An efficient protocol for micropropagation of <Emphasis Type="Italic">Melaleuca alternifolia</Emphasis> Cheel

Melaleuca alternifolia is cultivated for the production of an essential oil useful in the cosmetic and pharmaceutical industries. Despite the economic importance of this species, there is little knowledge about its in vitro propagation. The aim of this study was to establish an efficient protocol for micropropagation of M. alternifolia. With the goal of in vitro multiplication by axillary shoot proliferation, both solid and liquid MS and WPM media were tested with supplementation with BA at 0, 0.55, 1.11, 2.22, 3.33, and 4.44 μM. The best result for shoot multiplication was obtained when either 0.55 μM BA was added into solid MS medium or 1.11 μM BA was added into liquid MS medium, with 5.6 and 11.8 shoots per explant generated, respectively. On solid or liquid WPM medium supplemented with 0.55 μM BA, the proliferation rates were 5.5 and 4.7, respectively. Three auxins (NAA, IAA, and IBA) were tested at 0.53 and 2.64 μM during the rooting stage. Several sucrose concentrations (15, 30, and 45 g L⁻¹) were compared to a sucrose-free medium. Rooting performances on four culture media were then compared: MS, half-strength MS (MS/2), MS + activated charcoal (AC), and MS/2 + AC. The results showed that auxin addition to culture medium is not necessary for in vitro rooting. Rooted microcuttings from different culture media were acclimatized in a greenhouse, and the survival percentage was evaluated. All shoots cultured in an auxin-free MS medium supplemented with sucrose (30 g L⁻¹) produced roots, and all plants survived during acclimatization. Activated charcoal added in rooting medium reduced rooting rates.     

IgE-antibody-dependent immunotherapy of solid tumors: cytotoxic and phagocytic mechanisms of eradication of ovarian cancer cells

Abs have a paramount place in the treatment of certain, mainly lymphoid, malignancies, although tumors of nonhemopoietic origin have proved more refractory ones. We have previously shown that the efficacy of immunotherapy of solid tumors, in particular ovarian carcinoma, may be improved by the use of IgE Abs in place of the conventional IgG. An IgE Ab (MOv18 IgE) against an ovarian-tumor-specific Ag (folate binding protein), in combination with human PBMC, introduced into ovarian cancer xenograft-bearing mice, greatly exceeded the analogous IgG1 in promoting survival. In this study, we analyzed the mechanisms by which MOv18 IgE may exert its antitumor activities. Monocytes were essential IgE receptor-expressing effector cells that mediated the enhanced survival of tumor-bearing mice by MOv18 IgE and human PBMC. Monocytes mediated MOv18 IgE-dependent ovarian tumor cell killing in vitro by two distinct pathways, cytotoxicity and phagocytosis, acting respectively through the IgE receptors FcepsilonRI and CD23. We also show that human eosinophils were potent effector cells in MOv18 IgE Ab-dependent ovarian tumor cell cytotoxicity in vitro. These results demonstrate that IgE Abs can engage cell surface IgE receptors and activate effector cells against ovarian tumor cells. Our findings offer a framework for an improved immunotherapeutic strategy for combating solid tumors.     

Clip-phen conjugates for the specific cleavage of nucleic acids

For the first time Clip-Phen (1) was conjugated to oligonucleotides to provide very efficient tools for the cleavage of nucleic acids at specific positions. The synthesis of the conjugates as well as the cleavage experiments are reported.     

Force spectroscopy of hyaluronan by atomic force microscopy: from hydrogen-bonded networks toward single-chain behavior

The conformational behavior of hyaluronan (HA) polysaccharide chains in aqueous NaCl solution was characterized directly at the single-molecule level. This communication reports on one of the first single-chain atomic force microscopy (AFM) experiments performed at variable temperatures, investigating the influence of the temperature on the stability of the HA single-chain conformation. Through AFM single-molecule force spectroscopy, the temperature destabilization of a local structure was proven. This structure involved a hydrogen-bonded network along the polymeric chain, with hydrogen bonds between the polar groups of HA and possibly water, and a change from a nonrandom coil to a random coil behavior was observed when increasing the temperature from 29 +/- 1 to 46 +/- 1 degrees C. As a result of the applied force, this superstructure was found to break progressively at room temperature. The use of a hydrogen-bonding breaker solvent demonstrated the hydrogen-bonded water-bridged nature of the network structure of HA single chains in aqueous NaCl solution.     

Genetic variants modify the effect of age on APOE methylation in the Genetics of Lipid Lowering Drugs and Diet Network study

Although apolipoprotein E (APOE) variants are associated with age-related diseases, the underlying mechanism is unknown and DNA methylation may be a potential one. With methylation data, measured by the Infinium Human Methylation 450 array, from 993 participants (age ranging from 18 to 87 years) in the Genetics of Lipid Lowering Drugs and Diet Network (GOLDN) study, and from Encyclopedia of DNA Elements (ENCODE) consortium, combined with published methylation datasets, we described the methylation pattern of 13 CpG sites within APOE locus, their correlations with gene expression across cell types, and their relationships with age, plasma lipids, and sequence variants. Based on methylation levels and the genetic regions, we categorized the 13 APOE CpG sites into three groups: Group 1 showed hypermethylation (> 50%) and were located in the promoter region, Group 2 exhibited hypomethylation (< 50%) and were located in the first two exons and introns, and Group 3 showed hypermethylation (> 50%) and were located in the exon 4. APOE methylation was negatively correlated with gene expression (minimum r = −0.66, P = 0.004). APOE methylation was significantly associated with age (minimum P = 2.06E-08) and plasma total cholesterol (minimum P = 3.53E-03). Finally, APOE methylation patterns differed across APOE ε variants (minimum P = 3.51E-05) and the promoter variant rs405509 (minimum P = 0.01), which further showed a significant interaction with age (P = 0.03). These findings suggest that methylation may be a potential mechanistic explanation for APOE functions related to aging and call for further molecular mechanistic studies.     

The genome sequence of the leaf-cutter ant Atta cephalotes reveals insights into its obligate symbiotic lifestyle

Leaf-cutter ants are one of the most important herbivorous insects in the Neotropics, harvesting vast quantities of fresh leaf material. The ants use leaves to cultivate a fungus that serves as the colony's primary food source. This obligate ant-fungus mutualism is one of the few occurrences of farming by non-humans and likely facilitated the formation of their massive colonies. Mature leaf-cutter ant colonies contain millions of workers ranging in size from small garden tenders to large soldiers, resulting in one of the most complex polymorphic caste systems within ants. To begin uncovering the genomic underpinnings of this system, we sequenced the genome of Atta cephalotes using 454 pyrosequencing. One prediction from this ant's lifestyle is that it has undergone genetic modifications that reflect its obligate dependence on the fungus for nutrients. Analysis of this genome sequence is consistent with this hypothesis, as we find evidence for reductions in genes related to nutrient acquisition. These include extensive reductions in serine proteases (which are likely unnecessary because proteolysis is not a primary mechanism used to process nutrients obtained from the fungus), a loss of genes involved in arginine biosynthesis (suggesting that this amino acid is obtained from the fungus), and the absence of a hexamerin (which sequesters amino acids during larval development in other insects). Following recent reports of genome sequences from other insects that engage in symbioses with beneficial microbes, the A. cephalotes genome provides new insights into the symbiotic lifestyle of this ant and advances our understanding of host-microbe symbioses.     

Targeting of T/Tn antigens with a plant lectin to kill human leukemia cells by photochemotherapy

Photochemotherapy is used both for solid tumors and in extracorporeal treatment of various hematologic disorders. Nevertheless, its development in oncology remains limited, because of the low selectivity of photosensitizers (PS) towards human tumor cells. To enhance PS efficiency, we recently covalently linked a porphyrin (TrMPyP) to a plant lectin (Morniga G), known to recognize with high affinity tumor-associated T and Tn antigens. The conjugation allowed a quick uptake of PS by Tn-positive Jurkat leukemia cells and efficient PS-induced phototoxicity. The present study was performed: (i) to evaluate the targeting potential of the conjugate towards tumor and normal cells and its phototoxicity on various leukemia cells, (ii) to investigate the mechanism of conjugate-mediated cell death. The conjugate: (i) strongly increased (×1000) the PS phototoxicity towards leukemic Jurkat T cells through an O-glycan-dependent process; (ii) specifically purged tumor cells from a 1∶1 mixture of Jurkat leukemia (Tn-positive) and healthy (Tn-negative) lymphocytes, preserving the activation potential of healthy lymphocytes; (iii) was effective against various leukemic cell lines with distinct phenotypes, as well as fresh human primary acute and chronic lymphoid leukemia cells; (iv) induced mostly a caspase-independent cell death, which might be an advantage as tumor cells often resist caspase-dependent cell death. Altogether, the present observations suggest that conjugation with plant lectins can allow targeting of photosensitizers towards aberrant glycosylation of tumor cells, e.g. to purge leukemia cells from blood and to preserve the normal leukocytes in extracorporeal photochemotherapy.     

A novel neurotrophic drug for cognitive enhancement and Alzheimer's disease

Currently, the major drug discovery paradigm for neurodegenerative diseases is based upon high affinity ligands for single disease-specific targets. For Alzheimer''s disease (AD), the focus is the amyloid beta peptide (Aß) that mediates familial Alzheimer''s disease pathology. However, given that age is the greatest risk factor for AD, we explored an alternative drug discovery scheme that is based upon efficacy in multiple cell culture models of age-associated pathologies rather than exclusively amyloid metabolism. Using this approach, we identified an exceptionally potent, orally active, neurotrophic molecule that facilitates memory in normal rodents, and prevents the loss of synaptic proteins and cognitive decline in a transgenic AD mouse model.