Localization of vicilin genes via polymerase chain reaction on microisolated field bean chromosomes

A new technique is reported for the physical mapping of low copy DNA sequences on plant chromosomes. Individual chromosomes were microisolated and their DNA used as the target for the polymerase chain reaction in order to identify the chromosome carrying a specific gene sequence. The use of defined translocation chromosomes further refined the resolution of the method to a subchromosomal level. To demonstrate the applicability of the procedure genes have been localized coding for vicilin seed storage proteins on the field bean Vicia faba L. in a region which includes the centromere and the proximal parts of the short and the long arms of chromosome II.     

Traits of dominant plant species drive normalized difference vegetation index in grasslands globally

Aim

Theoretical, experimental and observational studies have shown that biodiversity–ecosystem functioning (BEF) relationships are influenced by functional community structure through two mutually non-exclusive mechanisms: (1) the dominance effect (which relates to the traits of the dominant species); and (2) the niche partitioning effect [which relates to functional diversity (FD)]. Although both mechanisms have been studied in plant communities and experiments at small spatial extents, it remains unclear whether evidence from small-extent case studies translates into a generalizable macroecological pattern. Here, we evaluate dominance and niche partitioning effects simultaneously in grassland systems world-wide.

Location

Two thousand nine hundred and forty-one grassland plots globally.

Time period

2000–2014.

Major taxa studied

Vascular plants.

Methods

We obtained plot-based data on functional community structure from the global vegetation plot database “sPlot”, which combines species composition with plant trait data from the “TRY” database. We used data on the community-weighted mean (CWM) and FD for 18 ecologically relevant plant traits. As an indicator of primary productivity, we extracted the satellite-derived normalized difference vegetation index (NDVI) from MODIS. Using generalized additive models and deviation partitioning, we estimated the contributions of trait CWM and FD to the variation in annual maximum NDVI, while controlling for climatic variables and spatial structure.

Results

Grassland communities dominated by relatively tall species with acquisitive traits had higher NDVI values, suggesting the prevalence of dominance effects for BEF relationships. We found no support for niche partitioning for the functional traits analysed, because NDVI remained unaffected by FD. Most of the predictive power of traits was shared by climatic predictors and spatial coordinates. This highlights the importance of community assembly processes for BEF relationships in natural communities.

Main conclusions

Our analysis provides empirical evidence that plant functional community structure and global patterns in primary productivity are linked through the resource economics and size traits of the dominant species. This is an important test of the hypotheses underlying BEF relationships at the global scale.     

Secondary structure of proteins associated in thin films

The solid state secondary structure of myoglobin, RNase A, concanavalin A (Con A), poly(L -lysine), and two linear heterooligomeric peptides were examined by both far-uv CD spectroscopy¹ and by ir spectroscopy. The proteins associated from water solution on glass and mica surfaces into noncrystalline, amorphous films, as judged by transmission electron microscopy of carbon-platinum replicas of surface and cross-fractured layer. The association into the solid state induced insignificant changes in the amide CD spectra of all α-helical myoglobin, decreased the molar ellipticity of the α/β RNase A, and increased the molar ellipticity of all-β Con A with no change in the positions of the bands' maxima. High-temperature exposure of the films induced permanent changes in the conformation of all proteins, resulting in less α-helix and more β-sheet structure. The results suggest that the protein α-helices are less stable in films and that the secondary structure may rearrange into β-sheets at high temperature. Two heterooligomeric peptides and poly (L -lysine), all in solution at neutral pH with “random coil” conformation, formed films with variable degrees of their secondary structure in β-sheets or β-turns. The result corresponded to the protein-derived Chou-Fasman amino acid propensities, and depended on both temperature and solvent used. The ir and CD spectra correlations of the peptides in the solid state indicate that the CD spectrum of a “random” structure in films differs from random coil in solution. Formic acid treatment transformed the secondary structure of the protein and peptide films into a stable α-helix or β-sheet conformations. The results indicate that the proteins aggregate into a noncrystalline, glass-like state with preserved secondary structure. The solid state secondary structure may undergo further irreversible transformations induced by heat or solvent. © 1993 John Wiley & Sons, Inc.     

Differential modulation of white adipose tissue endocannabinoid levels by n-3 fatty acids in obese mice and type 2 diabetic patients

n-3 polyunsaturated fatty acids (n-3 PUFA) might regulate metabolism by lowering endocannabinoid levels. We examined time-dependent changes in adipose tissue levels of endocannabinoids as well as in parameters of glucose homeostasis induced by n-3 PUFA in dietary-obese mice, and compared these results with the effect of n-3 PUFA intervention in type 2 diabetic (T2DM) subjects. Male C57BL/6J mice were fed for 8, 16 or 24?weeks a high-fat diet alone (cHF) or supplemented with n-3 PUFA (cHF?+?F). Overweight/obese, T2DM patients on metformin therapy were given for 24?weeks corn oil (Placebo; 5?g/day) or n-3 PUFA concentrate as above (Omega-3; 5?g/day). Endocannabinoids were measured by liquid chromatography-tandem mass-spectrometry. Compared to cHF-fed controls, the cHF?+?F mice consistently reduced 2-arachidonoylglycerol (up to ~2-fold at week 24) and anandamide (~2-fold) in adipose tissue, while the levels of endocannabinoid-related anti-inflammatory molecules N-eicosapentaenoyl ethanolamine (EPEA) and N-docosahexaenoyl ethanolamine (DHEA) increased more than ~10-fold and ~8-fold, respectively. At week 24, the cHF?+?F mice improved glucose tolerance and fasting blood glucose, the latter being positively correlated with adipose 2-arachidonoylglycerol levels only in obese cHF-fed controls, like fasting insulin and HOMA-IR. In the patients, n-3 PUFA failed to reduce 2-arachidonoylglycerol and anandamide levels in adipose tissue and serum, but they increased both adipose tissue and serum levels of EPEA and DHEA. In conclusion, the inability of n-3 PUFA to reduce adipose tissue and serum levels of classical endocannabinoids might contribute to a lack of beneficial effects of these lipids on glucose homeostasis in T2DM patients.     

Changes in the activity of some metabolic enzymes in the heart of SHR rat incurred by transgenic expression of CD36

Hypertension, dyslipidemia, and insulin resistance in the spontaneously hypertensive rat (SHR) can be alleviated by rescuing CD36 fatty acid translocase. The present study investigated whether transgenic rescue of CD36 in SHR could affect mitochondrial function and activity of selected metabolic enzymes in the heart. These analyses were conducted on ventricular preparations derived from SHR and from transgenic strain SHR-Cd36 that expresses a functional wild-type CD36. Our respirometric measurements revealed that mitochondria isolated from the left ventricles exhibited two times higher respiratory activity than those isolated from the right ventricles. Whereas, we did not observe any significant changes in functioning of the mitochondrial respiratory system between both rat strains, enzyme activities of total hexokinase, and both mitochondrial and total malate dehydrogenase were markedly decreased in the left ventricles of transgenic rats, compared to SHR. We also detected downregulated expression of the succinate dehydrogenase subunit SdhB (complex II) and 70 kDa peroxisomal membrane protein in the left ventricles of SHR-Cd36. These data indicate that CD36 may affect in a unique fashion metabolic substrate flexibility of the left and right ventricles.     

Nitrogen acquisition,net production and allometry of <Emphasis Type="Italic">Alnus fruticosa</Emphasis> at a young moraine in Koryto Glacier Valley,Kamchatka, Russian Far East

Alders (Alnus spp.) often dominate at nutrient-poor sites by symbiotic relations with atmospheric nitrogen-fixing bacteria. However, little is known about quantitative relationships between root nodule as a nitrogen acquisition organ and leaf as a carbon acquisition organ. To examine carbon allocation, nitrogen acquisition and net production in nutrient-poor conditions, we examined allocation patterns among organs of shrub Alnus fruticosa at a young 80-year-old moraine in Kamchatka. Slopes of double-log allometric equations were significantly smaller than 1.0 for the root mass, leaf mass and root nodule mass against stem mass, and for the root nodule mass against root mass, indicating that smaller individuals invested disproportionally more biomass into resource-acquiring leaf and root tissues than to supportive tissues compared to older individuals. The slope of allometric equation of root depth against stem height was 0.542, indicating that smaller/younger individuals allocate disproportionally more biomass into root length growth than stem height growth. On the contrary, the root nodule mass isometrically scaled to leaf mass. The whole-plant nitrogen content also isometrically scaled to root nodule mass, indicating that a certain ratio of nitrogen acquisition depended on root nodules, irrespective of plant size. Although the net production per plant increased with the increase in stem mass, the slope of the double-log regression was smaller than 1.0. On the contrary, the net production per plant isometrically increased with leaf mass, root nodule mass and leaf nitrogen content per plant. Since the leaf mass isometrically scaled to root nodule mass, growth of each individual occurred at the leaves and root nodules in a coordinated manner. It is suggested that their isometric increase contributes to the increase in net production per plant for A. fruticosa in nutrient-poor conditions.     

Coxsackievirus B3 entry into the host cell interferes with G-protein-mediated transmembrane signalling

In the present work we used various cell lines in order to study the possible effect of coxsackievirus B3 (CVB3) entry on the adenylyl cyclase transmembrane signalling system. A significant decrease (by about 10–20%) was found in forskolin-augmented as well as in AlF ₄ ^– - and GTPS-sensitive adenylyl cyclase activity in plasma membranes isolated from HeLa, HEp-2, Vero and green monkey kidney cells shortly (up to 60 min) preincubated with CVB3 (5 PFU/cell). Moreover, the ability of G-proteins derived from plasma membranes of infected cells to reconstitute AC activity in the cyc^– mutant of S49 cells was also reduced. Content of G-protein subunits, however, remained unchanged after CVB3 attachment. Functional alterations in the G-protein-mediated adenylyl cyclase signalling system were accompanied by a marked decrease (by about 20–40%) of intracellular cAMP levels in virus-affected cells. These findings demonstrate clearly that CVB3 may affect functioning of the G-protein regulated adenylyl cyclase transmembrane signalling system in virus-sensitive cells as early as during the first period of its contact with the cellular plasma membrane.     

Knockdown of human Oxa1l impairs the biogenesis of F1Fo-ATP synthase and NADH:ubiquinone oxidoreductase

The Oxa1 protein is a founding member of the evolutionarily conserved Oxa1/Alb3/YidC protein family, which is involved in the biogenesis of membrane proteins in mitochondria, chloroplasts and bacteria. The predicted human homologue, Oxa1l, was originally identified by partial functional complementation of the respiratory growth defect of the yeast oxa1 mutant. Here we demonstrate that both the endogenous human Oxa1l, with an apparent molecular mass of 42 kDa, and the Oxa1l-FLAG chimeric protein localize exclusively to mitochondria in HEK293 cells. Furthermore, human Oxa1l was found to be an integral membrane protein, and, using two-dimensional blue native/denaturing PAGE, the majority of the protein was identified as part of a 600-700 kDa complex. The stable short hairpin (sh)RNA-mediated knockdown of Oxa1l in HEK293 cells resulted in markedly decreased steady-state levels and ATP hydrolytic activity of the F₁F_o-ATP synthase and moderately reduced levels and activity of NADH:ubiquinone oxidoreductase (complex I). However, no significant accumulation of corresponding sub-complexes could be detected on blue native immunoblots. Intriguingly, the achieved depletion of Oxa1l protein did not adversely affect the assembly or activity of cytochrome c oxidase or the cytochrome bc₁ complex. Taken together, our results indicate that human Oxa1l represents a mitochondrial integral membrane protein required for the correct biogenesis of F₁F_o-ATP synthase and NADH:ubiquinone oxidoreductase.