Immuno‐ and Correlative Light Microscopy‐Electron Tomography Methods for 3D Protein Localization in Yeast

Compartmentalization of eukaryotic cells is created and maintained through membrane rearrangements that include membrane transport and organelle biogenesis. Three‐dimensional reconstructions with nanoscale resolution in combination with protein localization are essential for an accurate molecular dissection of these processes. The yeast Saccharomyces cerevisiae is a key model system for identifying genes and characterizing pathways essential for the organization of cellular ultrastructures. Electron microscopy studies of yeast, however, have been hampered by the presence of a cell wall that obstructs penetration of resins and cryoprotectants, and by the protein dense cytoplasm, which obscures the membrane details. Here we present an immuno‐electron tomography (IET) method, which allows the determination of protein distribution patterns on reconstructed organelles from yeast. In addition, we extend this IET approach into a correlative light microscopy‐electron tomography procedure where structures positive for a specific protein localized through a fluorescent signal are resolved in 3D. These new investigative tools for yeast will help to advance our understanding of the endomembrane system organization in eukaryotic cells.      

Integrative taxonomy and evolutionary history of a newly revealed spider Dysdera ninnii complex (Araneae: Dysderidae)

The spider genus Dysdera is a species‐rich clade of specialized woodlice predators, composed typically of complexes of sibling species. Here, we analyse the Dysdera ninnii complex, distinguishing three species that exhibit slight but constant differences in the morphology of their copulatory organs, and in their genetic background. We designate a neotype for D. ninnii and redescribe it. We consider Dysdera pavesii Thorell, 1873 to be a junior synonym of Dysdera ninnii Canestrini, 1868. In addition, we describe two new species ( D ysdera moravica sp. nov. and D ysdera microdonta sp. nov. ). All three species occur in the region of north‐eastern Italy, Slovenia, and north‐western Croatia. D ysdera moravica sp. nov. expanded to central Europe. The species occur allopatrically or parapatrically. All three species possess the same diploid number and X0 sex chromosome determination. In some individuals we found chromosome fusions, and such polymorphism is common in spiders with holokinetic chromosomes. The analysis of mitochondrial (cytochrome c oxidase subunit I, COI) and nuclear ribosomal (internal transcribed spacer 2, ITS2) DNA markers revealed two clades, one formed by D. ninnii and D . microdonta sp. nov. , and a second by D . moravica sp. nov. Species of the first clade are not well defined by DNA markers. We noticed only weak separation of maternally inherited COI, and even overlap of autosomally inherited ITS2 sequences. We suggest that either short speciation time, unfinished lineage sorting, or rare hybridization events caused this pattern. In one sample of D . microdonta sp. nov. we detected the coxA gene of a Rickettsia species, which is the first record of this parasitic bacteria from the spider family Dysderidae. D ysdera microdonta sp. nov. occurs at higher altitudes than D. ninnii, and their distribution ranges form a long contact zone. Remarkably, we did not record any overlap of the two distribution ranges, suggesting that the lack of a precopulatory interspecific barrier causes a loss of reproduction potential. We hypothesize that because of the unsolved interspecific barrier together with only tiny differences in morphology and COI sequences, and no differences in karyotypes and ITS2 sequences, the D. ninnii species complex is evolutionarily young. © 2014 The Linnean Society of London     

Synthesis and enantioselectivity of the enantiomers of PG9 and SM21, new potent analgesic and cognition-enhancing drugs

The enantiomers of two α-tropanyl esters, SM₂₁ (1) and PG₉ (2), derived from (+)-R-hyoscyamine, that act by increasing the central cholinergic tone, were obtained by esterification after resolution of the corresponding racemic acids [(−)-S-1, (−)-R-2 and (+)-S-2] and by stereospecific synthesis [(+)-R-1]. Their analgesic and cognition-enhancing activities were tested in mice and their ACh-releasing properties determined on rat parietal cortex. These compounds show enantioselectivity in analgesic and cognition-enhancing tests on mice, the eutomers being the isomers which possess the same spatial arrangement of the groups on the chiral atom as (+)-R hyoscyamine [(+)-R-SM₂₁, (+)-S-PG₉]. The ACh-releasing effect of the enantiomers of SM₂₁ in rats is in agreement with the results in mice, while PG₉ enantiomers do not show any appreciable enantioselectivity in this test. On the basis of the different effects of the 5-HT₄ antagonist SDZ 205557 on analgesia induced by the enantiomers of 1 and 2 and by (+)-R-hyoscyamine and the α-tropanyl ester of 2-phenylpropionic acid 3, a mechanism of action is proposed for this class of compounds. © 1996 Wiley-Liss, Inc.     

In vitro propagation ofMedicago andLotus species by node culture

Summary Nodes ofMedicago sativa, Lotus corniculatus, Lotus tenuis, andLotus pedunculatus were cultured on MS basal media with different growth regulators. InM. sativa each node produced one shoot and the apical dominance was unaffected by high levels of cytokinins, and subsequent cycles of culture. Shoot development was stimulated by the presence ofN ⁶-isopentenyl-adenine in the culture medium and was dependent on the genotype of the explant. Shoot development was not affected by the original position of the node on the plant nor by the plant age. Shoots rooted in MS medium gelled with starch and containing 2 mg·liter⁻¹ indol-3-acetic acid. In the threeLotus species, node culture was a more effective technique than inM. sativa. The number of shoots per node increased with the time of culture and with the presence of 0.05 mg·liter⁻¹ of 6-benzylaminopurine. The highest number of shoots derived from one node was achieved inL. pedunculatus and inL. tenuis by culturing single nodes, whereas inL. corniculatus stem segments had to be totally covered by the medium for success. Rooting was easily achieved in MS medium with or without auxins.     

Characterization of trotter horses urine metabolome by means of proton nuclear magnetic resonance spectroscopy

Background

Metabolomics has been recognized as a powerful approach for disease screening. In order to highlight potential health issues in subjects, a key factor is the possibility to compare quantitatively the metabolome of their biofluids with reference values from healthy individuals. Such efforts towards the systematic characterization of the metabolome of biofluids in perfect health conditions, far from concluded for humans, have barely begun on horses.

Objectives

The present work attempts, for the first time, to give reference quantitative values for the molecules mostly represented in the urine metabolome of horses at rest and under light training, as observable by ¹H-NMR.

Methods

The metabolome of ten trotter horses, four male and six female, ranging from 3 to 8 years of age, has been observed by ¹H-NMR spectroscopy before and after three training sessions.

Results

We could characterize and quantify 54 molecules in trotter horse urine, originated from diet, protein digestion, energy generation or gut-microbial co-metabolism.

Conclusion

We were able to describe how gender, age and exercise affected their concentration, by means of a two steps protocol based on univariate and robust principal component analysis.

     

Selenium Utilization by GPX4 Is Required to Prevent Hydroperoxide-Induced Ferroptosis

1. Download high-res image (131KB)
2. Download full-size image

     

Oxidative events in neuronal and glial cell-enriched fractions of rat cerebral cortex

The aim of this work was to investigate how neurons and glial cells separated from rat brain cortex respond to “in vitro” oxidative stress induced by incubation of the cellular fractions in the presence of prooxidant mixtures; in addition, the endogenous enzymatic antioxidant capacity of the purified fractions was investigated. Neuronal and glial cell-enriched fractions were obtained from rat cerebral cortex following passages of the tissue through meshes and centrifugations. The following parameters were evaluated: antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPx), and glucose-6-phosphate dehydrogenase (G6PDH); lipid peroxidation products (TBARS) prior to (basal) and after (iron-stimulated) incubation with a mixture of iron and ascorbic acid; intracellular production of reactive oxygen species (ROS) using a fluorescent probe, dichlorofluorescin-diacetate, in basal, iron-stimulated, and menadione stimulated conditions. SOD and GSHPx activities showed no significant changes between neurons and glia, whereas CAT and G6PDH activities were found to be significantly lower in glia than in neurons. TBARS levels were significantly lower in the glial fraction than in neurons, both in basal and iron-stimulated conditions. ROS production showed no differences between neurons and glia in both basal and menadione-stimulated conditions. Iron-stimulation produced a marked increase in ROS production, limited to the neuronal fraction, with the glial values being similar to the basal ones. Our conclusion is that glia and neurons isolated from rat cerebral cortex show a similar pattern of the most important antioxidant enzymes and of their basal ROS production, whereas glia is more resistant in “oxidative stress” conditions.     

Instrumental and Non-Instrumental Evaluation of 4-Meter Walking Speed in Older Individuals

BackgroundManual measurement of 4-meter gait speed by a stopwatch is the gold standard test for functional assessment in older adults. However, the accuracy of this technique may be biased by several factors, including intra- and inter-operator variability. Instrumental techniques of measurement using accelerometers may have a higher accuracy. Studies addressing the concordance between these two techniques are missing. The aim of the present community-based observational study was to compare manual and instrumental measurements of 4-meter gait speed in older individuals and to assess their relationship with other indicators of physical performance.MethodsOne-hundred seventy-two (69 men, 103 women) non-disabled community-dwellers aged ≥65 years were enrolled. They underwent a comprehensive geriatric assessment including physical function by Short Physical Performance Battery (SPPB), hand grip strength, and 6-minute walking test (6MWT). Timed usual walking speed on a 4-meter course was assessed by using both a stopwatch (4-meter manual measurement, 4-MM) and a tri-axial accelerometer (4-meter automatic measurement, 4-MA). Correlations between these performance measures were evaluated separately in men and women by partial correlation coefficients.ResultsIn both genders, 4-MA was associated with 4-MM (men r = 0.62, p<0.001; women r = 0.73, p<0.001), handgrip strength (men r = 0.40, p = 0.005; women r = 0.29, p = 0.001) and 6MWT (men r = 0.50, p = 0.0004; women r = 0.22, p = 0.048). 4-MM was associated with handgrip strength and 6MWT in both men and women. Considering gait speed <0.6 m/s as diagnostic of dismobility syndrome, the two methods of assessment disagreed, with a different categorization of subjects, in 19% of men and 23% of women. The use of accelerometer resulted in 29 (13 M, 16 F) additional diagnoses of dismobility, compared with the 4-MM.ConclusionsIn an older population, the concordance of gait speeds manually or instrumentally assessed is not optimal. The results suggest that manual measures might lead to misclassification of a substantial number of subjects. However, longitudinal studies using standardized and validated procedures aimed at the comparison of different techniques are needed before recommending the use of accelerometers in comprehensive geriatric assessment.