Phytoplasma distribution in coconut palms affected by lethal yellowing disease

Lethal yellowing (LY), the most devastating disease affecting the coconut palm in America, is caused by phytoplasmas known to be distributed in different parts of infected plants. However, no comprehensive reports exist on the phytoplasma distribution. This study refers to the detection of LY phytoplasma DNA using PCR in different coconut plant parts, throughout the development of the disease. Sample analysis of positive palms taken at different stages of disease development (either symptomatic or symptomless) showed differences in the percentage of LY detection between plant parts. Some parts showed a very high level of LY DNA (stem, young leaves, inflorescences, stem apex and root apex), low levels were found in the intermediate leaves and roots without apex, whereas no LY phytoplasma DNA was detected in mature leaves. The detection percentage of LY phytoplasma DNA was lowest in symptomless‐infected palms for all parts, except the stem, where phytoplasma accumulations were consistently detected. This pattern of detection among parts is consistent with the hypothesis that phytoplasmas move from photosynthate source tissues to sink tissues via the phloem mass flow process. The accumulations in the (lower) stem, prior to the appearance of symptoms, suggest that this part of the palm is where phytoplasmas first move from leaves after foliar feeding by vectors and in which they probably multiply and distribute to other palm parts, including roots. Embryos from infected palms were analysed by nested‐PCR and 28% of 394 embryos were positive. Phytoplasma DNA was detected in embryos from fruit on any of the fruiting bunches regardless the age, but no pattern of quantitative distribution throughout the bunch developmental stages was observed. Germination of seeds from LY‐positive symptomatic palms was 58% and from LY‐negative symptomless palms were 71%. No phytoplasma was detected in seedlings tested from both symptomatic and non‐symptomatic palms. Seedlings tested after 2 years did not develop LY symptoms or eventually died.     

Histochemical Detection of Quinone Reductase Activity In Situ Using LY 83583 Reduction and Oxidation

Abstract: The application of enzymatic staining techniques, using tetrazolium dyes, to aldehyde-treated brain sections has revealed the presence of NADPH-diaphorase activity attributed to nitric oxide synthase. When evaluating the specificity of the putative guanylyl cyclase inhibitor LY 83583, a robust and novel staining pattern was noted in epithelial, endothelial, and astrocytic cells when LY 83583 was included in the NADPH-diaphorase histochemical reaction. This LY 83583-dependent staining could be blocked by the NAD(P)H:quinone oxidoreductase inhibitor dicumarol. Based on its quinone structure, we hypothesized that LY 83583 was a substrate for the enzyme NAD(P)H:quinone oxidoreductase. Transfection of human embryonic kidney 293 cells with the rat liver isoform of NAD(P)H:quinone oxidoreductase resulted in robust NADPH- and LY 83583-dependent staining that was completely blocked by dicumarol and was not observed in untransfected cells. Analysis of transfected cell extracts and brain homogenates indicated that LY 83583 was a substrate for NAD(P)H:quinone oxidoreductase, with a K _m similar to the well-characterized substrate menadione. Sensitivity of the nitroblue tetrazolium reduction to superoxide dismutase indicated that the reduction of LY 83583 by NAD(P)H:quinone oxidoreductase leads to superoxide generation. The localization of NAD(P)H:quinone oxidoreductase activity to astrocytic cells suggests a role for glia in combating oxidative insults to brain and in activating quinone-like drugs such as LY 83583.     

Dopamine Depletion Preferentially Impairs D1 over D2-Receptor Regulation of Striatal In Vivo Acetylcholine Release

The roles of D2 and D1 dopaminergic receptors on the regulation of striatal acetylcholine (ACh) release in vivo were examined for a period of 120 min after acute (2 h) or prolonged (16 h) depletion of brain dopamine (DA) by alpha-methyl-p-tyrosine. The reduction of DA transmission did not affect basal ACh output after 2 h but markedly lowered ACh release by 16 h (50%). Acute alpha-methyl-p-tyrosine pretreatment prevented the reduction of ACh release by the D1 antagonist SCH 23390 and its increase by the D2 antagonist, remoxipride, consistent with a drastic reduction of DA transmission at both DA receptors. However, 16 h after alpha-methyl-p-tyrosine, the effect of remoxipride on ACh release was restored, but SCH 23390 still had no effect, suggesting that the D2 inhibitory tone on ACh release had recovered, whereas the reduction of the D1 facilitatory influence persisted. The D1 facilitatory control of ACh neurotransmission thus appears to be more sensitive than the D2 inhibitory control to a reduction in DA transmission. The new model of DA-ACh interaction resulting from these data casts fresh light on the relationship between changes in DA transmission and extrapyramidal motor function.     

Nitric oxide-induced cell death of cerebrocortical murine astrocytes is mediated through p53- and Bax-dependent pathways

We have investigated the mechanism by which nitric oxide (NO) induces the death of mouse astrocytes. We show that NO (from donor diethylenetriamine-NO adduct) induces death with several features of apoptosis, including chromatin condensation, phosphatidylserine exposure on the outer leaflet of the plasma membrane, Bax translocation to the mitochondria and cytochrome c release, but no caspase activation or nuclear fragmentation is observed. Nitric oxide also elevates p53 expression, causing a concomitant increase in p53 serine 18 phosphorylation and p53 translocation from the cytoplasm to the nucleus. Activation of Bax and p53 is important for NO-induced apoptosis-like cell death because Bax- or p53-deficient astrocytes are much more resistant than wild-type cells to the same NO treatment. We further demonstrate that LY294002-sensitive kinases are responsible for controlling serine 18 phosphorylation of p53, thereby regulating the pro-apoptotic activity of p53 in astrocytes. While apoptosis is suppressed in the presence of LY294002, however, death by necrosis is increased, suggesting that LY294002-sensitive kinases additionally suppress a latent necrotic response to NO. We conclude that NO-induced death in astrocytes is mediated by p53- and Bax-dependent mechanisms, although full manifestation of apoptosis is aborted by concomitant inhibition of caspase activation. More generally, our data suggest that apoptotic mediators should be evaluated as the cause of cell death even in cases where a full apoptotic phenotype is lacking.     

Development of a plasma high-performance liquid chromatographic assay for LY303366, a lipopeptide antifungal agent, and its application in a dog pharmacokinetic study

An HPLC assay for plasma analysis of LY303366 (I), a semi-synthetic lipopeptide antifungal related to echinocandin B (ECB), was developed to support the selection and subsequent preclinical development of I. The method involved extraction of I from plasma with the aid of solid-phase extraction (SPE) cartidges followed by reversed-phase HPLC with UV detection at 300 nm. The method is simple, selective and is applicable to dog, rat, mouse and rabbit plasma. Validation studies using dog plasma showed that the values obtained for parameters of linearity, precision and accuracy were within acceptable limits. Based on analysis of 0.3 ml of plasma, the lower limit of quantitation was 20 ng/ml. The method has been successfully applied to determine the pharmacokinetic parameters of I in the dog following intravenous (i.v.) and oral administration. Compared to first generation ECB antifungal agents, the results of the i.v. dog study indicated a 50% reduction in clearance of the drug from plasma (0.1 l/h/kg) and an 18-fold increase in the volume of distribution at steady state (1.8 l/kg). When administered orally, compound I had an absolute bioavailability of 9%; however, plasma levels remained above the MIC for C. albicans (0.005 μg/ml) through 48 h. Given the excellent potency of I and its broad spectrum of activity relative to first generation ECB antifungal agents, the assay results for I indicate the potential for its use as a broad spectrum i.v. and oral antifungal agent.     

Pharmacological inhibition of S6K1 impairs self‐renewal and osteogenic differentiation of bone marrow stromal cells

mTORC1 signaling not only plays important physiological roles in the regulation of proliferation and osteogenic differentiation of BMSCs, but also mediates exogenous Wnt‐induced protein anabolism and osteoblast differentiation. However, the downstream effectors of the mTORC1 signaling in the above processes are still poorly understood. In this study, we explored the specific role of S6K1, one of the major targets of the mTORC1 pathway, in BMSCs self ‐ renewal and osteogenic differentiation. We first found that S6K1 was active in primary mouse bone marrow stromal cells, and further activated upon osteogenic induction. We then determined the effects of S6K1 inhibition by LY2584702 Tosylate, a selective inhibitor of S6K1 (hereafter S6KI), using both primary mouse bone marrow stromal cells and ST2 cells. Colony‐Forming Unit‐Fibroblast (CFU‐F) assays showed that S6KI dramatically reduced the total number of colonies formed in primary BMSCs cultures. Under the basal osteogenic culture condition, S6KI significantly inhibited mRNA expression of osteoblast marker genes (Sp7, Bglap, Ibsp, and Col1a1), ALP activity and matrix mineralization. Upon Wnt3a treatments, S6KI inhibited Wnt3a‐induced osteoblast differentiation and expression of protein anabolism genes in ST2 cells, but to a much lesser degree than rapamycin (a specific inhibitor of mTORC1 signaling). Collectively, our findings have demonstrated that pharmacological inhibition of S6K1 impaired self ‐ renewal and osteogenic differentiation of BMSCs, but only partially suppressed exogenous Wnt3a‐induced osteoblast differentiation and protein anabolism.     

UV-induced mutagenesis at the hypoxanthine—guanine phosphoribosyl transferase locus in two L5178Y mouse lymphoma cell strains with different UV sensitivities

Two strains of L5178Y mouse lymphoma cells, L5178Y-R (LY-R) and L5178Y-S (LY-S), differ markedly in their sensitivity to 254 nm UV radiation (D₀ = 0.7 and 5.5 J/m²; n = 6.0 and 2.0 for LY-R and LY-S cells, respctively). In this study, the frequency o hypoxanthine-guanine-phosporibosyl-transferase-deficient mutants was determined, using 6-thioguanine (TG) as a selective agent, in populations of LY-R and LY-S cells exposed to various fluences of UV radiation. The spontaneous mutation frequency for LY-R cells was (3.7 ± 0.6) × 10^?5 TG^r mutants per viable cell, and the UV induction rate was (2.2 ± 0.8) × 10^?4 TG^r mutants per viable cell, per J/m². Both spontaneous and induced mutantion frequencies were much lower for LY-S cells. The sopntaneous mutation frequency for these cells were too low to make its measurement practicable ( < 0.0013 × 10^?5 TG^r mutants per viable cell). Mutation induction rate was (4.2 ± 2.2) × 10^?7 TG^r mutants per viable cell, per J/m². These differences in mutability do not appear to be due to gene duplication in LY-S cells, or to selective growth disadvantage of LY-S-derived TG-resistant mutants. Possible mechanisms underlying the differences in mutability of LY-R and LY-S cells are considered.     

LY411575, a potent γ-secretase inhibitor,suppresses osteoclastogenesis in vitro and LPS-induced calvarial osteolysis in vivo

A series of osteolytic bone diseases are usually related to excessive bone resorption and osteoclast formation. Thus, agents or drugs which can target osteoclast development and attenuate bone loss are potentially considerable in preventing and treating of bone lytic diseases. In recent years, many studies have reported that Notch signaling has substantial impacts on the process of osteoclast differentiation, maturation, and bone destruction. In the present study, we showed that LY411575, a γ-secretase inhibitor, could potently suppress osteoclast differentiation, osteoclast-specific gene expression, and bone resorption via suppressing Notch/HES1/MAPK (ERK and p38)/Akt-mediated NFATc1 induction in vitro. Consistent with in vitro results, LY411575 exhibited protective effects in lipopolysaccharides-induced calvarial bone destruction in vivo. Collectively, these results indicate that LY411575 may have therapeutic potential in the treatment of osteoclast-mediated osteolytic bone diseases.     

Centelloside accumulation in leaves of Centella asiatica is determined by resource partitioning between primary and secondary metabolism while influenced by supply levels of either nitrogen,phosphorus or potassium

In the present study we aimed to investigate the relevance of either N, P or K supply for herb and leaf yield and for centelloside concentrations in Centella asiatica L. Urban leaves. In this regard, we elucidated the causal relationship between assimilation rate, leaf N, P and K concentrations, herb and leaf production, and centelloside accumulation. The experiments were conducted consecutively in a greenhouse where C. asiatica was grown in hydroponic culture and fertigated with nutrient solutions at either 0, 30, 60, 100 or 150% of the N, P or K amount in a standard Hoagland solution. In general, the increase in N, P or K supply enhanced assimilation rate and herb and leaf yield. However, exceeding specific thresholds, the high availability of one single nutrient caused lower leaf N concentrations and a decline in assimilation rate and plant growth. Irrespective of N, P and K supply, the leaf centelloside concentrations were negatively associated with herb and leaf yield, which is in accordance with the assumptions of the carbon/nutrient balance and the growth differentiation balance hypotheses. Moreover, we found strong negative correlations between saponins and leaf N concentrations, while the respective sapogenins were negatively correlated with K concentrations. Using C. asiatica as model system, our experiments reveal for the first time that the accumulation of saponins and sapogenins is affected by resource allocation between primary and secondary metabolism and that besides carbon, also nutrient availability is relevant for the regulation of the centelloside synthesis. Finally, our results highlight the huge potential of optimized and carefully controlled mineral nutrition of medicinal plants for steering the bio-production of high-quality natural products.     

EGF induces efficient Cx43 gap junction endocytosis in mouse embryonic stem cell colonies via phosphorylation of Ser262, Ser279/282, and Ser368

Gap junctions (GJs) traverse apposing membranes of neighboring cells to mediate intercellular communication by passive diffusion of signaling molecules. We have shown previously that cells endocytose GJs utilizing the clathrin machinery. Endocytosis generates cytoplasmic double-membrane vesicles termed annular gap junctions or connexosomes. However, the signaling pathways and protein modifications that trigger GJ endocytosis are largely unknown. Treating mouse embryonic stem cell colonies – endogenously expressing the GJ protein connexin43 (Cx43) – with epidermal growth factor (EGF) inhibited intercellular communication by 64% and activated both, MAPK and PKC signaling cascades to phosphorylate Cx43 on serines 262, 279/282, and 368. Upon EGF treatment Cx43 phosphorylation transiently increased up to 4-fold and induced efficient (66.4%) GJ endocytosis as evidenced by a 5.9-fold increase in Cx43/clathrin co-precipitation.