Functional analysis of the uL11 protein impact on translational machinery

The ribosomal GTPase associated center constitutes the ribosomal area, which is the landing platform for translational GTPases and stimulates their hydrolytic activity. The ribosomal stalk represents a landmark structure in this center, and in eukaryotes is composed of uL11, uL10 and P1/P2 proteins. The modus operandi of the uL11 protein has not been exhaustively studied in vivo neither in prokaryotic nor in eukaryotic cells. Using a yeast model, we have brought functional insight into the translational apparatus deprived of uL11, filling the gap between structural and biochemical studies. We show that the uL11 is an important element in various aspects of ‘ribosomal life’. uL11 is involved in ‘birth’ (biogenesis and initiation), by taking part in Tif6 release and contributing to ribosomal subunit-joining at the initiation step of translation. uL11 is particularly engaged in the ‘active life’ of the ribosome, in elongation, being responsible for the interplay with eEF1A and fidelity of translation and contributing to a lesser extent to eEF2-dependent translocation. Our results define the uL11 protein as a critical GAC element universally involved in trGTPase ‘productive state’ stabilization, being primarily a part of the ribosomal element allosterically contributing to the fidelity of the decoding event.     

Heteroplasmy and paternally oriented shift of the organellar DNA composition in barley-wheat hybrids during backcrosses with wheat parents.

Mitochondrial (mt) and chloroplast (ct) genome inheritance was studied in barley-wheat hybrids, as were their progenies obtained from backcrosses with different common wheat cultivars, by monitoring the composition of 4 mtDNA (coxI, a 5'-flanking region of cob, nad3-orf156, and 5'-upstream region of 18S/5S) and 2 ctDNA (simple-sequence repeat locus downstream of trnS and a 3'-flanking region of rbcL) loci. In male sterile F1 and BC1 plants, maternal barley mtDNA fragments were mainly detected and very low levels of paternal wheat fragments were occasionally detected by PCR in coxI, a 5'-flanking region of cob and nad3-orf156, whereas a 5'-upstream region of 18S/5S showed clear heteroplasmy, containing both maternal and paternal copies, with maternal copies prevailing. Plants showing such heteroplasmic mtDNA composition remained either semisterile or became completely sterile in the later backcross generations. Only maternal ctDNA copies were detected in these plants. In 3 stable, self-fertile, and vigourous lines obtained in the advanced backcross generations and possessing recombinant wheat nuclear genome, however, only mt- and ctDNA copies of wheat parents were detected; thus, the original alloplasmic condition appeared to be lost. Our results suggest that transmission followed by selective replication of the paternal wheat organellar DNA leads to a paternally oriented shift of the organellar DNA composition in barley-wheat hybrids, which correlates with the restoration of fertility and plant vigour. These 2 processes seem to be related to nucleocytoplasmic compatibility and to be under the control of the nuclear genome composition.     

Flavonoid biosynthesis in tomato fruit cuticles after in vivo incorporation of 3 H-phenylalanine precursor

Radiolabelling of epicuticular waxes and cutin of isolated tomato fruit cuticles were determined after fruit surface application of 3 H-phenylalanine precursor. During fruit ripening, the precursor is incorporated in different phenolic components: the flavanone naringenin was found to be the major compound in the epicuticular waxes, while the amount of the labelled flavonoid in the cutin matrix was progressively increased throughout fruit ripening. Confocal microscopy, together with experimental estimation of the mobility (diffusion coefficient, D ) and affinity (partition coefficient, K ) of the flavonoids naringenin and chalconaringenin for the different cuticular components, indicate that these compounds are extruded to the outer surface of tomato fruits, forming molecular clusters.     

Kinetic Studies on the Interaction of Ferricytochrome c with Anionic Surfactants

The kinetics of absorbance and fluorescence changes of cytochrome c as induced by an aqueous solution of the anionic surfactant sodium dodecyl sulfate (SDS) or sodium bis(2-ethylhexyl)sulfosuccinate (AOT) are studied. The results are compared with far-UV circular dichroism (CD) spectra. Both surfactants cause similar alterations in the secondary structure of cytochrome c, while their influence on the heme environment of cytochrome c is different. In the presence of AOT below and above critical micellar concentration a conversion of the low-spin native cytochrome c to a denatured low-spin protein not having methionine ligand takes place. In the presence of SDS micelles conversion of the native protein to a denatured mixed-spin form occurs. The changes in the heme group induced by both surfactants occur independently of the alterations in tertiary structure.     

The effects of modulation of gamma-glutamyl transpeptidase activity in HepG2 cells on thiol homeostasis and caspase-3-activity

The present studies aimed to elucidate how the modulation of gamma-glutamyl transpeptidase (gammaGT) activity in human hepatoma (HepG2) cell line influences H(2)O(2) production, caspase 3 activity, protein S-thiolation by glutathione (GSH), cysteinyl-glycine (Cys-Gly) and cysteine (Cys), and the level of other redox forms of these thiols. The experiments showed that 1-h stimulation of gammaGT elevated H(2)O(2) production, leading to prooxidant conditions. After 24-h stimulation, H(2)O(2) concentration was at the control level, while Cys-Gly-, Cys- and GSH-dependent S-thiolation was markedly increased, which was accompanied by a drop in caspase-3 activity. The inhibition of gammaGT activity by acivicin led to H(2)O(2) decrease after 1-h incubation which still persisted after 24 h. The inhibition of gammaGT activity in HepG2 cells was also connected with the lowering of S-thiolation with Cys and Cys-Gly and with increasing of caspase-3 activity. The results of our studies indicate that the modulation of gammaGT activity can be used to change cellular redox status, and can affect Cys- and Cys-Gly-dependent S-thiolation and caspase-3 activity. We suggest that the role of high gammaGT activity in HepG2 cells can be connected with production of reactive oxygen species and with S-thiolation with Cys and Cys-Gly that can influence activity of caspase 3.     

Measuring activity patterns using actigraphy in multiple sclerosis

Multiple sclerosis (MS) is a demyelinating disease resulting in impairments in motor and mental performance and restrictions in activities. Self-report instruments are commonly used to measure activity patterns; alternatively, actigraphs can be placed on several parts of the body. The aims of this study were to evaluate the superiority and specificity of actigraph placement (wrist vs. ankle) in subjects with MS and healthy controls and explore the relationship between self-report and objective activity patterns. A total of 19 subjects with definite MS and 10 healthy volunteers wore actigraphs on the non-dominant wrist and ankle for three days while they kept a log to register performed activities every .5 h. Wrist and ankle actigraphs produced similar activity patterns during the most active hours (09:00-20:30 h) (ANOVA, time×location interaction: F=.901, df=23, p=.597) in individuals with MS and healthy controls (between subjects factor F=3.275, p=.083). Wrist placement of the actigraphs was better tolerated than ankle placement. Wrist actigraph data corresponded to a higher degree with self-reported activities of the upper limbs in the early afternoon, whereas ankle data seem to reflect better whole body movements in the later afternoon/early evening. Overall, actigraph data correlated moderately with self-reported activity (r=.57 for ankle and r=.59 for wrist). The regression model revealed that self-reported activities explained 44% of the variance in ankle and 50% of wrist data. Wrist and ankle actigraphs produce similar activity patterns in subjects with MS and in healthy controls; however, the placement of actigraphs on the wrist is better tolerated. Ankle actigraphs reflect general movement but underestimate upper body activity. Subjective registration of activity level partly matches with objective actigraph measurement. A combination of both objective and subjective activity registration is recommended to evaluate the physical activity pattern of subjects with MS.     

Stem aqueous extracts of accumulator Bidens tripartita L. strongly promoted Solanum nigrum L. Cd hyperaccumulation from soil

Plant and Soil - Cover crops may restore soil functions lost with single summer crop sequences enhancing soil organic carbon and nitrogen (SOC and SON) stocks. We investigated the effect of cover...