Peroxisomes were visualized for the first time in living fission yeast cells. In small, newly divided cells, the number of peroxisomes was low but increased in parallel with the increase in cell ...length/volume that accompanies cell cycle progression. In cells grown in oleic acid, both the size and the number of peroxisomes increased. The peroxisomal inventory of cells lacking the dynamin-related proteins Dnm1 or Vps1 was similar to that in wild type. By contrast, cells of the double mutant dnm1Δ vps1Δ contained either no peroxisomes at all or a small number of morphologically aberrant organelles. Peroxisomes exhibited either local Brownian movement or longer-range linear displacements, which continued in the absence of either microtubules or actin filaments. On the contrary, directed peroxisome motility appeared to occur in association with mitochondria and may be an indirect function of intrinsic mitochondrial dynamics. We conclude that peroxisomes are present in fission yeast and that Dnm1 and Vps1 act redundantly in peroxisome biogenesis, which is under cell cycle control. Peroxisome movement is independent of the cytoskeleton but is coupled to mitochondrial dynamics.
Pexophagy is a selective autophagy process that degrades damaged and/or superfluous peroxisomes in the yeast vacuole or in mammalian lysosomes. The molecular mechanisms of pexophagy are well studied ...in yeast. Peroxisomes can be rapidly induced by oleate in the budding yeast, Saccharomyces cerevisiae, and by oleate or methanol in the methylotrophic yeast, Pichia pastoris. A number of peroxisomal matrix enzymes, such as 3-ketoacyl CoA thiolase (thiolase) and alcohol oxidase (AOX), are upregulated correspondingly to meet metabolic demands of the cells. Removal of these peroxisome-inducing carbon sources creates conditions wherein peroxisomes are superfluous and results in pexophagy and the degradation of these peroxisomal matrix enzymes. In this chapter, we discuss different assays to monitor pexophagy in yeast. These assays rely on tracking the localization of the BFP-SKL protein (a peroxisomally targeted version of the blue fluorescent protein) by microscopy, biochemical analysis of the degradation of peroxisomal matrix proteins, thiolase and AOX, and/or measuring the reduction of AOX activity during pexophagy.
A biennial experiment (2009 and 2010) was conducted at Calvi (Benevento, Southern Italy) to evaluate the effect of compost by organic fraction municipal solid waste (OFMSW), in combination with ...mineral nitrogen (N) fertilization, on yield and quality of three Dark Fire-cured (Kentucky) tobacco cultivars commonly cultivated at Benevento province (Campania region, Southern Italy). Six N fertilization treatments (N0 = soil N reserves available for plant growth; MIN = 135 kg ha−1 of N applied as mineral fertilizer; C10 = 10 Mg d.w. ha−1 compost; C10N = 10 Mg d.w. ha−1 compost + 50% MIN; C20 = 20 Mg d.w. ha−1 compost; C20N = 20 Mg d.w. ha−1 compost + 50% MIN) were combined with the following cultivars: (i) Foiano, medium early maturing; (ii) Riccio Beneventano (local ecotype), medium maturing; (iii) SKL, medium maturing. Yield of cured leaves (Mg ha−1) and growth components (number of leaves per plant, mean individual leaf area, leaf area per plant, specific leaf weight, stem diameter and height) and color parameters (L*, a*/b*) were measured. Leaf quality traits (nitrates, total N and alkaloids contents, score) and N use efficiency were also determined. The best growth and yield performance was reached in 2010 when plants were taller, developed both stems that were more robust and leaves having greater individual leaf area, and showed a higher leaf area per plant than in the first year. Regardless of the form of applied N (compost, mineral fertilizer, or a combination of both), tobacco plants appeared to be directly and positively influenced by increasing quota of readily available N received by each treatment, which was determined at the beginning of field growth by N soil balance and taking into account the percentage of N supplied by organic (compost) and mineral fertilizers. Results obtained with compost treatments, particularly when combined with mineral fertilizer (at C10N more than C20N), appeared comparable or sometimes better than those of full mineral fertilization although N fertilization by synthetic products was applied at very low doses.
The characteristics of the 5th Supercomputer Nurion Knights Landing (KNL) system of the Korea Institute of Science and Technology Information (KISTI) were analyzed by developing ultra-high resolution ...atmospheric and ocean numerical circulation models. These models include the Weather Research and Forecasting System (WRF), Regional Ocean Modeling System (ROMS), and Unstructured Grid Finite Volume Community Ocean Model (FVCOM). Ideal and real-case experiments were simulated for each model according to the number of parallelized cores used for comparing performances. Identical experiments were performed on a general multicore system (Skylake and a general cluster system) for a performance comparison with the Nurion KNL system. Although the KNL system has more than twice as many cores per node as the Skylake system, the KNL system demonstrated 1/3 of the performance rate of the Skylake system. However, the performance rate of the Nurion KNL system was approximately 43% for all experiments. Reducing the number of cores per node in the KNL system by half (36 cores) is the most efficient method when the total number of cores is less than 256 cores, while it is more economical to use all cores when using more than 256 cores. In all experiments, the performance was continuously improved even for a maximum core experiment (1024 cores), thereby indicating that the KNL system can effectively simulate ultra-high resolution numerical circulation models.
Although rice (Oryza sativa L.) produces little glycine betaine (GB), it has two betaine aldehyde dehydrogenase (BADH; EC 1.2.1.8) gene homologs (OsBADH1 and OsBADH2). We found that OsBADH1 catalyzes ...the oxidation of acetaldehyde efficiently, while the activity of OsBADH2 is extremely low. The accumulation of OsBADH1 mRNA decreases following submergence treatment, but quickly recovers after re-aeration. We confirmed that OsBADH1 localizes in peroxisomes. In this paper, a possible physiological function of OsBADH1 in the oxidation of acetaldehyde produced by catalase in rice plant peroxisomes is discussed.
Emp24 is a member of the p24 protein family, which was initially localized to the endoplasmic reticulum, Golgi and COP vesicles, but has recently shown to be associated with
Saccharomyces cerevisiae ...peroxisomes as well. Using cell fractionation and electron- and fluorescence microscopy, we show that in the yeast
Hansenula polymorpha, Emp24 also associates with peroxisomes. In addition, we show that peroxisome numbers are strongly decreased in
H. polymorpha cells lacking two proteins of the p24 complex, Emp24 and Erp3. Detailed fluorescence microscopy analyses suggest that
emp24.erp3 cells are disturbed in peroxisome fission and inheritance.
We used the dye N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl) pyridinium dibromide (FM4-64) and a fusion protein, consisting of the green fluorescent protein appended to the ...peroxisomal targeting signal, Ser-Lys-Leu (SKL), to label the vacuolar membrane and the peroxisomal matrix, respectively, in living Pichia pastoris cells and followed by fluorescence microscopy the morphological and kinetic intermediates in the vacuolar degradation of peroxisomes by microautophagy and macroautophagy. Structures corresponding to the intermediates were also identified by electron microscopy. The kinetics of appearance and disappearance of these intermediates is consistent with a precursor-product relationship between intermediates, which form the basis of a model for microautophagy. Inhibitors affecting different steps of microautophagy did not impair peroxisome delivery to the vacuole via macroautophagy, although inhibition of vacuolar proteases affected the final vacuolar degradation of green fluorescent protein (S65T mutant version GFP)-SKL via both autophagic pathways. P. pastoris mutants defective in peroxisome microautophagy (pag mutants) were isolated and characterized for the presence or absence of the intermediates. These mutants, comprising 6 complementation groups, support the model for microautophagy. Our studies indicate that the microautophagic degradation of peroxisomes proceeds via specific intermediates, whose generation and/or processing is controlled by PAG gene products, and shed light on the poorly understood phenomenon of peroxisome homeostasis.
Objectives. To determine whether labor is associated with alterations of the levels of soluble c-kit ligand (sKL) and endothelin-1 (ET-1) in maternal plasma and umbilical cord blood.
Methods. The sKL ...and ET-1 levels were investigated in umbilical cord and maternal plasma on the day of delivery in 18 pregnant women with vaginal delivery during labor, 18 non-pregnant women and 9 pregnant women before cesarean delivery, using an ELISA assay.
Results. Umbilical cord plasma sKL levels were significantly higher than the maternal plasma in both types of delivery (p = 0.0001, p < 0.0001, respectively). However, maternal plasma ET-1 levels in the presence of labor were significantly higher than the cesarean delivery group (p < 0.0001). No difference was noted for sKL and ET-1 in umbilical cord vessels of both groups. Furthermore, a highly significant inverse correlation was documented between the individual levels of cord plasma ET-1 and the levels of cord plasma sKL (r = −0.6269, p = 0.0054).
Conclusions. The sKL levels found in umbilical cord plasma are consistent with the pleiotropic effects of sKL in facilitating the transition of the fetus to the neonatal stage. The reduced ET-1 maternal plasma levels, compared to non-pregnant women, probably are indicative of a putative mechanism for embryo protection from vasoconstriction sequelae. This assumption is strengthened by the corresponding ET-1 levels in umbilical cord plasma.
A 25 kb segment of genomic DNA from Trypanosoma cruzi, the causative agent of Chagas' disease, was sequenced. It contains five genes, pyr1, pyr2, pyr3, pyr4, and pyr6-5, encoding all six enzymes ...involved in de novo pyrimidine biosynthesis, glutamine-dependent carbamoyl-phosphate synthetase, aspartate carbamoyltransferase, dihydroorotase, dihydroorotate dehydrogenase, and orotidine-5'-phosphate decarboxylase linked with orotate phosphoribosyltransferase, respectively. The pyr genes constitute a polycistronic transcription unit on an 800 kb chromosomal DNA in the order of pyr1, pyr3, pyr6-5, pyr2, and pyr4 from the 5' terminus, with intervening sequences of 2.2, 0.4, 8.1, and 0.8 kb. The amino acid sequences deduced from the trypanosomatid pyr genes, except for pyr6, showed closer similarities to mammalian and yeast sequences, and less similarity to archaeal and bacterial sequences. The last two enzymes encoded by a single gene, pyr6-5, are covalently linked in the order opposite to mammalian pyr5-6, and possess a putative glycosomal targeting signal tripeptide, serine-lysine-leucine, at the C terminus. The calculated isoelectric points of 9.3 and 9.9 are also diagnostic of the glycosomal localization of these enzymes. We conclude that the T. cruzi pyr gene organization represents an early progenitor in de novo pyrimidine biosynthesis in eukaryotic lineage, and that the independent pyr genes may have evolved before the gene fusion events that resulted in the three mammalian-type genes, pyr1-3-2, pyr4, and pyr5-6, for UMP synthesis. Peculiarities in the trypanosomatid pyr6-5 gene product are discussed.
A column-switching high-performance liquid chromatography (HPLC) method is described for the simultaneous determination of loganin and sweroside, which are the active ingredients of purified herbal ...extract from
Lonicera japonica (SKL JI), in rat plasma using column-switching and ultraviolet (UV) absorbance detection. Plasma was simply filtrated prior to injection to the HPLC system consisting of a clean-up column, a concentrating column, and an analytical column, which were connected with two six-port switching valves. Detection of loganin and sweroside was accurate and repeatable, with a limit of quantitation of 0.05
μg
ml
−1 in plasma. The calibration curves for both loganin and sweroside were linear over the concentration ranges of 0.05–40.0 and 0.02–40.0
μg
ml
−1 in rat plasma, respectively. The intra- and inter-day precision over the concentration range for loganin and sweroside were lower than 8.1 and 10.9% (relative standard deviation, R.S.D.), and accuracy was between 94.7 and 113.5 and 95.0 and 113.1%, respectively. This method has been successfully applied to determine the levels of loganin and sweroside in rat plasma samples from pharmacokinetics studies.
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