Natural daucane sesquiterpenes with antiproliferative and proapoptotic activity against human tumor cells

Plants of the genera Ferula and Ferulago are known for their complex content in bioactive secondary metabolites such as coumarins, phenylpropanoids, and sesquiterpenes. We used the ground parts of Ferula communis subsp. communis, Ferula glauca subsp. glauca and Ferulago campestris as natural sources for the isolation of four coumarins (CU-1 to CU-4), two phenylpropanoids (PE-1 and PE-2), one polyacetylene (PA-1) and 16 daucane esters (DE-1 to DE-16). The cytotoxic activity of the isolated compounds was evaluated against a panel of seven human tumor cell lines. Fourteen of the daucane derivatives showed antiproliferative activity at least against one of the human tumor cell lines tested, four compounds (DE-5, DE-8, DE-11, and DE-16) were active against all the tested cell lines. Among them DE-11 was the most cytotoxic compound against HeLa (4.4 ± 0.7 μM), A549 (2.8 ± 1.4 μM), HL-60 (2.6 ± 0.4 μM), K562 (26.5 ± 6.0 μM) RS 4;11 (1.7 ± 0.3 μM) and SEM (2.4 ± 0.1 μM) cell lines, while DE-8 was the most active against Jurkat (3.3 ± 0.8 μM). Preliminary structure-activity relationship suggests that the most active compounds in the daucane series present the trans fusion of the penta- and hepta-atomic cycles, and lipophylic ester groups linked to position 6. Isomeric derivatives such as DE-8 and DE-9 or DE-3, DE-4, and DE-5 exhibited significant differences in their IC(50) supporting that the β orientation for the ester group in the position 2 enhances the cytotoxic activity. Furthermore, the pro-apoptotic effect of the most active compounds evaluated in Jurkat cell line showed that these compounds are able to induce apoptosis in a time and concentration-dependent manner. Our findings suggest the potential role of daucane derivatives as models for the development of proapoptotic compounds.     

Solid-phase microextraction (SPME) analysis of six Italian populations of Ephedra nebrodensis Tineo ex Guss. subsp. nebrodensis

Headspace solid-phase microextraction (HS-SPME) coupled with GC/FID and GC/MS was applied for the first time in the analysis of the volatile fraction of an Ephedra species. Notably, six Italian populations (Marche, Abruzzo, and Sardinia) of Ephedra nebrodensis subsp. nebrodensis, covering almost the entire Italian area, were investigated to examine the chemical variability and to support the taxonomy of the species. A fiber screening with polymethylsiloxane (PDMS), Carboxen(TM) /polymethylsiloxane (CAR/PDMS), and polymethylsiloxane/divinylbenzene (PDMS/DVB) coatings, together with an optimization of the extraction conditions were carried out before analysis of the six populations. A total of 119 volatiles were identified in the headspace of different samples, accounting for 63.35-100.00% of the total volatiles. A great variability was found in the qualitative composition of different samples, since only 18 components were in common among all populations. The headspace composition was dominated by sesquiterpene hydrocarbons (52.30-88.32%), with β-maaliene (traces-7.49%), β-patchoulene (traces-1.29%), β-panasinsene (traces-6.85%), α-isocomene (traces-31.25%), α-trans-bergamotene (traces-6.95%), alloaromadendrene (traces-33.20%), α-acoradiene (traces-9.41%), and γ-muurolene (0.61-16.33%) being the most abundant constituents. Noteworthy is the occurrence in a sample of two major unknown sesquiterpenes, one hydrocarbon (24.49%, RI: 1396) and one oxygenated compound (10.37%, RI: 1591), whose mass spectra were reported for the first time. Multivariate chemometric techniques, such as cluster analysis (CA) and principal component analysis (PCA), were used to characterize the samples according to the geographical origin.     

Cloning the full-length IL-2/15 receptor-beta cDNA sequence from mouse brain: evidence of enrichment in hippocampal formation neurons

Numerous studies have implicated interleukin-2 (IL-2) in various brain processes, and more recently, several studies have also attributed neurobiological actions to interleukin-15 (IL-15). On lymphocytes, receptors for IL-2 and IL-15 share a common subunit, the IL-2/15 receptor-beta (IL-2/15Rbeta) that is essential for intracellular signaling. Although a short segment of IL-2/15Rbeta has been cloned (0.35 kb) from normal brain cells, attempts to isolate the full-length cDNA have been unsuccessful, suggesting the possibility that the genes expressed by brain cells and lymphocytes may differ. Using conventional and anchored PCR cloning strategies, we isolated the full-length cDNA of IL-2/15Rbeta (2038 bp) from well-perfused, normal mouse forebrain. The coding sequence and the adjacent 5' and 3' UTR sequences from brain and lymphocyte were found to be fully homologous. Although evidence of expression of IL-2/15Rbeta can be found in many brain regions using PCR, clear evidence of gene expression by in situ hybridization was detectable only in the hippocampal formation, habenula and piriform cortex. This same pattern of mRNA expression in situ was also observed for the common gamma subunit shared by IL-2 and IL-15. In the hippocampus, IL-2/15Rbeta expression was localized to neurons by high resolution in situ hybridization and evidence of IL-2 receptor protein expression was also detected by radioligand receptor binding using hippocampal homogenates. Comparison of undifferentiated and differentiated, immortalized H19-7 hippocampal neurons showed that IL-2/15Rbeta was constitutively expressed across disparate stages of hippocampal neuronal differentiation. These data indicate that IL-2/15Rbeta may serve to modulate neuronal processes in the hippocampus and associated limbic brain regions.     

Ascorbic acid decreases [3H]dopamine binding in striatum without inhibiting dopamine-sensitive adenylate cyclase

Ascorbic acid is found in very high concentrations in cells of neural crest origin such as the central nervous system and the adrenal gland. A variety of evidence has been marshalled to support a role for ascorbate as a chemical messenger. One of the first non-biosynthetic biochemical effects ascribed to ascorate in the CNS was its ability to inhibit dopamine-stimulated adenylate cyclase (DA-ACase) in homogenates from striata of Long Evans rats (J. Neurochem.28, 663, 1977). Using an adenylate cyclase assay based on preparative HPLC, we were unable to detect any inhibition of DA-ACase by ascorbate at concentrations as high as 1 mM. Moreover, this failure to find inhibitory effects of ascorbate on DA-ACase occurred not only when striatal homogenates from Long-Evans rats were used, but also when tissue from Sprague-Dawley rats of N.C. Board of Health mice was tested. Although ascorbate may play a neuromodulatory role, it does not appear that its effects are mediated through effects on cAMP biosynthesis. Despite our inability to detect effects of ascorbate on DA-ACase, we did confirm that ascorbate significantly altered the binding of [³H]dopamine to striatal membranes. Thus, it is clear that the sites binding [³H]dopamine that are affected by ascorbate are unlikely to be the same ones coupled to aenylate cyclase.     

Morphine-induced immune alterations in vivo

The high incidence of human immunodeficiency virus (HIV) seropositivity among drug abusers prompted us to examine in an animal model the effects of morphine on aspects of the immune system that may be specifically related to HIV infection. We now report a robust, sustained elevation in the ratio of CD4+/CD8+ cells in the spleen and thymus of mice chronically treated with morphine. Since CD4+ cells have been reported to be target cells for HIV, these alterations, in concert with a marked cellular atrophy that appears to be restricted to organs of the immune system, suggest that opiates may serve as cofactors in altering the immune status of the host and thus contribute to the increased susceptibility to HIV infection and eventual development of AIDS in opiate abusers.     

Validation of a functional method for the estimation of hip joint centre location

The present study assesses the accuracy with which the subject specific coordinates of the hip joint centre (HJC) in a pelvic anatomical frame can be estimated using different methods. The functional method was applied by calculating the centre of the best sphere described by the trajectory of markers placed on the thigh during several trials of hip rotations. Different prediction methods, proposed in the literature and in the present investigation, which estimate the HJC of adult subjects using regression equations and anthropometric measurements, were also assessed. The accuracy of each of the above-mentioned methods was investigated by comparing their predictions with measurements obtained on a sample of 11 male adult able-bodied volunteers using roentgen stereophotogrammetric analysis (RSA), assumed to provide the true HJC locations. Prediction methods estimated the HJC location at an average rms distance of 25-30 mm. The functional method performed significantly better and estimated HJCs within a rms distance of 13 mm on average. This result may be confidently generalised if the photogrammetric experiment is carefully conducted and an optimal analytical approach used. The method is therefore suggested for use in motion analysis when the subject's hip range of motion is not limited. In addition, the facts that it is not an invasive technique and that it has relatively small and un-biased errors, make it suitable for regression equations identification with no limit to sample size and population typology.     

Fingerspelling as a Novel Gateway into Reading Fluency in Deaf Bilinguals

Studies have shown that American Sign Language (ASL) fluency has a positive impact on deaf individuals’ English reading, but the cognitive and cross-linguistic mechanisms permitting the mapping of a visual-manual language onto a sound-based language have yet to be elucidated. Fingerspelling, which represents English orthography with 26 distinct hand configurations, is an integral part of ASL and has been suggested to provide deaf bilinguals with important cross-linguistic links between sign language and orthography. Using a hierarchical multiple regression analysis, this study examined the relationship of age of ASL exposure, ASL fluency, and fingerspelling skill on reading fluency in deaf college-age bilinguals. After controlling for ASL fluency, fingerspelling skill significantly predicted reading fluency, revealing for the first-time that fingerspelling, above and beyond ASL skills, contributes to reading fluency in deaf bilinguals. We suggest that both fingerspelling—in the visual-manual modality—and reading—in the visual-orthographic modality—are mutually facilitating because they share common underlying cognitive capacities of word decoding accuracy and automaticity of word recognition. The findings provide support for the hypothesis that the development of English reading proficiency may be facilitated through strengthening of the relationship among fingerspelling, sign language, and orthographic decoding en route to reading mastery, and may also reveal optimal approaches for reading instruction for deaf and hard of hearing children.