Identification of an EcoRI restriction site for a rapid and precise determination of beta-asarone-free Acorus calamus cytotypes

Calamus (Acorus calamus L., Araceae) is an aromatic herb, indigenous to Central Asia and Eastern Europe. The fragrant oils obtained by alcoholic extraction of the rhizome are mainly used in the pharmaceutical and oenological industries. Nevertheless, the occurrence of beta-asarone [(Z)-1,2,4-trimethoxy-5-prop-1-enyl-benzene] limits the possibility of its use due to the carcinogenic properties of this compound. The aim of this work was to identify a diploid beta-asarone-free A. calamus by using chemical and molecular approaches. For these purposes alcoholic extracts of both diploid and triploid A. calamus were analyzed by gas chromatography-mass spectrometry (GC-MS) and comparison of the 700 bp sequence of the non-transcribed spacer (NTS) in the 5S-rRNA gene was also performed. Alcoholic extracts of the triploid A. calamus were characterized by a higher percentage of beta-asarone (11%), which was the main compound, followed by higher percentages of camphene (2.27%), E-beta-ocimene (3.28%), camphor (1.54%), calarene (1.42%), alpha-selinene (5.02%) and tau-cadinol (2.00%), when compared to the diploid A. calamus. The latter had higher percentages of iso-shyobunone (8.62%), beta-sesquiphellandrene (3.28%), preiso calamendiol (22.81%) and acorone (26.33%), and completely lacked of beta-asarone. The 5S-rRNA spacer region of both diploid and triploid A. calamus were amplified by PCR using a pair of primers located at the 3' and 5' ends of the coding sequence of 5S-rRNA gene. The resulting PCR products (about 700 bp) were gel purified, subcloned into pGEM-T Easy vector and sequenced. By aligning the isolated nucleotide sequences of the two varieties and the sequences from different A. calamus chemotypes present in Genbank, sequence diversities were found in the spacer region. Furthermore, the PCR products were digested by using EcoRI. The restriction profile of the spacer domain resulted different for the two cytotypes. Along with chemical analysis of alcoholic extracts, sequence analysis coupled to restriction mapping was demonstrated to represent a powerful tool to distinguish the A. calamus diploid cytotype from the others. The security and effective usage of the diploid beta-asarone-free A. calamus was also discussed.     

SAR and biological evaluation of SEN12333/WAY-317538: Novel alpha 7 nicotinic acetylcholine receptor agonist

Alpha 7 nicotinic acetylcholine receptor (α₇ nAChR) agonists are promising therapeutic candidates for the treatment of cognitive impairment associated with a variety of disorders including Alzheimer’s disease and schizophrenia. Alpha 7 nAChRs are expressed in brain regions associated with cognitive function, regulate cholinergic neurotransmission and have been shown to be down regulated in both schizophrenia and Alzheimer’s disease. Herein we report a novel, potent small molecule agonist of the alpha 7 nAChR, SEN12333/WAY-317538. This compound is a selective agonist of the α₇ nAChR with excellent in vitro and in vivo profiles, excellent brain penetration and oral bioavailability, and demonstrates in vivo efficacy in multiple behavioural cognition models. The SAR and biological evaluation of this series of compounds are discussed.     

129-Derived Mouse Strains Express an Unstable but Catalytically Active DNA Polymerase Iota Variant

Mice derived from the 129 strain have a nonsense codon mutation in exon 2 of the polymerase iota (Polι) gene and are therefore considered Polι deficient. When we amplified Polι mRNA from 129/SvJ or 129/Ola testes, only a small fraction of the full-length cDNA contained the nonsense mutation; the major fraction corresponded to a variant Polι isoform lacking exon 2. Polι mRNA lacking exon 2 contains an open reading frame, and the corresponding protein was detected using a polyclonal antibody raised against the C terminus of the murine Polι protein. The identity of the corresponding protein was further confirmed by mass spectrometry. Although the variant protein was expressed at only 5 to 10% of the level of wild-type Polι, it retained de novo DNA synthesis activity, the capacity to form replication foci following UV irradiation, and the ability to rescue UV light sensitivity in Polι^−/− embryonic fibroblasts derived from a new, fully deficient Polι knockout (KO) mouse line. Furthermore, in vivo treatment of 129-derived male mice with Velcade, a drug that inhibits proteasome function, stabilized and restored a substantial amount of the variant Polι in these animals, indicating that its turnover is controlled by the proteasome. An analysis of two xeroderma pigmentosum-variant (XPV) cases corresponding to missense mutants of Polη, a related translesion synthesis (TLS) polymerase in the same family, similarly showed a destabilization of the catalytically active mutant protein by the proteasome. Collectively, these data challenge the prevailing hypothesis that 129-derived strains of mice are completely deficient in Polι activity. The data also document, both for 129-derived mouse strains and for some XPV patients, new cases of genetic defects corresponding to the destabilization of an otherwise functional protein, the phenotype of which is reversible by proteasome inhibition.     

Spatial scale and meiobenthic copepod recolonisation: testing the effect of disturbance size in a seagrass habitat

A central problem in relating disturbance to community structure lies in determining how community structure is affected by the size of disturbance events. In soft-bottom habitats, recovery rate and patterns of macrobenthic community are usually affected by the spatial scale of disturbance. Thus far, no studies have explicitly addressed these issues for meiobenthic copepods. To test the effects of the size of hypoxia/anoxia disturbance on the recovery of meiobenthic copepod communities in a vegetated (Ruppia cirrhosa) sediment, a field experiment was set up in Valle Smarlacca, a brackish lagoon on the northern Adriatic coast of Italy. Plots of three different sizes—small (40×40 cm), medium (80×80 cm) and large (160×160 cm)—were exposed to experimentally induced hypoxia/anoxia for 5 days. Control plots of 40×40 cm were added, for comparison with ambient abundance. Recolonisation and community recovery were then observed for 12 days, with samplings on days 0, 1, 3, 5, 7, 9 and 12. Sampling was designed to focus on the distance from source pools of colonists. The induced anoxia had a severe impact on the copepods, but the impact of the disturbance was independent of plot size. Copepod abundance increased linearly over time, and no differences in recolonisation rate among the differently sized plots were observed. Recolonisation comprised two phases: until day 3, abundances were low and very similar in all plot sizes; from day 5 onwards, abundances of the dominant species (which were the same in control and disturbed plots) increased, and a more diversified pattern among disturbance sizes was observed. Multivariate analyses showed a gradual response of community structure to disturbance size: copepod assemblages in small plots attained the same structure of the control plots at day 5, while for larger-sized plots this occurred later and was observed on the following sampling date. However, clear differences among the three disturbance sizes were never detected. Variability in community structure seemed to respond more to the overall impact of disturbance than to the size of the disturbed area. In the seagrass meadows of the Valle Smarlacca, several factors seem to influence the structural organisation of meiobenthic copepod communities during recolonisation processes. Among others, the recovery of the vegetated habitat to suitable conditions seems to play a much more relevant role than the size of the disturbed patches.     

Citrus Allergy from Pollen to Clinical Symptoms

Allergy to citrus fruits is often associated with pollinosis and sensitization to other plants due to a phenomenon of cross-reactivity. The aims of the present study were to highlight the cross-reactivity among citrus and the major allergenic pollens/fruits, throughout clinical and molecular investigations, and to evaluate the sensitization frequency to citrus fruits in a population of children and adults with pollinosis. We found a relevant percentage of sensitisation (39%) to citrus fruits in the patients recruited and in all of them the IgE-mediated mechanism has been confirmed by the positive response to the prick-to-prick test. RT-PCR experiments showed the expression of Cit s 1, Cit s 3 and a profilin isoform, already described in apple, also in Citrus clementine pollen. Data of multiple sequence alignments demonstrated that Citrus allergens shared high percentage identity values with other clinically relevant species (i.e. Triticum aestivum, Malus domestica), confirming the possible cross-allergenicity citrus/grasses and citrus/apple. Finally, a novelty of the present work has been the expression of two phospholipaseA2 isoforms (PLA2 α and β) in Citrus as well as in Triticum pollens; being PLA2 able to generate pro-inflammatory factors, this enzyme could participate in the activation of the allergenic inflammatory cascade.     

Opposite Roles of NMDA Receptors in Relapsing and Primary Progressive Multiple Sclerosis

Synaptic transmission and plasticity mediated by NMDA receptors (NMDARs) could modulate the severity of multiple sclerosis (MS). Here the role of NMDARs in MS was first explored in 691 subjects carrying specific allelic variants of the NR1 subunit gene or of the NR2B subunit gene of this glutamate receptor. The analysis was replicated for significant SNPs in an independent sample of 1548 MS subjects. The C allele of rs4880213 was found to be associated with reduced NMDAR-mediated cortical excitability, and with increased probability of having more disability than the CT/TT MS subjects. MS severity was higher in the CC group among relapsing-remitting MS (RR-MS) patients, while primary progressive MS (PP-MS) subjects homozygous for the T allele had more pronounced clinical worsening. Mean time to first relapse, but not to an active MRI scan, was lower in the CC group of RR-MS patients, and the number of subjects with two or more clinical relapses in the first two years of the disease was higher in CC compared to CT/TT group. Furthermore, the percentage of relapses associated with residual disability was lower in subjects carrying the T allele. Lesion load at the MRI was conversely unaffected by the C or T allele of this SNP in RR-MS patients. Axonal and neuronal degeneration at the optical coherence tomography was more severe in the TT group of PP-MS patients, while reduced retinal nerve fiber thickness had less consequences on visual acuity in RR-MS patients bearing the T allele. Finally, the T allele was associated with preserved cognitive abilities at the Rao’s brief repeatable neuropsychological battery in RR-MS. Signaling through glutamate NMDARs enhances both compensatory synaptic plasticity and excitotoxic neurodegeneration, impacting in opposite ways on RR-MS and PP-MS pathophysiological mechanisms.     

Removal of Vanadium(III) and Molybdenum(V) from Wastewater Using Posidonia oceanica (Tracheophyta) Biomass

The use of dried and re-hydrated biomass of the seagrass Posidonia oceanica was investigated as an alternative and –low-cost biomaterial for removal of vanadium(III) and molybdenum(V) from wastewaters. Initial characterisation of this biomaterial identified carboxylic groups on the cuticle as potentially responsible for cation sorption, and confirmed the toxic-metal bioaccumulation. The combined effects on biosorption performance of equilibrium pH and metal concentrations were investigated in an ideal single-metal system and in more real-life multicomponent systems. There were either with one metal (vanadium or molybdenum) and sodium nitrate, as representative of high ionic strength systems, or with the two metals (vanadium and molybdenum). For the single-metal solutions, the optimum was at pH 3, where a significant proportion of vanadium was removed (ca. 70%) while there was ca. 40% adsorption of molybdenum. The data obtained from the more real-life multicomponent systems showed that biosorption of one metal was improved both by the presence of the other metal and by high ionic strength, suggesting a synergistic effect on biosorption rather than competition. There data ware used for the development of a simple multi-metal equilibrium model based on the non-competitive Langmuir approach, which was successfully fitted to experimental data and represents a useful support tool for the prediction of biosorption performance in such real-life systems. Overall, the results suggest that biomass of P. oceanica can be used as an efficient biosorbent for removal of vanadium(III) and molybdenum(V) from aqueous solutions. This process thus offers an eco-compatible solution for the reuse of the waste material of leaves that accumulate on the beach due to both human activities and to storms at sea.     

Perthamide C Inhibits eNOS and iNOS Expression and Has Immunomodulating Activity In Vivo

Here we have characterized perthamide C, a cyclopeptide from a Solomon Lithistid sponge Theonella swinhoei, which displays an anti-inflammatory/immunomodulatory activity. The study has been performed using the carragenan-induced mouse paw edema that displays an early (0–6 h) and a late phase (24–96 h). Perthamide C significantly inhibits neutrophils infiltration in tissue both in the early and late phases. This effect was coupled to a reduced expression of the endothelial nitric oxide synthase (eNOS) in the early phase while cyclooxygenase-1 and 2 (COX-1, COX-2), and inducible NOS (iNOS) expression were unaffected. In the late phase perthamide C reduced expression of both NOS isoforms without affecting COXs expression. This peculiar selectivity toward the two enzymes deputed to produce NO lead us to investigate on a possible action of perthamide C on lymphocytes infiltration and activation. We found that perthamide C inhibited the proliferation of peripheral lymphocytes, and that this effect was secondary to its metabolic activation in vivo. Indeed, in vitro perthamide C did not inhibit proliferation as opposite to its metabolite perthamide H.In conclusion, perthamide C selectively interferes with NO generation triggered by either eNOS or iNOS without affecting either COX-1 or COX-2. This in turn leads to modulation of the inflammatory response through a reduction of vascular permeability, neutrophil infiltration as well as lymphocyte proliferation.     

Modulation of the cell-mediated immune function by interferon α, β or γ can partially reverse the immunosuppression induced by human T-cell leukemia virus I in human cord blood cultures

Summary Infection with human T-cell leukemia virus type I (HTLV-I) is associated in vitro and in vivo with a remarkable depression of cell-mediated immune functions. In the present report it is shown that early events following virus-induced suppression of the cell-mediated immune response of freshly isolated cord blood mononuclear cells (CBL) infected with HTLV-I can be partially counteracted by treatment with interferons , or (IFN). All three types of IFN exerted a protective effect on CBL cultures exposed to the virus. This resulted in: (a) a reduced number of virus-positive cells until 4 weeks of culture; (b) delay in the clonal expansion of infected cells (IFN and ); (c) increased natural killer cell activity of CBL, 1 week post-infection (p.i.), mediated by IFN; (d) increase of allospecific recognition of infecting and priming HTLV-I donor MT-2 cells by CBL in a cytotoxic-T-lymphocyte-like response, mediated by IFN and particularly by IFN; (e) phenotype distribution of CBL subpopulations, tested 4 days p.i., more similar to that of non-infected CBL cultures.In contrast, the overall CBL proliferation, that is profoundly depressed during the first week p.i., was not restored by IFN treatments, suggesting that boosting of the cell-mediated killing induced by IFN might involve the maturation of undifferentiated precursor cells rather than stimulation of their proliferation. The improvement of the efficiency of the antiviral immune response induced by treatment with IFN is likely to contribute to the clearance of virus-positive cells during the early phase of infection. This would provide experimental evidence to support an immunopharmacological approach contributing to the conversion of HTLV-I carriers from positive to negative.