 produced no resting spores while 7 produced >1000 resting spores/ml. Resting spore production was not affected when isolates were mixed. Glycerol (used for fungal storage), trehalose, and selected amino acids each inhibited resting spore formation. Fetal bovine serum was required for spore production but the presence of >5% yielded lower resting spore densities. A large surface area:volume ratio (12.5 cm(2):ml versus 4.2 cm(2):ml) was required for abundant formation of resting spores. At present, resting spores have only been produced in small volumes with a maximum of 3 x 10(4) resting spores/ml.