Multifunctional liposomes for nasal delivery of the anti-Alzheimer drug tacrine hydrochloride

The purpose of this study was the development of multifunctional liposomes for nasal administration of tacrine hydrochloride. Liposomes were prepared using traditional excipients (cholesterol and phosphatidylcholine), partly enriched with α-tocopherol and/or Omega3 fatty acids. This approach was chosen in order to obtain at the same time two positive results: an enhanced drug permeation through nasal mucosa and a concomitant neuroprotective effect. Several liposome formulations were prepared using the Reverse Phase Evaporation technique followed by membrane filter extrusion. In particular, liposome capacity to enhance drug permeation was evaluated by means of membrane permeation and cellular uptake studies. Furthermore, liposome effect on neuronal viability and intracellular ROS production was evaluated as well as their cytoprotective effect against oxidative stress. All liposome formulations showed a mean diameter in the range of 175?nm to 219?nm with polydispersity index lower than 0.22, a lightly negative zeta potential and excellent encapsulation efficiency. Moreover, along with good mucoadhesive properties, multifunctional liposomes showed a markedly increase in tacrine permeability, which can be related to liposome fusion with cellular membrane, a hypothesis, which was also supported by cellular uptake studies. Finally, the addition of α-tocopherol without Omega3 fatty acids, was found to increase the neuroprotective activity and antioxidant properties of liposomes.     

Tetrastylis ovalis: A second case of bat-pollinated passionflower (Passifloraceae)

The floral biology ofTetrastylis ovalis and its pollination by bats was studied in southeastern Brazil. The flowers ofT. ovalis seem more fitted to bat-pollination than those ofPassiflora mucronata, the other known chiropterophilous passionflower. Two species of bats were observed visiting flowers ofT. ovalis: the phyllostomidsGlossophaga soricina andRhinophylla pumilio. Due to the attributes ofTetrastylis, its presumed primitiveness and a better defined bat-pollination syndrome we suggest that chiropterophily evolved independently in the former genus and inPassiflora.     

Different structure of polytene chromosomes ofPhaseolus coccineus suspensors during early embryogenesis

Summary The pattern of DNA and RNA puffs in pair VII of polytene chromosomes has been investigated in the suspensor ofPhaseolus coccineus during early embryo development. The pattern of³H-TdR and³H-U incorporation has been also detected. Collected data indicate that: 1. both heterochromatic regions, p₁₁ and q_(111+112), of chromosome pair VII, organize large DNA puffs; 2. DNA puffs of both regions are specific of different embryo differentiation steps; 3. a seasonal influence on the DNA puffing seems also to be present, as demonstrated by the comparison of the results collected in two different crops; 4. the incorporation experiment by³H-TdR evidences that not all DNA puffs show clustered labeling; 5. the RNA puffing of the two regions seems also to be specific of determined embryo stages.     

In vitro mimicry of CaMBr1 tumor-associated antigen by synthetic oligosaccharides

The murine monoclonal antibody (Mab) MBr1, raised against thebreast cancer cell line MCF7, recognizes a saccharidic epitopeoverexpressed on a high percentage of human breast, ovary, andlung carcinomas. This antigen was originally identified on theimmunogen as a globo-series glycosphingolipid with an H-likedeterminant at its terminus (globo-H). We report here the biologicalcharacterization of the entire globo-H hexasaccharide and fivesynthetic oligosaccharides representing fragments of the entirestructure andlor different anomeric configurations. Using competitivebinding assays on live cells, we identified the residues andthe linkages essential for mimicry of the cellular antigensrecognized by Mab MBr1 on the breast carcinoma cell line MCF7and small cell lung cancer cell line POVD. The terminal tetrasaccharidicfragment of globo-H is the oligosaccharide that most resemblesthe MBr1-defined epitope both on glycolipids and on glycoproteins.This information will help in the rational design of a highlyspecific reagent for active specific immunotherapy of carcinomasoverexpressing the MBr1-defined antigen. CaMBr1 immunotherapy monoclonal antibody oligosaccharides tumor-associated antigen     

The Evolution of MHC Diversity by Segmental Duplication and Transposition of Retroelements

Sequence analysis of a 237 kb genomic fragment from the central region of the MHC has revealed that the HLA-B and HLA-C genes are contained within duplicated segments peri-B (53 kb) and peri-C (48 kb), respectively, and separated by an intervening sequence (IF) of 30 kb. The peri-B and peri-C segments share at least 90% sequence homology except when interrupted by insertions/deletions including Alu, L1, an endogenous retrovirus, and pseudogenes. The sequences of peri-B, IF, and peri-C were searched for the presence of Alu elements to use as markers of evolution, chromosomal rearrangements, and polymorphism. Of 29 Alu elements, 14 were identified in peri-B, 11 in peri-C, and 4 in IF. The Alu elements in peri-B and peri-C clustered phylogenetically into two clades which were classified as ``preduplication' and ``postduplication' clades. Four Alu J elements that are shared by peri-B and peri-C and are flanked by homologous sequences in their paralogous locations, respectively, clustered into a ``preduplication' clade. By contrast, the majority of Alu elements, which are unique to either peri-B or peri-C, clustered into a postduplication clade together with the Alu consensus subfamily members ranging from platyrrhine-specific (Spqxcg) to catarrhine-specific Alu sequences (Y). The insertion of platyrrhine-specific Alu elements in postduplication locations of peri-B and peri-C implies that these two segments are the products of a duplication which occurred in primates prior to the divergence of the New World primate from the human lineage (35–44 mya). Examination of the paralogous Alu integration sites revealed that 9 of 14 postduplication Alu sequences have produced microsatellites of different length and sequence within the Alu 3′-poly A tail. The present analysis supports the hypothesis that HLA-B and HLA-C genes are products of an extended segmental duplication between 44 and 81 million years ago (mya), and that subsequent diversification of both genomic segments occurred because of the mobility and mutation of retroelements such as Alu repeats. Received: 21 May 1997 / Accepted: 9 July 1997     

Sister chromatid exchanges in Drosophila melanogaster cell lines in vitro

The frequency of sister chromatid exchanges (SCEs) in two cell lines of Drosophila melanogaster with different karyotypes (XX and XY) was determined, considering (1) the distribution of SCEs within each chromosome, with reference to eu- and heterochromatin and (2) the distribution of SCEs in different chromosomes. A comparison was made between chromosome pairs within each karyotype and between the two different karyotypes. The following results were obtained. The SCEs are not randomly distributed along chromosomes, since exchanges were never observed in heterochromatin. SCEs are more frequent in XY than in XX cells; moreover, in both cell types there exists a significantly higher frequency of SCEs in the X chromosome than in the autosomes. These findings are discussed in relation to chromosome aberrations and mitotic recombination.     

Vacuolar targeting of recombinant antibodies in Nicotiana benthamiana

Plant‐based platforms are extensively used for the expression of recombinant proteins, including monoclonal antibodies. However, to harness the approach effectively and leverage it to its full potential, a better understanding of intracellular processes that affect protein properties is required. In this work, we examined vacuolar (vac) targeting and deposition of the monoclonal antibody (Ab) 14D9 in Nicotiana benthamiana leaves. Two distinct vacuolar targeting signals (KISIA and NIFRGF) were C‐terminal fused to the heavy chain of 14D9 (vac‐Abs) and compared with secreted and ER‐retained variants (sec‐Ab, ER‐Ab, respectively). Accumulation of ER‐ and vac‐Abs was 10‐ to 15‐fold higher than sec‐Ab. N‐glycan profiling revealed the predominant presence of plant typical complex fucosylated and xylosylated GnGnXF structures on sec‐Ab while vac‐Abs carried mainly oligomannosidic (Man 7‐9) next to GnGnXF forms. Paucimannosidic glycans (commonly assigned as typical vacuolar) were not detected. Confocal microscopy analysis using RFP fusions showed that sec‐Ab‐RFP localized in the apoplast while vac‐Abs‐RFP were exclusively detected in the central vacuole. The data suggest that vac‐Abs reached the vacuole by two different pathways: direct transport from the ER bypassing the Golgi (Ab molecules containing Man structures) and trafficking through the Golgi (for Ab molecules containing complex N‐glycans). Importantly, vac‐Abs were correctly assembled and functionally active. Collectively, we show that the central vacuole is an appropriate compartment for the efficient production of Abs with appropriate post‐translational modifications, but also point to a reconsideration of current concepts in plant glycan processing.