Pyrrolidine dithiocarbamate induces apoptosis by a cytochrome c-dependent mechanism

Pyrrolidine dithiocarbamate (PDTC) is a synthetic antioxidant molecule, which has been recently proposed as an antitumoral agent on the basis of its capability of inducing apoptosis. We investigated the effect of PDTC on the proliferation and survival of the promyelocitic cell line HL-60. Concentration as low as 10 microM of PDTC induces a significant reduction of the growth rate and the contemporaneous activation of the apoptotic process. Programmed cell death was demonstrated by biochemical analyses, including the activation of procaspase 3 and the cleavage of poly(ADP-ribose) polymerase (PARP). PDTC-dependent apoptosis was associated with an early release of cytochrome c from mitochondria, while the involvement of pathways due to cell death receptors engagement was ruled out by detailed time-course analyses of caspases 3 and 8 activation. Moreover, no up-regulation of p21(CIP1) level, a pivotal cyclin-dependent kinase inhibitor, occurred at PDTC concentration able to induce apoptosis. Finally, in vitro incubation of purified mitochondria with PDTC demonstrated that the molecule is directly able to induce cytochrome c release from the intermembrane space, thus confirming that mitochondria are a primary cellular target of the molecule.     

Genotyping of Vitis vinifera L. within the Slovak national collection of genetic resources

Microsatellites were used as a very effective tool for genetic diversity analysis and characterization of 51 grapevine (Vitis vinifera L.) accessions from the national collection of genetic resources. Genetic diversity was relatively high, 8.91 alleles were detected per analysed microsatellite locus in average, and fifty-one accessions were distinguished into 45 groups. Distribution of recent Slovak cultivars across the dendrogram accented both their genetic diversity and the effectiveness of the national breeding program in maintaining genetic diversity and generating new genetic variants. Each cultivar was different from the others and twelve of them contained 77.6% of the total genetic diversity of the whole analysed set. Microsatellite patterns were also able to confirm parentage in selected Slovak cultivars. An unusual phenomenon of triallelism was also detected in one of the analysed accessions. The present study has initiated molecular characterization within the national grapevine genetic resource collection and their comparison with well-established international cultivars.     

UPLC-MS-based urine metabolomics reveals indole-3-lactic acid and phenyllactic acid as conserved biomarkers for alcohol-induced liver disease in the Ppara-null mouse model

Since the development and prognosis of alcohol-induced liver disease (ALD) vary significantly with genetic background, identification of a genetic background-independent noninvasive ALD biomarker would significantly improve screening and diagnosis. This study explored the effect of genetic background on the ALD-associated urinary metabolome using the Ppara-null mouse model on two different backgrounds, C57BL/6 (B6) and 129/SvJ (129S), along with their wild-type counterparts. Reversed-phase gradient UPLC-ESI-QTOF-MS analysis revealed that urinary excretion of a number of metabolites, such as ethylsulfate, 4-hydroxyphenylacetic acid, 4-hydroxyphenylacetic acid sulfate, adipic acid, pimelic acid, xanthurenic acid, and taurine, were background-dependent. Elevation of ethyl-β-d-glucuronide and N-acetylglycine was found to be a common signature of the metabolomic response to alcohol exposure in wild-type as well as in Ppara-null mice of both strains. However, increased excretion of indole-3-lactic acid and phenyllactic acid was found to be a conserved feature exclusively associated with the alcohol-treated Ppara-null mouse on both backgrounds that develop liver pathologies similar to the early stages of human ALD. These markers reflected the biochemical events associated with early stages of ALD pathogenesis. The results suggest that indole-3-lactic acid and phenyllactic acid are potential candidates for conserved and pathology-specific high-throughput noninvasive biomarkers for early stages of ALD.     

Does soil organic carbon quality or quantity govern relative temperature sensitivity in soil aggregates?

Soil aggregates govern soil organic carbon (SOC) sequestration. But, sparse understanding about the process leads to inaccuracy in predicting potential of soil to stabilize C in warming world. We appraised effects of 43 years of fertilization on relative temperature sensitivity of SOC decomposition (Q₁₀) in soil aggregates to know whether SOC quality or quantity governs Q₁₀. Treatments were: fallow, control, 100% recommended dose of nitrogen (N), N and phosphorus (NP), N, P and potassium (NPK), and NPK + farmyard manure (FYM) (NPK + FYM). Macroaggregates, microaggregates and silt + clay (s + c) fractions were incubated for 16 weeks at 25, 35 and 45 °C, SOC quality (R₀) and Q₁₀ were computed. SOC mineralization from macro- and micro- aggregates were 34 and 28% higher than s + c across the treatments. The s + c fraction of NPK + FYM had ~ 41, 40 and 24% higher C decay rate than NPK plots at 25, 35 and 45 °C, respectively. For s + c fraction Q₁₀ increased over other aggregates. Mean Q₁₀ of s + c fraction was ~ 18.3 and 17.5% higher than macro and micro-aggregate-C, respectively. R₀ was the lowest for NPK + FYM, suggesting long-term manuring with balanced NPK significantly enhance recalcitrance of C. We observed Q₁₀ of macroaggregates and s + c fraction is controlled by C quality but C quantity governs Q₁₀ of microaggregates in Vertisol. Specifically, microaggregates of NPK + FYM were more temperature sensitive, and could be vulnerable to C loss. Hence, practices facilitating microaggregate formation should be avoided. Thus, we recommend manure application for facilitating C sequestration.

     

Hydrogen sulfide and L-cysteine increase phosphatidylinositol 3,4,5-trisphosphate (PIP3) and glucose utilization by inhibiting phosphatase and tensin homolog (PTEN) protein and activating phosphoinositide 3-kinase (PI3K)/serine/threonine protein kinase (AKT)/protein kinase Cζ/λ (PKCζ/λ) in 3T3l1 adipocytes

This work examined the novel hypothesis that reduced levels of H(2)S or L-cysteine (LC) play a role in the impaired glucose metabolism seen in diabetes. 3T3L1 adipocytes were treated with high glucose (HG, 25 mM) in the presence or absence of LC or H(2)S. Both LC and H(2)S treatments caused an increase in phosphatidylinositol-3,4,5 trisphosphate (PIP3), AKT phosphorylation, and glucose utilization in HG-treated cells. The effect of LC on PIP3 and glucose utilization was prevented by propargylglycine, an inhibitor of cystathionine γ-lyase that catalyzes H(2)S formation from LC. This demonstrates that H(2)S mediates the effect of LC on increased PIP3 and glucose utilization. H(2)S and LC caused phosphatidylinositol 3-kinase activation and PTEN inhibition. Treatment with LC, H(2)S, or PIP3 increased the phosphorylation of IRS1, AKT, and PKCζ/λ as well as GLUT4 activation and glucose utilization in HG-treated cells. This provides evidence that PIP3 is involved in the increased glucose utilization observed in cells supplemented with LC or H(2)S. Comparative signal silencing studies with siAKT2 or siPKCζ revealed that PKCζ phosphorylation is more effective for the GLUT4 activation and glucose utilization in LC-, H(2)S-, or PIP3-treated cells exposed to HG. This is the first report to demonstrate that H(2)S or LC can increase cellular levels of PIP3, a positive regulator of glucose metabolism. The PIP3 increase is mediated by PI3K activation and inhibition of PTEN but not of SHIP2. This study provides evidence for a molecular mechanism by which H(2)S or LC can up-regulate the insulin-signaling pathways essential for maintenance of glucose metabolism.     

TRPC5 channel sensitivities to antioxidants and hydroxylated stilbenes

Transient receptor potential canonical 5 (TRPC5) forms cationic channels that are polymodal sensors of factors including oxidized phospholipids, hydrogen peroxide, and reduced thioredoxin. The aim of this study was to expand knowledge of the chemical-sensing capabilities of TRPC5 by investigating dietary antioxidants. Human TRPC5 channels were expressed in HEK 293 cells and studied by patch clamp and intracellular Ca(2+) recording. GFP- and HA-tagged channels were used to quantify plasma membrane localization. Gallic acid and vitamin C suppressed TRPC5 activity if it was evoked by exogenous hydrogen peroxide or lanthanide ions but not by lysophosphatidylcholine or carbachol. Catalase mimicked the effects, suggesting that lanthanide-evoked activity depended on endogenous hydrogen peroxide. Trans-resveratrol, by contrast, inhibited all modes of TRPC5, and its effect was additive with that of vitamin C, suggesting antioxidant-independent action. The IC(50) was ～10 μM. Diethylstilbestrol, a related hydroxylated stilbene, inhibited TRPC5 with a similar IC(50), but its action contrasted sharply with that of resveratrol in outside-out membrane patches where diethylstilbestrol caused strong and reversible inhibition and resveratrol had no effect, suggesting indirect modulation by resveratrol. Resveratrol did not affect channel surface density, but its effect was calcium-sensitive, indicating an action via a calcium-dependent intermediate. The data suggest previously unrecognized chemical-sensing properties of TRPC5 through multiple mechanisms: (i) inhibition by scavengers of reactive oxygen species because a mode of TRPC5 activity depends on endogenous hydrogen peroxide; (ii) direct channel blockade by diethylstilbestrol; and (iii) indirect, antioxidant-independent inhibition by resveratrol.