Synthesis and characterization of oligodeoxynucleotides containing the mutagenic base analogue 4-O-ethylthymine.

A method for the preparation of oligonucleotides containing the mutagenic base 4-O-ethylthymine is described for the first time. Use of p-nitrophenylethyl type base protecting groups together with phosphitetriester solid-phase methodology makes possible the rapid and efficient preparation of oligonucleotides bearing 4-O-ethylthymine, while standard base protecting groups are not compatible with the presence of this base. Possible applications of this methodology are discussed.     

The relevance of imidazole tautomerism for the hormonal activity of histidine-containing peptides

Substitution of 5-nitro- -histidine for -histidine is proposed as a useful tool to study the relationships among tautomerism, acid-base properties, and biological activity of peptide hormones. This approach is illustrated by an analog of the tripeptide thyroliberin, [5-nitro- -histidine]²-thyroliberin, which has been prepared by solid-phase peptide synthesis. The acid-base properties of the hormone analog and the position of the imidazole ring tautomeric equilibrium have been investigated by spectroscopic methods. Correlation of these properties with the biological activity of the nitrated tripeptide strongly supports the idea that imidazole ring tautomerism is a key factor for hormonal activity and that the N^τ-H tautomer must be considered the biologically active form of thyroliberin.     

Fusion of enveloped viruses with cells and liposomes

The fusion of viruses with cells and liposomes is reviewed with focus on the analysis of the final extents and kinetics of fusion.Influenza virus andSendai virus exhibit 100% of fusion capacity with cells at pH 5 and pH 7.5, respectively. On the other hand, there may be in certain cases, a limit on the number of virions that can fuse with a single cell, that is significantly below the limit on binding. It still remains to be resolved whether this limit reflects a limited number of possible fusion sites, or a saturation limit on the amount of viral glycoproteins that can be incorporated in the cellular membrane, like the case of virus fusion with pure phospholipid vesicles, in which the fusion products were shown to consist of a single virus and several liposomes. Both viruses demonstrate incomplete fusion activity towards liposomes of a variety of compositions. In the case ofSendai virus, fusion inactive virions bind essentially irreversibly to liposomes. Yet, preliminary results revealed that such bound, unfused virions can be released by sucrose gradient centrifugation. The separated unfused virions subsequently fuse when incubated with a “fresh” batch of liposomes. We conclude, therefore, that the fraction of initially bound unfused virions does not consist of dective particles, but rather of particles bound to liposomes via “inactive” sites. Details of the low pH inactivation of fusion capacity ofinfluenza virus towards cells and liposomes are presented. This inactivation is caused by protonation and exposure of the hydrophobic segment of HA₂, and affects primarily the fusion rate constants. Some degree of inactivation also occurs when virions are bound to cellular membranes.     

Factors controlling somatic embryogenesis

Histological and ultrastructural, molecular and elemental distribution changes were investigated during the induction of direct somatic embryogenesis using theCamellia japonica leaf culture system. In this culture system, direct somatic embryogenesis is induced in a controlled way in a specific leaf region (leaf blade) within a leaf. Embryogenic and non-embryogenic leaf regions have characteristic energy-dispersive X-ray spectra already before induction. According to these results electron probe X-ray microanalysis (EPMA) can be a tool for early diagnosis of embryogenic competence. Histological studies showed that severe fluctuations in the number of calcium oxalate crystals and in starch accumulation occur after induction but only in induced tissues. Changes in the cell wall composition of competent cells occur shortly after the induction treatment. The induction of morphogenesis is linked to the appearance of callose covering the surface cells of induced leaves and calluses. A 2^nd deposition of material (cutin) is necessary for normal somatic embryogenesis to occur. The involvement of lipid transfer proteins in the appearance of cutin in the embryogenic regions of the explant is suggested.     

Phosphitylation of Primary Carboxamides. Synthesis of Peptide-Oligonucleotide Conjugates with Acylphosphoramidate Linkages

Abstract

N-acylphosphoramidates can be obtained from the reaction of phosphitylated primary carboxamides and an alcohol in the presence of an acid catalyst such as tetrazole and subsequent oxidation. The reaction is useful for the preparation of peptide-oligonucleotide conjugates.     

Courtship Sounds Advertise Species Identity and Male Quality in Sympatric Pomatoschistus spp. Gobies

Acoustic signals can encode crucial information about species identity and individual quality. We recorded and compared male courtship drum sounds of the sand goby Pomatoschistus minutus and the painted goby P. pictus and examined if they can function in species recognition within sympatric populations. We also examined which acoustic features are related to male quality and the factors that affect female courtship in the sand goby, to determine whether vocalisations potentially play a role in mate assessment. Drums produced by the painted goby showed significantly higher dominant frequencies, higher sound pulse repetition rates and longer intervals between sounds than those of the sand goby. In the sand goby, male quality was predicted by visual and acoustic courtship signals. Regression analyses showed that sound amplitude was a good predictor of male length, whereas the duration of nest behaviour and active calling rate (i.e. excluding silent periods) were good predictors of male condition factor and fat reserves respectively. In addition, the level of female courtship was predicted by male nest behaviour. The results suggest that the frequency and temporal patterns of sounds can encode species identity, whereas sound amplitude and calling activity reflects male size and fat reserves. Visual courtship duration (nest-related behaviour) also seems relevant to mate choice, since it reflects male condition and is related to female courtship. Our work suggests that acoustic communication can contribute to mate choice in the sand goby group, and invites further study.     

Traffic of Leukocytes and Cytokine Up-regulation in the Central Nervous System in a Murine Model of Neuroparacoccidioidomycosis

Background

Paracoccidioidomycosis is the most important systemic mycosis in South America. In the last decades, it was observed that central nervous system involvement is frequent, occurring in 12.5 % of the cases. The aim of this study was to report the early inflammatory changes associated with an experimental model of neuroparacoccidioidomycosis (NPCM).

Methods

C57BL/6 mice were infected by intracranial route with 10⁶ yeast cells of PB18 strain of Paracoccidioides brasiliensis. Leukocyte–endothelium interactions were assessed by intravital microscopy 1, 2, 4, and 8 weeks post-infection (p.i.). Chemokine/cytokine levels in the brain and histopathological changes were assessed 4 and 8 weeks p.i..

Results

Intravital microscopy analysis revealed a progressive increase in leukocyte recruitment in the vessels of pia mater with a peak 4 weeks p.i. The chemokine CXCL9 was increased at 4 and 8 weeks p.i., while CCL2, CCL3, and CCL5 were increased at 8 weeks p.i. Histopathological analysis revealed the infiltration of inflammatory cells and the development of progressive granulomatous meningoencephalitis. CCL3 levels correlated with clinical manifestations of disease, as measured by the SHIRPA battery.

Conclusions

The experimental model of NPCM showed increased leukocyte recruitment associated with increased expression of chemokines and nervous tissue inflammation which correlated with clinical manifestations of disease.     

Biological individuality: the case of biofilms

This paper examines David Hull’s and Peter Godfrey-Smith’s accounts of biological individuality using the case of biofilms. Biofilms fail standard criteria for individuality, such as having reproductive bottlenecks and forming parent-offspring lineages. Nevertheless, biofilms are good candidates for individuals. The nature of biofilms shows that Godfrey-Smith’s account of individuality, with its reliance on reproduction, is too restrictive. Hull’s interactor notion of individuality better captures biofilms, and we argue that it offers a better account of biological individuality. However, Hull’s notion of interactor needs more precision. We suggest some ways to make Hull’s notion of interactor and his account of individuality more precise. Generally, we maintain that biofilms are a good test case for theories of individuality, and a careful examination of biofilms furthers our understanding of biological individuality.     

Gene delivery mediated by cationic liposomes: from biophysical aspects to enhancement of transfection

Cationic liposomes complexed with DNA have been used extensively as non-viral vectors for the intracellular delivery of reporter or therapeutic genes in culture and in vivo. However, the relationship between the features of the lipid-DNA complexes (`lipoplexes') and their mode of interaction with cells, the efficiency of gene transfer and gene expression remain to be clarified. To gain insights into these aspects, the size and zeta potential of cationic liposomes (composed of 1,2-dioleoyl-3- (trimethylammonium) propane (DOTAP) and its mixture with phosphatidylethanolamine (PE)), and their complexes with DNA at different (+/-) charge ratios were determined. A lipid mixing assay was used to assess the interaction of liposomes and lipoplexes with monocytic leukaemia cells. The use of inhibitors of endocytosis indicated that fusion of the cationic liposomes with cells occurred mainly at the plasma membrane level. However, very limited transfection of these cells was achieved using the above complexes. It is possible that the topology of the cationic liposome-DNA complexes does not allow the entry of DNA into cells through a fusion process at the plasma membrane. In an attempt to enhance transfection mediated by lipoplexes composed of DOTAP and its equimolar mixture with dioleoylphosphatidylethanolamine (DOPE) two different strategies were explored: (i) association of a targeting ligand (transferrin) to the complexes to promote their internalization, presumably by receptor-mediated endocytosis; and (ii) association of synthetic fusogenic peptides (GALA or the influenza haemagglutinin Nterminal peptide HA-2) to the complexes to promote endosomal destabilization and release of the genetic material into the cytoplasm. These strategies were effective in enhancing transfection in a large variety of cells, including epithelial and lymphoid cell lines, as well as human macrophages, especially with the use of optimized lipid/ DNA (+/-) charge ratios. Besides leading to high levels of transfection, the ternary complexes of cationic liposomes, DNA, and protein or peptide, have the advantages of being active in the presence of serum and being non-toxic. Moreover, such ternary complexes present a net negative charge and, thus, are likely to alleviate the problems associated with the use of highly positively charged complexes in vivo, such as avid complexation with serum proteins. Overall, the results indicate that these complexes, and their future derivatives, may constitute viable alternatives to viral vectors for gene delivery in vivo.