Gamma proteobacteria can nodulate legumes of the genus Hedysarum

The bacteria hosted in the root nodules of the three Mediterranean wild legume species Hedysarum carnosum, Hedysarum spinosissimum subsp. capitatum, and Hedysarum pallidum, growing in native stands in different habitats in Algeria were isolated. Bacteria were recovered on yeast-mannitol-agar or on minimal media from a total of 52 nodules. Isolates were analyzed by Amplified Ribosomal DNA Restriction Analysis (ARDRA) using the enzyme CfoI, and further sorted by RAPD fingerprinting. A total of ten different types were found and their amplified 16S rDNA was sequenced and compared to databases. The BLAST alignment indicates that all the species whose sequences share 98 to 100% identity to the bacteria found in these nodules belong to the class Gammaproteobacteria and include Pantoea agglomerans, Enterobacter kobei, Enterobacter cloacae, Leclercia adecarboxylata, Escherichia vulneris, and Pseudomonas sp. No evidence of any rhizobial-like sequence was found even upon amplifying from the bulk of microbial cells obtained from the squashed nodules, suggesting that the exclusive occupants of the nodules formed by the three plants tested are members of the orders Enterobacteriales or Pseudomonadales. This is the first report of Gammaproteobacteria associated with legume nodules. Despite the presence of the related crop plant Hedysarum coronarium, specifically nodulated by Rhizobium sullae, these three Hedysarum species demonstrate to have undergone a separate path in terms of endophytic interactions with bacteria. An hypothesis to account for differences between the symbiotic relationships engaged by man-managed legumes, and those found in plants whose ecology is independent from human action, is discussed.     

Amplified androstenedione enzymeimmunoassay for the diagnosis of cryptorchidism in the male horse: comparison with testosterone and estrone sulphate methods

An amplified enzymeimmunoassay (EIA) was validated for androstenedione in the serum of male horses. We will use the assay as a tool for the diagnosis of equine cryptorchidism. We will compare androstenedione EIA to the currently used methods (testosterone and estrone sulphate determinations). The study was conducted on 115 horses of pure Spanish and Arabian breeds, that included 30 geldings, 60 bilateral cryptorchids and 25 stallions. Androstenedione standard curve covered a range between 0 and 1 ng per well. Low detection limit was 1.54 pg/ml. Intra- and inter-assay coefficients of variation (CV%) were <8.2 and <9.3, respectively (n=10). Recovery rate of known androstenedione concentrations averaged from 96.62+/-2.69 to 97.63+/-1.87%. Androstenedione mean+/-S.E. serum concentrations were 10.52+/-1.36 ng/ml in stallions (n=25), 0.51+/-0.04 ng/ml in cryptorchids (n=60), and 0.03+/-0.01 ng/ml in geldings (n=30). Diagnostic validation parameters in basal samples showed for estrone sulphate the lower positive predictive value (0.85) with the higher number of false positives, and lower specificity (0.84). Testosterone showed the higher number of false negatives with a negative predictive value of 0.85, and lower sensitivity (0.85). Among the three hormones evaluated, androstenedione presented the best results with the smaller number of horses diagnosed as false positives (0.93) or negatives (0.91). This technique also resulted in higher sensitivity, specificity and efficiency over the other two methods assayed. We concluded that our amplified EIA is a highly sensitive and specific assay that provides a rapid, simple, and inexpensive alternative to other methods.     

Easy preparation of dehydroalanine building blocks equipped with oxazolidin-2-one chiral auxiliaries,and applications to the stereoselective synthesis of substituted tryptophans

Chiral dehydroamino acid building blocks are versatile starting materials for the preparation of optically active unusual amino acids and other compounds of pharmacological interest. Herein we disclose the expedient preparation of dehydroalanines (ΔAla) equipped with oxazolidin-2-one (Oxd) chiral auxiliaries, Ts-Oxd-ΔAla-OMe. These compounds have been obtained in high yields from dipeptides Ts-Ser/Thr/phenylSer-Ser-OMe by the one-pot cyclization–elimination reaction with N,N-disuccinimidyl carbonate and catalytic DIPEA. To test the efficacy of the chiral auxiliaries in controlling asymmetric transformations, the Friedel–Crafts alkylations of indoles carrying diverse substituents were performed in the presence of Lewis and Brønsted acids. The reactions proceeded with good to excellent diastereomeric ratios giving (S)- or (R)-tryptophan derivatives, isolated very conveniently by simple flash chromatography. To verify the utility of this approach, optically pure (S)-2-methyltryptophan and (S)-5-fluorotryptophan were obtained and utilized to prepare analogues of endogenous opioid peptide endomorphin-1, H-Tyr-Pro-Trp-PheNH₂.     

A new ditopic GdIII complex functionalized with an adamantyl moiety as a versatile building block for the preparation of supramolecular assemblies

A dimeric GdAAZTA-like complex (AAZTA is 6-amino-6-methylperhydro-1,4-diazepinetetraacetic acid) bearing an adamantyl group (Gd₂ L1) able to form strong supramolecular adducts with specific hosts such as β-cyclodextrin (β-CD), poly-β-CD, and human serum albumin (HSA) is reported. The relaxometric properties of Gd₂ L1 were investigated in aqueous solution by measuring the ¹H relaxivity as a function of pH, temperature, and magnetic field strength. The relaxivity of Gd₂ L1 (per Gd atom) at 40 MHz and 298 K is 17.6 mM^?1 s^?1, a value that remains almost constant at higher fields owing to the great compactness and rigidity of the bimetallic chelate, resulting in an ideal value for the rotational correlation time for high-field MRI applications (1.5–3.0 T). The noncovalent interaction of Gd₂ L1 with β-CD, poly-β-CD, and HSA and the relaxometric properties of the resulting host–guest adducts were investigated using ¹H relaxometric methods. Relaxivity enhancements of 29 and 108 % were found for Gd₂ L1–β-CD and Gd₂ L1–poly-β-CD, respectively. Binding of Gd₂ L1 to HSA (K _A = 1.2 × 10⁴ M^?1) results in a remarkable relaxivity of 41.4 mM^?1 s^?1 for the bound form (+248 %). The relaxivity is only limited by the local rotation of the complex within the binding site, which decreases on passing from Gd₂ L1–β-CD to Gd₂ L1–HSA. Finally, the applicability of Gd₂ L1 as tumor-targeting agent through passive accumulation of the HSA-bound adduct was evaluated via acquisition of magnetic resonance images at 1 T of B16-tumor-bearing mice. These experiments indicate a considerable signal enhancement (+160 %) in tumor after 60 min from the injection and a very low hepatic accumulation.     

The single‐subunit RING‐type E3 ubiquitin ligase RSL1 targets PYL4 and PYR1 ABA receptors in plasma membrane to modulate abscisic acid signaling

Membrane‐delimited events play a crucial role for ABA signaling and PYR/PYL/RCAR ABA receptors, clade A PP2Cs and SnRK2/CPK kinases modulate the activity of different plasma membrane components involved in ABA action. Therefore, the turnover of PYR/PYL/RCARs in the proximity of plasma membrane might be a step that affects receptor function and downstream signaling. In this study we describe a single‐subunit RING‐type E3 ubiquitin ligase RSL1 that interacts with the PYL4 and PYR1 ABA receptors at the plasma membrane. Overexpression of RSL1 reduces ABA sensitivity and rsl1 RNAi lines that impair expression of several members of the RSL1/RFA gene family show enhanced sensitivity to ABA. RSL1 bears a C‐terminal transmembrane domain that targets the E3 ligase to plasma membrane. Accordingly, bimolecular fluorescent complementation (BiFC) studies showed the RSL1–PYL4 and RSL1–PYR1 interaction is localized to plasma membrane. RSL1 promoted PYL4 and PYR1 degradation in vivo and mediated in vitro ubiquitylation of the receptors. Taken together, these results suggest ubiquitylation of ABA receptors at plasma membrane is a process that might affect their function via effect on their half‐life, protein interactions or trafficking.     

Myrtucommulone production by a strain of Neofusicoccum australe endophytic in myrtle (Myrtus communis)

Myrtucommulones are acylphloroglucinol compounds reported from myrtle (Myrtus communis) and a few more plant species belonging in the Myrtaceae that have recently attracted the attention of pharmacologists for their anti-oxidant, anti-inflammatory and anti-tumor properties. An endophytic strain of Neofusicoccum australe recovered from a myrtle branch was selected based on the bioactivity of its culture extracts, and found to produce myrtucommulones A and D. A mixture of these compounds induced anti-proliferative effects on the human prostatic cancer cell lines DU145 and PC3, with a IC₅₀ of respectively 4.64 and 3.11 mg/l. Along the lines of recent evidences of the ability by endophytic fungi to produce bioactive compounds originally extracted from their host plants, this is the first report of myrtucommulones as secondary metabolites of an endophytic fungal strain. The availability of a microbial strain to be cultured in vitro may provide access to more substantial amounts of these products for further investigations in view of their possible pharmaceutical use.     

A General and Concise Enantioselective Divergent Approach to 13‐Alkyl‐Substituted Ionones

A novel enantioselective divergent route to 13‐alkyl derivatives of α‐ and γ‐ionone, important components of perfumes and fragrances, is reported. This relatively short and convenient methodology takes advantage of the use of a common intermediate, easily obtained from highly enantiomerically enriched (S)‐α‐ionone, which avoids the separate installation of the butenone side chain at C(6) for each analog. Olfactory evaluation of synthesized compounds reconfirmed the influence of the hydrophobic interactions of alkyl substituents at C(5) with olfactory receptors (ORs) in the chemoreception of ionones, and suggested that a synperiplanar orientation of C(13) and the lateral chain is the better geometry fitting OR's cavity.     

Hypoxic blackwater event severely impacts Murray crayfish (Euastacus armatus) populations in the Murray River,Australia

Prolonged flooding in 2010/11 ended a decade of drought and produced a large‐scale hypoxic blackwater event across the southern Murray‐Darling Basin, Australia. The hypoxic conditions caused fish kills and Murray crayfish Euastacus armatus to emerge from the water onto the river banks to avoid the poor water quality. This study examined the medium‐term impact of this blackwater event on Murray crayfish populations in the Murray River, where approximately 1800 km of the main channel were affected by hypoxia. Murray crayfish populations were surveyed in July 2012, along a 1100‐km section of the Murray River at 10 sites affected by hypoxic blackwater and six sites that were not affected, and data were compared with surveys of the same sites undertaken in July 2010, four months before the hypoxic blackwater event (before‐after‐control‐impact experimental design). Murray crayfish abundance in 2012 (post‐blackwater) was significantly lower at blackwater affected sites (81% reduction from 2010), but not at non‐affected sites. The hypoxic blackwater impacted Murray crayfish of both sexes and all size‐classes in a similar manner. The results demonstrate that prolonged periods of hypoxia can markedly impact populations of the long‐lived and slow‐growing Murray crayfish despite the species ability to emerge from hypoxic water. The findings highlight important challenges for the management of both the recreational fishery for this species and riverine flows in relation to hypoxic blackwater events.