Influence of liposomal local anesthetics on platelet aggregation in vitro

We assessed the effect of local anesthetics (LA) from different families such as esters (benzocaine), linear aminoamides (lidocaine) and cyclic aminoamides (bupivacaine) on the platelet aggregation induced by ADP. Liposomal formulations of the three LA, prepared with egg phosphatidylcholine:cholesterol alpha-tocopherol, were also tested. The three LA were able to inhibit platelet aggregation induced by ADP, in the following order: bupivacaine > lidocaine > benzocaine. After encapsulation into liposomes the inhibitory effect increased for all anesthetics studied, showing that aggregation tests could be used to assess the toxicity of new drug formulations.     

Enantiomeric resolution of kielcorin derivatives by HPLC on polysaccharide stationary phases using multimodal elution

Analytical HPLC methods using carbamate chiral stationary phases of polysaccharide derivatives were developed for the enantiomeric resolution of five racemic mixtures of xanthonolignoids: rac-trans-kielcorin C, rac-cis-kielcorin C, rac-trans-kielcorin D, rac-trans-isokielcorin D, and rac-trans-kielcorin E. The separations were evaluated with the stationary phases cellulose tris-3,5-dimethylphenylcarbamate, amylose tris-3,5-dimethylphenylcarbamate, amylose tris-(S)-1-phenylethylcarbamate, and amylose tris-3,5-dimethoxyphenylcarbamate under normal, reversed-phase, and polar organic elution conditions. Chiral recognition of those chiral stationary phases, the influence of mobile phases on the enantiomers separation, and the effects of structural features of the solutes on the chiral discrimination observed are discussed. The best performance was achieved on an amylose tris-3,5-dimethylphenylcarbamate phase. Polar organic conditions gave shorter retention factors and better resolutions and were a valuable alternative to the alcohol-hexane or reversed-phase conditions.     

Reactive phenotypes after acute and chronic NK-cell activation

Several phenotypic changes have been shown to occur after NK-cell stimulation, involving molecules that have been proved to regulate NK-cell migration into tissues and NK-cell activation and proliferation as well as target cell recognition and killing. Here, we review the reactive phenotypes observed in vivo after acute and chronic NK-cell activation.     

Relationships in subtribe Diocleinae (Leguminosae; Papilionoideae) inferred from internal transcribed spacer sequences from nuclear ribosomal DNA

The complete sequences of nuclear ribosomal DNA (nrDNA) internal transcribed spacer regions (ITS/5.8S) were determined for species belonging to six genera from the subtribe Diocleinae as well as for the anomalous genera Calopogonium and Pachyrhizus. Phylogenetic trees constructed by distance matrix, maximum parsimony and maximum likelihood methods showed that Calopogonium and Pachyrhizus were outside the clade Diocleinae (Canavalia, Camptosema, Cratylia, Dioclea, Cymbosema, and Galactia). This finding supports previous morphological, phytochemical, and molecular evidence that Calopogonium and Pachyrhizus do not belong to the subtribe Diocleinae. Within the true Diocleinae clade, the clustering of genera and species were congruent with morphology-based classifications, suggesting that ITS/5.8S sequences can provide enough informative sites to allow resolution below the genus level. This is the first evidence of the phylogeny of subtribe Diocleinae based on nuclear DNA sequences.     

Farnesyl protein transferase inhibitors targeting the catalytic zinc for enhanced binding

Successful efforts to make farnesyl transferase (FT) inhibitors with appropriately tethered ligands designed to interact with a catalytic zinc that exist in the enzyme have been realized. Thus, by introducing either a pyridylmethylamino or propylaminolimidazole amide moieties off the 2-position of the piperidine ring, FT inhibitors with activities in the picomolar range have been achieved as exemplified by compounds 12a and 12b. An X-ray structure of 11b bound to FT shows the enhanced activity is a result of interacting with the active-site zinc.     

Involvement of peptidorhamnomannan in the interaction of Pseudallescheria boydii and HEp2 cells

Pseudallescheria boydii is an emerging fungal pathogen that has a worldwide distribution. Virulence mechanisms of P. boydii are largely unknown. We studied the interaction between P. boydii and HEp2 cells and demonstrated that conidia of P. boydii attached to, and were ingested by, HEp2 cells in a time-dependent process. After 2 h of interaction, the conidia produced a germ-tube like projection, which was able to penetrate the epithelial cell membrane. Recently, our group characterized a peptidorhamnomannan (PRM) antigen on the cell surface of P. boydii. In order to better understand the role played by this surface glycoconjugate during cell adhesion and endocytosis, inhibition assays were performed using intact PRM and anti-PRM polyclonal antibody. When HEp2 cells were pre-treated with whole PRM molecule, the adhesion and endocytic indices were, respectively, 50% and 60% lower than in non-treated epithelial cells. Moreover, when the conidial cells were pre-incubated with anti-PRM antibodies, the adherence and endocytosis processes were inhibited in a dose-dependent manner. As PRM influenced the conidia P. boydii-HEp2 cell interaction, we also performed inhibition assays in order to observe which PRM moieties could be involved in this process. Treatment of PRM with proteinase K promoted a slight inhibition of adhesion. However, the de-O-glycosylated PRM molecule as well as the monosaccharide mannose was able to efficiently inhibit the adhesion and endocytic processes. In addition, our results indicate for the first time that P. boydii PRM binds to a polypeptide of 25 kDa on the HEp2 cell surface.     

Hidden biodiversity of the extremophilic Cyanidiales red algae

The Cyanidiales is a group of asexual, unicellular red algae, which thrive in acidic and high temperature conditions around hot springs. These unicellular taxa have a relatively simple morphology and are currently classified into three genera, Cyanidium, Cyanidioschyzon and Galdieria. Little is known, however, about the biodiversity of Cyanidiales, their population structure and their phylogenetic relationships. Here we used a taxonomically broadly sampled three-gene data set of plastid sequences to infer a robust phylogenetic framework for the Cyanidiales. The phylogenetic analyses support the existence of at least four distinct Cyanidiales lineages: the Galdieria spp. lineage (excluding Galdieria maxima), the Cyanidium caldarium lineage, a novel monophyletic lineage of mesophilic Cyanidium spp. and the Cyanidioschyzon merolae plus Galdieria maxima lineage. Our analyses do not support the notion of a mesophilic ancestry of the Cyanidiales and suggest that these algae were ancestrally thermo-acidotolerant. We also used environmental polymerase chain reaction (PCR) for the rbcL gene to sample Cyanidiales biodiversity at five ecologically distinct sites at Pisciarelli in the Phlegrean Fields in Italy. This analysis showed a high level of sequence divergence among Cyanidiales species and the partitioning of taxa based on environmental conditions. Our research revealed an unexpected level of genetic diversity among Cyanidiales that revises current thinking about the phylogeny and biodiversity of this group. We predict that future environmental PCR studies will significantly augment known biodiversity that we have discovered and demonstrate the Cyanidiales to be a species-rich branch of red algal evolution.     

Effect of abietane diterpenes from Plectranthus grandidentatus on T- and B-lymphocyte proliferation

Five known abietane diterpenes of the royleanone and coleon type, namely, fatty acid esters of 7alpha-acyloxy-6beta-hydroxyroyleanone (1), grandidone A (2), 7alpha-acetoxy-6beta-hydroxyroyleanone (3), 6beta,7alpha-dihydroxyroyleanone (4) and coleon U (5), isolated from Plectranthus grandidentatus, were evaluated for their effect on the proliferation of human lymphocytes induced by the mitogen PHA. All except 4, showed a dose-dependent suppressor effect, with 3 yielding the most potent antiproliferative activity, followed by 5. These two compounds, that represent diterpenes of the royleanone and coleon type respectively, were also shown to be potent inhibitors of mouse splenocyte proliferation induced by ConA or LPS mitogens. However, the sensitivity of ConA-stimulated splenocytes to their suppressive effect was higher, suggesting a preferential inhibition of T-lymphocyte proliferation. The antiproliferative activity of 3 seemed to be exerted without affecting the expression of the lymphocyte activation marker CD69. On the contrary, 5 was shown to reduce the expression of CD69 of TCD8(+) and B-cells, suggesting a relationship between its antiproliferative effect and the expression of this early marker of activation on these cell populations. The capacity of 5 to induce apoptosis on ConA-stimulated splenocytes could also be related with its antiproliferative activity.     

Induction of CD8+ T cells to an HIV-1 antigen through a prime boost regimen with heterologous E1-deleted adenoviral vaccine carriers

E1-deleted adenoviral recombinants most commonly based on the human serotype 5 (AdHu5) have been shown thus far to induce unsurpassed transgene product-specific CD8(+) T cell responses. A large percentage of the adult human population carries neutralizing Abs due to natural exposures to AdHu5 virus. To circumvent reduction of the efficacy of adenovirus (Ad) vector-based vaccines by neutralizing Abs to the vaccine carrier, we developed E1-deleted adenoviral vaccine carriers based on simian serotypes. One of these carriers, termed AdC68, expressing a codon-optimized truncated form of gag of HIV-1 was shown previously to induce a potent transgene product-specific CD8(+) T cell response in mice. We constructed a second chimpanzee adenovirus vaccine vector, termed AdC6, also expressing the truncated gag of HIV-1. This vector, which belongs to a different serotype than the AdC68 virus, induces high frequencies of gag-specific CD8(+) T cells in mice including those pre-exposed to AdHu5 virus. Generation of an additional E1-deleted adenoviral vector of chimpanzee origin allows for sequential booster immunizations with heterologous vaccine carriers. In this study, we show that such heterologous prime boost regimens based on E1-deleted adenoviral vectors of different serotypes expressing the same transgene product are highly efficient in increasing the transgene product-specific CD8(+) T cell response. They are equivalent to sequential vaccinations with an E1-deleted Ad vector followed by booster immunization with a poxvirus vector and they surpass regimens based on DNA vaccine prime followed by a recombinant adenoviral vector boost.