Enantioselective catalytic reduction of ketoimines with trichlorosilane promoted by readily available chiral Lewis bases

The straightforward synthesis of a series of enantiomerically pure Lewis basic amides by simple condensation of commercially available enantiopure diamines with picolinic acid is reported. These compounds were shown to promote the enantioselective reduction of ketoimines with trichlorosilane. Working with the model substrate N-phenyl benzophenone imine, the new organocatalysts led to the formation of the corresponding amine, with excellent chemical efficiency (up to 99% chemical yield) and good stereoselectivity (up to 73% ee). Up to 83% of enantioselectivity was reached in the reduction of differently substituted imines.     

Genetic diversity and geographic differentiation of disjunct Atlantic and Amazonian populations of <Emphasis Type="Italic">Psychotria ipecacuanha</Emphasis> (Rubiaceae)

Ipecac (Psychotria ipecacuanha) is a perennial, medicinal herb that grows in the understory of semi-deciduous tropical forests in the Neotropics. Ipecacs present a widely disjunct distribution, with two of its three ranges occurring in Brazil. The Amazonian populations are at least 1600 km from the nearest Atlantic populations. This work used ISSR markers to compare the genetic diversity and structure of populations from the two Brazilian ranges. Lower genetic diversity in Amazon populations (P = 60.11%, Hs = 0.18) and higher genetic diversity in Atlantic populations (P = 73.94%, Hs = 0.20) were detected. Differentiation between ranges were high (θ ^B = 0.6838, G_ST-B = 0.6665). AMOVA revealed that 65.3% of the total molecular variance can be attributed to regional differences between the two ranges. Principal coordinate analyses and cluster analyses organized ipecacs at either individual or population level into two exclusive groups that correspond each to one of the two disjunct ranges, without exception. The results do not support a scenario that postulates human-mediated, long-distance dispersal events as a plausible origin for the distribution of the Brazilian ipecacs, but indicate geographic isolation as a long-standing barrier to genetic exchange and connectivity among populations from different ranges. Conservation implications are discussed.     

Differential ultrastructural changes in tomato hormonal mutants exposed to cadmium

Cadmium (Cd) is a toxic heavy metal, which can cause severe damage to plant development. The aim of this work was to characterize ultrastructural changes induced by Cd in miniature tomato cultivar Micro-Tom (MT) mutants and their wild-type counterpart. Leaves of diageotropica (dgt) and Never ripe (Nr) tomato hormonal mutants and wild-type MT were analysed by light, scanning and transmission electron microscopy in order to characterize the structural changes caused by the exposure to 1 mM CdCl₂. The effect of Cd on leaf ultrastructure was observed most noticeably in the chloroplasts, which exhibited changes in organelle shape and internal organization, of the thylakoid membranes and stroma. Cd caused an increase in the intercellular spaces in Nr leaves, but a decrease in the intercellular spaces in dgt leaves, as well as a decrease in the size of mesophyll cells in the mutants. Roots of the tomato hormonal mutants, when analysed by light microscopy, exhibited alterations in root diameter and disintegration of the epidermis and the external layers of the cortex. A comparative analysis has allowed the identification of specific Cd-induced ultrastructural changes in wild-type tomato, the pattern of which was not always exhibited by the mutants.     

Mitochondria Are Linked to Calcium Stores in Striated Muscle by Developmentally Regulated Tethering Structures

Bi-directional calcium (Ca²⁺) signaling between mitochondria and intracellular stores (endoplasmic/sarcoplasmic reticulum) underlies important cellular functions, including oxidative ATP production. In striated muscle, this coupling is achieved by mitochondria being located adjacent to Ca²⁺ stores (sarcoplasmic reticulum [SR]) and in proximity of release sites (Ca²⁺ release units [CRUs]). However, limited information is available with regard to the mechanisms of mitochondrial-SR coupling. Using electron microscopy and electron tomography, we identified small bridges, or tethers, that link the outer mitochondrial membrane to the intracellular Ca²⁺ stores of muscle. This association is sufficiently strong that treatment with hypotonic solution results in stretching of the SR membrane in correspondence of tethers. We also show that the association of mitochondria to the SR is 1) developmentally regulated, 2) involves a progressive shift from a longitudinal clustering at birth to a specific CRU-coupled transversal orientation in adult, and 3) results in a change in the mitochondrial polarization state, as shown by confocal imaging after JC1 staining. Our results suggest that tethers 1) establish and maintain SR–mitochondrial association during postnatal maturation and in adult muscle and 2) likely provide a structural framework for bi-directional signaling between the two organelles in striated muscle.     

Anaesthesia of free-ranging Northern chamois (<Emphasis Type="Italic">Rupicapra rupicapra</Emphasis>) with xylazine/ketamine and reversal with atipamezole

One-hundred and fifty-five free-ranging Northern chamois (Rupicapra rupicapra) were anaesthetised in the course of a restocking programme using xylazine plus ketamine. Mean ± SD dosages for xylazine and ketamine were 1.9 ± 0.5 and 2.2 ± 0.7 mg/kg, respectively. In 57 chamois, sedation was reversed using 0.3 ± 0.1 mg/kg atipamezole. Although all the anaesthetic dosages tested immobilised free-ranging Northern chamois, shorter induction times (4.8 ± 2.6 min), deeper sedation with no reaction to handling in >90% of the animals and quick reversal (4.0 ± 2.7 min) were obtained using 2.5 mg/kg xylazine plus 3.0 mg/kg ketamine reversed with 0.25 mg/kg atipamezole. Under the conditions of this study, suggested standard doses are 63 mg/animal xylazine plus 76 mg/animal ketamine reversed by 6.3 mg/animal atipamezole. This anaesthetic protocol improves the results from the previous study of Dematteis et al. (Vet Rec 163:184–189, 2008) using xylazine alone.     

The Tnt1 family member Retrosol copy number and structure disclose retrotransposon diversification in different <Emphasis Type="Italic">Solanum</Emphasis> species

Eukaryotic genome expansion/retraction caused by LTR-retrotransposon activity is dependent on the expression of full length copies to trigger efficient transposition and recombination-driven events. The Tnt1 family of retrotransposons has served as a model to evaluate the diversity among closely related elements within Solanaceae species and found that members of the family vary mainly in their U3 region of the long terminal repeats (LTRs). Recovery of a full length genomic copy of Retrosol was performed through a PCR-based approach from wild potato, Solanum oplocense. Further characterization focusing on both LTR sequences of the amplified copy allowed estimating an approximate insertion time at 2 million years ago thus supporting the occurrence of transposition cycles after genus divergence. Copy number of Tnt1-like elements in Solanum species were determined through genomic quantitative PCR whereby results sustain that Retrosol in Solanum species is a low copy number retrotransposon (1–4 copies) while Retrolyc1 has an intermediate copy number (38 copies) in S. peruvianum. Comparative analysis of retrotransposon content revealed no correlation between genome size or ploidy level and Retrosol copy number. The tetraploid cultivated potato with a cellular genome size of 1,715 Mbp harbours similar copy number per monoploid genome than other diploid Solanum species (613–884 Mbp). Conversely, S. peruvianum genome (1,125 Mbp) has a higher copy number. These results point towards a lineage specific dynamic flux regarding the history of amplification/activity of Tnt1-like elements in the genome of Solanum species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Role of Tryptophan 95 in substrate specificity and structural stability of Sulfolobus solfataricus alcohol dehydrogenase

A mutant of the thermostable NAD⁺-dependent (S)-stereospecific alcohol dehydrogenase from Sulfolobus solfataricus (SsADH) which has a single substitution, Trp95Leu, located at the substrate binding pocket, was fully characterized to ascertain the role of Trp95 in discriminating between chiral secondary alcohols suggested by the wild-type SsADH crystallographic structure. The Trp95Leu mutant displays no apparent activity with short-chain primary and secondary alcohols and poor activity with aromatic substrates and coenzyme. Moreover, the Trp → Leu substitution affects the structural stability of the archaeal ADH, decreasing its thermal stability without relevant changes in secondary structure. The double mutant Trp95Leu/Asn249Tyr was also purified to assist in crystallographic analysis. This mutant exhibits higher activity but decreased affinity toward aliphatic alcohols, aldehydes as well as NAD⁺ and NADH compared to the wild-type enzyme. The crystal structure of the Trp95Leu/Asn249Tyr mutant apo form, determined at 2.0 Å resolution, reveals a large local rearrangement of the substrate site with dramatic consequences. The Leu95 side-chain conformation points away from the catalytic metal center and the widening of the substrate site is partially counteracted by a concomitant change of Trp117 side chain conformation. Structural changes at the active site are consistent with the reduced activity on substrates and decreased coenzyme binding.