Partial Purification and Characterization of a 3'- Phosphoadenosine 5' -Phosphosulfate: Desulfoglucosinolate Sulfotransferase from Cress (Lepidium sativum)

A 3′ -phosphoadenosine 5′ -phosphosulfate (PAPS):desulfoglucosinolate sulfotransferase (EC 2.8.2-) was extensively purified from light-grown cress (Lepidium sativum L.) seedlings by gel filtration and concanavalin A-Sepharose 4B, Matrex Gel Green A, and Mono Q fast protein liquid chromatography. The purified enzyme, which required bovine serum albumin for stabilization, had a native molecular weight of 31,000 ± 5,000 and an apparent isoelectric point of 5.2. Using PAPS (K_m 60 micromolar) as sulfur donor, it catalyzed the sulfation of desulfobenzylglucosinolate (K_m 82 micromolar), desulfo-p-hydroxybenzylglucosinolate (K_m 670 micromolar), and desulfoallylglucosinolate (K_m 6.5 millimolar) at an optimal pH of 9.0. All other potential substrates tested, including flavonoids, flavonoid glycosides, cinnamic acids, and phenylacetaldoxime, were not sulfated. Sulfotransferase activity was stimulated by MgCl₂, MnCl₂ and reducing agents and inhibited by ZnCl₂, PbNO₃ NiCl₂ and the reaction product PAP. The thiol reagents N-ethylmaleimide, p-chloromercuriphenylsulfonic acid, and 5,5′ -dithio-bis-(2-nitrobenzoic acid) were also potent inhibitors, but the enzyme was protected from covalent modification by β-mercaptoethanol. The kinetics of desulfobenzylglucosinolate sulfation were consistent with a rapid equilibrium ordered mechanism with desulfobenzylglucosinolate binding first and PAPS second.     

Germ-line deletions of mtDNA in mitochondrial myopathy.

mtDNA encodes subunits of the electron transport chain and is exclusively maternally inherited in mammals. It has been suggested that mtDNA might be the site of some of the mutations causing a group of human disorders called the "mitochondrial myopathies," because these may both be (1) accompanied by defects in the electron transport chain and (2) display a maternal pattern of inheritance. However, all of the deletions and duplications of mtDNA which occur in these patients have been sporadic, apart from families in whom affected members all carry different deletions suggesting a mutant autosomal dominantly inherited nuclear gene with de novo deletions in each individual. We present the first evidence for the presence of deleted mtDNAs in the germ line in these disorders. The patient carries a higher level of deleted mtDNAs than do his relatives, corresponding to severity of symptoms and consistent with a predicted dosage effect. "Selfishness" of deleted mtDNAs is probably one of the factors over and above random segregation of a small number of "founder" mtDNAs (the bottleneck hypothesis) which may be invoked to explain the usual distribution of mtDNAs in different tissues of patients with mtDNA deletions.     

Purification and properties of S-adenosyl-L-methionine: caffeic acid O-methyltransferase from leaves of spinach beet (Beta vulgaris L).

1. An enzyme catalysing the methylation of caffeic acid to ferulic acid, using S-adenosyl-L-methionine as methyl donor, has been extracted from leaves of spinach beet and purified 75-fold to obtain a stable preparation. 2. The enzyme showed optimum activity at pH 6.5, and did not require the addition of Mg2+ for maximum activity. 3. It was most active with caffeic acid, but showed some activity with catechol, protocatechuic acid and 3,4-dihydroxybenzaldehyde. The Km for caffeic acid was 68 muM. 4. 4. The Km for S-adenosyl-L-methionine was 12.5 muM. S-Adenosyl-L-homocystein (Ki = 4.4 muM) was a competitive inhibitor of S-adenosyl-L-methionine. 5. The synthesis of S-adenosyl-L-homocysteine from adenosine and L-homocysteine and its consequent effect on caffeic acid methylation were demonstrated with a partially-purified preparation from spinach-beet leaves, which possessed both S-adenosyl-L-homocysteine hydrolase (EC 3.3.1.1) and adenosine nucleosidase (EC 3.2.2.7) activities. This preparation was also able to catalyse the rapid breakdown of S-adenosyl-L-homocysteine to adenosine and adenine; the possible significance of this reaction in relieving the inhibition of caffeic acid methylation by S-adenosyl-L-homocystein is discussed.     

Effect of Castanospermine and Related Polyhydroxyalkaloids on Purified Myrosinase from Lepidium sativum Seedlings

Myrosinase (β-thioglucoside glucohydrolase, EC 3.2.3.1) was purified to apparent homogeneity from light-grown cress (Lepidium sativum L.) seedlings. This enzyme, which catalyzes hydrolysis of the glucosinolate sinigrin (K_m, 115 micromolar) at an optimum pH of 5.5 in sodium citrate buffer, had a native molecular weight of 130 ± 5 kilodaltons and an isoelectric point of 4.7 to 4.9. SDS-PAGE revealed two polypeptides with molecular weights of 62 and 65 kilodaltons. Both subunits contained carbohydrate as shown by periodic acid-Schiff staining. The purified enzyme hydrolyzed p-nitrophenyl-β-d-glucoside (K_m, 2.0 millimolar) at an optimum pH of 6.5 in phosphate buffer. The indolizidine alkaloid castanospermine, a known inhibitor of O-glycosidases, competitively inhibited the hydrolyses of sinigrin (thioglucosidase activity) and p-nitrophenyl-β-d-glucoside (O-glucosidase activity) with K_i values of 5 and 6 micromolar, respectively. In contrast, the related polyhydroxyalkaloids swainsonine and deoxynojirimycin were without effect upon these hydrolyses.     

CD1d-restricted NKT cells: an interstrain comparison

CD1d-restricted Valpha14-Jalpha281 invariant alphabetaTCR(+) (NKT) cells are well defined in the C57BL/6 mouse strain, but they remain poorly characterized in non-NK1.1-expressing strains. Surrogate markers for NKT cells such as alphabetaTCR(+)CD4(-)CD8(-) and DX5(+)CD3(+) have been used in many studies, although their effectiveness in defining this lineage remains to be verified. Here, we compare NKT cells among C57BL/6, NK1.1-congenic BALB/c, and NK1.1-congenic nonobese diabetic mice. NKT cells were identified and compared using a range of approaches: NK1.1 expression, surrogate phenotypes used in previous studies, labeling with CD1d/alpha-galactosylceramide tetramers, and cytokine production. Our results demonstrate that NKT cells and their CD4/CD8-defined subsets are present in all three strains, and confirm that nonobese diabetic mice have a numerical and functional deficiency in these cells. We also highlight the hazards of using surrogate phenotypes, none of which accurately identify NKT cells, and one in particular (DX5(+)CD3(+)) actually excludes these cells. Finally, our results support the concept that NK1.1 expression may not be an ideal marker for CD1d-restricted NKT cells, many of which are NK1.1-negative, especially within the CD4(+) subset and particularly in NK1.1-congenic BALB/c mice.     

Soil moisture mediates association between the winter North Atlantic Oscillation and summer growth in the Park Grass Experiment

Several aspects of terrestrial ecosystems are known to be associated with the North Atlantic Oscillation (NAO) through effects of the NAO on winter climate, but recently the winter NAO has also been shown to be correlated with the following summer climate, including drought. Since drought is a major factor determining grassland primary productivity, the hypothesis was tested that the winter NAO is associated with summer herbage growth through soil moisture availability, using data from the Park Grass Experiment at Rothamsted, UK between 1960 and 1999. The herbage growth rate, mean daily rainfall, mean daily potential evapotranspiration (PE) and the mean and maximum potential soil moisture deficit (PSMD) were calculated between the two annual cuts in early summer and autumn for the unlimed, unfertilized plots. Mean and maximum PSMD were more highly correlated than rainfall or PE with herbage growth rate. Regression analysis showed that the natural logarithm of the herbage growth rate approximately halved for a 250 mm increase in maximum PSMD over the range 50-485 mm. The maximum PSMD was moderately correlated with the preceding winter NAO, with a positive winter NAO index associated with greater maximum PSMD. A positive winter NAO index was also associated with low herbage growth rate, accounting for 22% of the interannual variation in the growth rate. It was concluded that the association between the winter NAO and summer herbage growth rate is mediated by the PSMD in summer.     

MPV17 Loss Causes Deoxynucleotide Insufficiency and Slow DNA Replication in Mitochondria

MPV17 is a mitochondrial inner membrane protein whose dysfunction causes mitochondrial DNA abnormalities and disease by an unknown mechanism. Perturbations of deoxynucleoside triphosphate (dNTP) pools are a recognized cause of mitochondrial genomic instability; therefore, we determined DNA copy number and dNTP levels in mitochondria of two models of MPV17 deficiency. In Mpv17 ablated mice, liver mitochondria showed substantial decreases in the levels of dGTP and dTTP and severe mitochondrial DNA depletion, whereas the dNTP pool was not significantly altered in kidney and brain mitochondria that had near normal levels of DNA. The shortage of mitochondrial dNTPs in Mpv17^-/- liver slows the DNA replication in the organelle, as evidenced by the elevated level of replication intermediates. Quiescent fibroblasts of MPV17-mutant patients recapitulate key features of the primary affected tissue of the Mpv17^-/- mice, displaying virtual absence of the protein, decreased dNTP levels and mitochondrial DNA depletion. Notably, the mitochondrial DNA loss in the patients’ quiescent fibroblasts was prevented and rescued by deoxynucleoside supplementation. Thus, our study establishes dNTP insufficiency in the mitochondria as the cause of mitochondrial DNA depletion in MPV17 deficiency, and identifies deoxynucleoside supplementation as a potential therapeutic strategy for MPV17-related disease. Moreover, changes in the expression of factors involved in mitochondrial deoxynucleotide homeostasis indicate a remodeling of nucleotide metabolism in MPV17 disease models, which suggests mitochondria lacking functional MPV17 have a restricted purine mitochondrial salvage pathway.     

Detection of mitochondrial DNA depletion in living human cells using PicoGreen staining

Human mitochondria DNA (mtDNA) is arranged within the mitochondria into discrete DNA-protein complexes, termed nucleoids. The size of the human mitochondrial genome is less than that of yeast and is more difficult to visualise by fluorescent DNA stains such as DAPI and Hoescht. We have developed a simple yet effective method to visualise mtDNA in situ within living cells using the fluorescent stain PicoGreen. Quantitative analysis shows that PicoGreen can be used to estimate the degree of mtDNA depletion within living cells. We have used this approach to study the arrangement and fluorescence of nucleoids in cells depleted of mtDNA by treatment with the anti-viral nucleoside analogue, 2',3'-dideoxycytidine. We also studied the distribution of mtDNA in fibroblasts cultured from patients with mitochondrial disease. Combining PicoGreen staining with histochemical and immunocytochemical approaches enabled us to examine the effects of mtDNA depletion on mtDNA-related components at the level of single cells. This method is able to detect an intermediate degree of mtDNA depletion in living cells, and can be used to detect mtDNA free cells (rho0 cells) in culture even at very low numbers. We have also adapted the technique to efficiently sort rho0 cells from populations of normal cells by fluorescent-assisted cell sorting (FACS), without the need for selection of respiratory competence. This should be useful for the construction of new trans-mitochondrial 'cybrid' cell lines.     

Capturing diversity of marine heterotrophic protists: one cell at a time

Recent applications of culture-independent, molecular methods have revealed unexpectedly high diversity in a variety of functional and phylogenetic groups of microorganisms in the ocean. However, none of the existing research tools are free from significant limitations, such as PCR and cloning biases, low phylogenetic resolution and others. Here, we employed novel, single-cell sequencing techniques to assess the composition of small (<10 μm diameter), heterotrophic protists from the Gulf of Maine. Single cells were isolated by flow cytometry, their genomes amplified, and 18S rRNA marker genes were amplified and sequenced. We compared the results to traditional environmental PCR cloning of sorted cells. The diversity of heterotrophic protists was significantly higher in the library of single amplified genomes (SAGs) than in environmental PCR clone libraries of the 18S rRNA gene, obtained from the same coastal sample. Libraries of SAGs, but not clones contained several recently discovered, uncultured groups, including picobiliphytes and novel marine stramenopiles. Clone, but not SAG, libraries contained several large clusters of identical and nearly identical sequences of Dinophyceae, Cercozoa and Stramenopiles. Similar results were obtained using two alternative primer sets, suggesting that PCR biases may not be the only explanation for the observed patterns. Instead, differences in the number of 18S rRNA gene copies among the various protist taxa probably had a significant role in determining the PCR clone composition. These results show that single-cell sequencing has the potential to more accurately assess protistan community composition than previously established methods. In addition, the creation of SAG libraries opens opportunities for the analysis of multiple genes or entire genomes of the uncultured protist groups.