hNK2 receptor antagonists. The use of intramolecular hydrogen bonding to increase solubility and membrane permeability

Starting from in-house capped tripeptide libraries, we have developed two series of compounds as potent antagonists of the hNK₂ receptor with a reduced peptide character. These two series maintained a crucial amide bond, which could not be methylated or substituted with classical isostere without a dramatic loss in binding affinity, very likely due conformational changes.We report here the planning, synthesis and evaluation of molecules belonging to the selected chemical series, which contain a strategically placed hydrogen bond acceptor. The aim of the work was to improve membrane permeability via the formation of an intramolecular hydrogen bonding, and at the same time to maintain the structural characteristics geometry and polarity of the amide linkage so as to retain a relevant binding affinity for the biological target.     

<Emphasis Type="Italic">St</Emphasis>CDPK2 expression and activity reveal a highly responsive potato calcium-dependent protein kinase involved in light signalling

Calcium-dependent protein kinases (CDPKs) are essential calcium sensors. In this work, we have studied StCDPK2 isoform from potato both at gene and protein level. StCdpk2 genomic sequence contains eight exons and seven introns, as was observed for StCdpk1. There is one copy of the gene per genome located in chromosome 7. StCDPK2 encodes an active CDPK of 515 aminoacids, with an apparent MW of 57 kDa, which presents myristoylation and palmitoylation consensus in its N-terminus. StCDPK2 is highly expressed in leaves and green sprouts; enhanced expression was detected under light treatment, which corresponds well with light responsive cis-acting elements found in its promoter sequence. Antibodies against the recombinant StCDPK2::6xHis protein detected this isoform in soluble and particulate fractions from leaves. StCDPK2 autophosphorylation and kinase activity are both calcium dependent reaching half maximal activation at 0.6 μM calcium. The active kinase is autophosphorylated on serine and tyrosine residues and its activity is negatively modulated by phosphatidic acid (PA). Our results reveal StCDPK2 as a signalling element involved in plant growth and development and show that its activity is tightly regulated.     

Root-ABA1 QTL affects root lodging, grain yield, and other agronomic traits in maize grown under well-watered and water-stressed conditions

A major QTL affecting root traits and leaf ABA concentration was identified in maize (Zea mays L.) and named root-ABA1. For this QTL, back-cross-derived lines (BDLs) homozygous either for the (+) or for the (-) allele increasing or decreasing, respectively, root size and leaf ABA concentration, were developed. This study was conducted to evaluate the QTL effects in various genetic backgrounds and at different water regimes. The (+/+) and (-/-) BDLs were crossed with five or 13 inbred tester lines of different origin, thus producing two sets of test-crosses that were evaluated in Italy and China, respectively. Testing was conducted under both well-watered and water-stressed conditions. In Italy, the test-crosses derived from (+/+) BDLs, as compared with those derived from (-/-) BDLs, showed, across both water regimes, higher leaf ABA concentration (on average 384 versus 351 ng g(-1) DW) and lower root lodging (28.0 versus 52.5%), and lower grain yield under water-stressed conditions (4.88 versus 6.27 Mg ha(-1)). In China, where root lodging did not occur, the test-crosses derived from (+/+) BDLs were less productive at both water regimes (on average, 6.83 versus 7.49 Mg ha(-1)). The lower grain yield of the test-crosses derived from (+/+) BDLs was due to a lower number of ears per plant and to lower kernel weight. The results indicate that the (+) root-ABA1 allele confers not only a consistently lower susceptibility to root lodging but also a lower grain yield, especially when root lodging does not occur.     

Alkaline titrations of poly(dG-dC).poly(dG-dC): microemulsion versus solution behavior

PolyGC was titrated with a strong base in the presence of increasing concentrations of NaCl (from 0.00 to 0.60M) either in water solution or with the polynucleotide solubilized in the aqueous core of reverse micelles, i.e., the cationic quaternary water-in-oil microemulsion CTAB/n-hexane/n-pentanol/water. The results for matched samples in the two media were compared. CD and UV spectroscopies and, for the solution experiments, pH measurements were used to follow the course of deprotonation. In both media the primary effect of the addition of base was denaturation of the polynucleotide, reversible by back-titration with a strong acid. In solution, the apparent pK(a) of the transition decreases with increasing the salt concentration and a roughly linear dependence of pK(a) on p[NaCl] has been found. A parallel monotonic decay with ionic strength has been found in solution for R(OH), defined as the number of hydroxyl ions required per monomeric unit of polyGC to reach half-transition. By contrast, in microemulsion, R(OH) has been found to be independent of the NaCl concentration (and 10 to 50 times lower than in solution). This result is proposed as an indirect evidence of the independence of pK(a) on the salt concentration in microemulsion, where the pH cannot be measured. A sort of buffering effect of the positive charges on the micellar wall and of their counter-ions on the ionic strength could well explain this discrepancy of behavior in the two media.     

Influence of salt stress on seed yield and oil quality of two sunflower hybrids

Sunflower is a major oil seed crop worldwide, and it is also an important crop in Mediterranean areas where salinity is an increasing problem. In this paper, the effect of saline irrigation water on seed yield and quality of sunflower was evaluated. A pot experiment was carried out over two crop seasons on two hybrids – a standard one (Carlos) and a high oleic one (Tenor) – submitted to five salinity levels of irrigation water (0.6, 3, 6, 9 and 12 dS m^?1). Soil salinity was monitored over the entire crop cycle, and leaf ion content was determined at maturity. Tenor showed higher Na⁺ and Mg²⁺ content but lower K⁺ values. No difference between the two hybrids was observed for Cl^? content. A progressive increase in leaf Na⁺, K⁺ and Cl^? contents and Na⁺/K⁺ ratio with increasing salinity level was observed. Seed weight per head, 1000 achene weight, number of seeds per plant and oil yield significantly decreased under salt stress in both hybrids. The percent seed yield decrease was higher per unit increase in electrical conductivity of irrigation water, ECw (8%), than per unit increase in electrical conductivity of saturated‐soil extracts, ECe (5%). Concerning oil fatty acid composition, the main significant difference as result of salt stress was a progressive increase in oleic acid content, from 82.2% to 86.7% for Tenor and from 21.8% to 27.3% for Carlos, which was consistent with a decrease in linoleic acid content, from 5.9% to 3% for Tenor and from 66% to 61.3% for Carlos. These results confirm the possible inhibition of oleate desaturase under salt stress.     

The morphology and activity of the extrafloral nectaries in Reynoutria × bohemica (Polygonaceae)

• Reynoutria × bohemica is an invasive species causing significant damage to native ecosystems in North America and Europe.
• In this work, we performed an in‐depth micromorphological characterisation of the extrafloral nectaries (EFN), during their secretory and post‐secretory phases, in combination with field monitoring of nectary activity over time and the qualitative pool of insect visitors.
• EFN consist of secretory trichomes and vascularised parenchyma. Polysaccharides, lipids and proteins were histochemically detected in all trichome cells; phenolic substances were detected in parenchyma cells. Our data indicate that all nectary regions are involved in nectar production and release, constituting a functional unit. Moreover, the main compound classes of nectar and their transfer change over time: first, granulocrine secretion for sugars prevails, then eccrine secretion of the lipophilic fraction takes place. Active nectaries are mainly located in the apical portion of the stem during the growth phase (April–May), when we detected the highest number of individuals visited by ants; from mid‐August onwards, during flowering, the number of active nectaries declined then ceased production (September), with a concomitant decrease in visits by the ants. The spectrum of nectar‐foraging ants mainly included representatives of the genera Formica, Lasius and Camponotus.
• Reynoutria × bohemica produces an attractive secretion able to recruit local ants that may potentially act as ‘bodyguards’ for protecting young shoots, reducing secretions during the blooming stage. This defence mechanism against herbivores is the same as that displayed by the parental species in its native areas.

     

Investigating the candidacy of LPS-based glycoconjugates to prevent invasive meningococcal disease: conjugates based on core oligosaccharides

In this study we have prepared glycoconjugates with core oligosaccharides (OS) from the lipopolysaccharide (LPS) of Neisseria meningitidis, thus avoiding the neo-epitopes of the deacylated lipid A region of the derived LPS molecule identified in our previous studies. A comprehensive investigation was performed with glycoconjugates prepared from the most extended to the most truncated core OS still maintaining the conserved inner core epitope. As previously, we have established reproducible bactericidal killing of the homologous antigen elaborating strain, but a failure to kill wild-type strains. In these studies it was evident that the linker molecules used in the conjugation methodologies were dominating the immune response. However, when galE core OS based conjugates were prepared without utilizing linkers, via direct reductive amination, we failed to generate an immune response to even the homologous antigen. We also identified that immunisation with the galE antigen via linker methodologies provoked an immune response that was dependent upon key residues of the conserved inner core OS structure, whereas the immune responses to lgtB and lgtA antigens did not involve the inner core OS. This comprehensive study has, despite our best efforts, cast significant doubt as to the utility of the conserved inner core region of the meningococcal LPS as a potential vaccine antigen.     

Mutations, clinical findings and survival estimates in South American patients with X-linked adrenoleukodystrophy

In this study, we analyzed the ABCD1 gene in X-linked adrenoleukodystrophy (X-ALD) patients and relatives from 38 unrelated families from South America, as well as phenotypic proportions, survival estimates, and the potential effect of geographical origin in clinical characteristics.

Methods

X- ALD patients from Brazil, Argentina and Uruguay were invited to participate in molecular studies to determine their genetic status, characterize the mutations and improve the genetic counseling of their families. All samples were screened by SSCP analysis of PCR fragments, followed by automated DNA sequencing to establish the specific mutation in each family. Age at onset and at death, male phenotypes, genetic status of women, and the effect of family and of latitude of origin were also studied.

Results

We identified thirty-six different mutations (twelve novel). This population had an important allelic heterogeneity, as only p.Arg518Gln was repeatedly found (three families). Four cases carried de novo mutations. Intra-familiar phenotype variability was observed in all families. Out of 87 affected males identified, 65% had the cerebral phenotype (CALD). The mean (95% CI) ages at onset and at death of the CALD were 10.9 (9.1–12.7) and 24.7 (19.8–29.6) years. No association was found between phenotypic manifestations and latitude of origin. One index-case was a girl with CALD who carried an ABCD1 mutation, and had completely skewed X inactivation.

Conclusions

This study extends the spectrum of mutations in X-ALD, confirms the high rates of de novo mutations and the absence of common mutations, and suggests a possible high frequency of cerebral forms in our population.