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.     

Sensing drought- and salinity-imposed stresses on tomato leaves by means of fluorescence techniques

In our study, we investigated whether multiple fluorescence indices may be used to sense physiological changes in tomato plants (Solanum lycopersicum L.) caused by salinity and water deficit as single or combined stresses. The fluorescence intensity in the blue (B), red (R) and far-red (FR) spectral regions and the pulse-amplitude-modulated (PAM) chlorophyll fluorescence, were recorded on a weekly basis in the scope of a long-term experiment. The results indicate the coefficient of photochemical quenching (qL), the B to FR fluorescence ratio and the logarithm of the FR fluorescence ratio after R and UV-light excitation as appropriate parameters to sense the response of plants to the imposed stress. The qL revealed the impact of water deficiency, whereas the two multispectral ratios revealed the influence of combined salinity and water shortage. Despite minor changes in the chlorophyll concentration, salinity and water deficit, when combined, had an additive impact on the chlorophyll fluorescence. Overall, the fluorescence signals of ‘Rio Grande’ were more affected by the induced stresses compared to ‘Harzfeuer’. The multiparametric fluorescence technique, confirming the trends obtained with the PAM-method, reveals promising perspectives for the ‘in situ’ evaluation of the physiological status of horticultural crops.     

Regulation of mTORC1 and mTORC2 Complex Assembly by Phosphatidic Acid: Competition with Rapamycin

mTOR, the mammalian target of rapamycin, is a critical node for control of cell growth and survival and has widely been implicated in cancer survival signals. mTOR exists in two complexes: mTORC1 and mTORC2. Phospholipase D (PLD) and its metabolite phosphatidic acid (PA) have been implicated in the regulation of mTOR; however, their role has been controversial. We report here that suppression of PLD prevents phosphorylation of the mTORC1 substrate S6 kinase (S6K) at Thr389 and the mTORC2 substrate Akt at Ser473. Suppression of PLD also blocked insulin-stimulated Akt phosphorylation at Ser473 and the mTORC2-dependent phosphorylation of PRAS40. Importantly, PA was required for the association of mTOR with Raptor to form mTORC1 and that of mTOR with Rictor to form mTORC2. The effect of PA was competitive with rapamycin—with much higher concentrations of rapamycin needed to compete with the PA-mTORC2 interaction than with PA-mTORC1. Suppressing PA production substantially increased the sensitivity of mTORC2 to rapamycin. Data provided here demonstrate a PA requirement for the stabilization of both mTORC1 and mTORC2 complexes and reveal a mechanism for the inhibitory effect of rapamycin on mTOR. This study also suggests that by suppressing PLD activity, mTORC2 could be targeted therapeutically with rapamycin.It has become apparent during the past decade that a critical aspect of tumor progression is the suppression of default apoptotic programs that constitute what is likely the most important protection against cancer. Cellular signals that suppress apoptosis have come to be known as “survival signals.” A common node for survival signals is mTOR, the mammalian target of rapamycin (5, 13, 14, 25). mTOR exists in two distinct complexes, mTORC1 and mTORC2 (21), that differ in their subunit composition and sensitivity to rapamycin. mTORC1 consists of a complex that includes mTOR and a protein known as Raptor (regulatory associated protein of mTOR), whereas mTORC2 consists of a complex that includes mTOR and a protein known as Rictor (rapamycin-insensitive companion of mTOR) (13, 14). There are also mTORC2 complexes that can be distinguished by association with different isoforms of mSin1 (9). While much is known about the regulation of mTORC1 (21), very little is known about the regulation of mTORC2.mTORC1 is highly sensitive to rapamycin, whereas mTORC2 is relatively insensitive to rapamycin (21). However, it was recently reported that long-term exposure to rapamycin prevented the formation of mTORC2 complexes and blocked the phosphorylation of the mTORC2 substrate Akt at Ser473 (24, 38). Rapamycin, in association with FK506 binding protein 12 (FKBP12), has been reported to interact with mTOR in a manner that is competitive with phosphatidic acid (PA), the metabolic product of phospholipase D (PLD) (2, 4). PLD, like mTOR, has been implicated in survival signals in several human cancer cell lines (1, 10, 11, 27, 32, 39). Since rapamycin-FKBP12 competes with PA for binding to mTOR, the sensitivity of mTORC2 complex formation to rapamycin suggests that PA facilitates the assembly of mTORC2—and ultimately the activation of mTORC2. We report here that the assembly of both mTORC1 and mTORC2 complexes is dependent upon PLD and its metabolite PA. The study also provides mechanistic insight into how rapamycin impacts on mTOR-mediated signals and how PLD regulates mTOR by facilitating the formation of mTOR complexes.     

Genomic DNA k-mer spectra: models and modalities

Background  

The empirical frequencies of DNA k-mers in whole genome sequences provide an interesting perspective on genomic complexity, and the availability of large segments of genomic sequence from many organisms means that analysis of k-mers with non-trivial lengths is now possible.     

Structural changes in bacteriorhodopsin following retinal photoisomerization from the 13-cis form

Bacteriorhodopsin (BR), a light-driven proton pump in Halobacterium salinarum, accommodates two resting forms of the retinylidene chromophore, the all-trans form (AT-BR) and the 13-cis,15-syn form (13C-BR). Both isomers are present in thermal equilibrium in the dark, but only the all-trans form has proton-pump activity. In this study, we applied low-temperature Fourier-transform infrared (FTIR) spectroscopy to 13C-BR at 77 K and compared the local structure around the chromophore before and after photoisomerization with that in AT-BR. Strong hydrogen-out-of-plane (HOOP) vibrations were observed at 964 and 958 cm(-)(1) for the K state of 13C-BR (13C-BR(K)) versus a vibration at 957 cm(-)(1) for the K state of AT-BR (AT-BR(K)). In AT-BR(K), but not in 13C-BR(K), the HOOP modes exhibit isotope shifts upon deuteration of the retinylidene at C15 and at the Schiff base nitrogen. Whereas the HOOP modes of AT-BR(K) were significantly affected by the mutation of Thr89, this was not the case for the HOOP modes of 13C-BR(K). These observations imply that, while the chromophore distortion is localized near the Schiff base in AT-BR(K), it is located elsewhere in 13C-BR(K). By use of [zeta-(15)N]lysine-labeled BR, we identified the N-D stretching vibrations of the 13C-BR Schiff base (in D(2)O) at 2173 and 2056 cm(-)(1), close in frequency to those of AT-BR. These frequencies indicate strong hydrogen bonding of the Schiff base in 13C-BR, presumably with a water molecule as in AT-BR. In contrast, the N-D stretching vibration appears at 2332 and 2276 cm(-)(1) in 13C-BR(K) versus values of 2495 and 2468 cm(-)(1) for AT-BR(K), suggesting that the rupture of the Schiff base hydrogen bond that occurs in AT-BR(K) does not occur in 13C-BR(K). Rotational motion of the Schiff base upon retinal isomerization is probably smaller in magnitude for 13C-BR than for AT-BR. These differences in the primary step are possibly related to the absence of light-driven proton pumping by 13C-BR.     

Melanin-concentrating hormone stimulates human growth hormone secretion: a novel effect of MCH on the hypothalamic-pituitary axis

Melanin-concentrating hormone (MCH), a 19-amino acid orexigenic (appetite-stimulating) hypothalamic peptide, is an important regulator of energy homeostasis. It is cleaved from its precursor prepro-MCH (ppMCH) along with several other neuropeptides whose roles are not fully defined. Because pituitary hormones such as growth hormone (GH), ACTH, and thyroid-stimulating hormone affect body weight and composition, appetite, insulin sensitivity, and lipoprotein metabolism, we investigated whether MCH exerts direct effects on the human pituitary to regulate energy balance using dispersed human fetal pituitaries (21-22 wk gestation) and cultured GH-secreting adenomas. We found that MCH receptor-1 (MCH-R1), but not MCH receptor-2, is expressed in both normal (fetal and adult) human pituitary tissues and in GH cell adenomas. MCH (10 nM) stimulated GH release from human fetal pituitary cultures by up to 62% during a 4-h incubation (P < 0.05). Interestingly, neuropeptide EI (10 nM), which is also cleaved from ppMCH, increased human GH secretion by up to 124% in fetal pituitaries. A milder, albeit significant, induction of GH secretion by MCH (20%) was seen in cultured GH-secreting pituitary adenomas. A comparable stimulation of GH secretion was seen when cultured mouse pituitary cells were treated with MCH. Treatment of cultured GH adenoma cells with MCH (100 nM) induced extracellular signal-regulated kinases 1 and 2 phosphorylation, suggesting activation of MCH-R1. In aggregate, these data suggest that MCH may regulate pituitary GH secretion and imply a potential cross-talk mechanism between appetite-regulating neuropeptides and pituitary hormones.     

Maximum likelihood molecular clock comb: analytic solutions.

Maximum likelihood (ML) is increasingly used as an optimality criterion for selecting evolutionary trees, but finding the global optimum is a hard computational task. Because no general analytic solution is known, numeric techniques such as hill climbing or expectation maximization (EM), are used in order to find optimal parameters for a given tree. So far, analytic solutions were derived only for the simplest model--three taxa, two state characters, under a molecular clock. Four taxa rooted trees have two topologies--the fork (two subtrees with two leaves each) and the comb (one subtree with three leaves, the other with a single leaf). In a previous work, we devised a closed form analytic solution for the ML molecular clock fork. In this work, we extend the state of the art in the area of analytic solutions ML trees to the family of all four taxa trees under the molecular clock assumption. The change from the fork topology to the comb incurs a major increase in the complexity of the underlying algebraic system and requires novel techniques and approaches. We combine the ultrametric properties of molecular clock trees with the Hadamard conjugation to derive a number of topology dependent identities. Employing these identities, we substantially simplify the system of polynomial equations. We finally use tools from algebraic geometry (e.g., Gr?bner bases, ideal saturation, resultants) and employ symbolic algebra software to obtain analytic solutions for the comb. We show that in contrast to the fork, the comb has no closed form solutions (expressed by radicals in the input data). In general, four taxa trees can have multiple ML points. In contrast, we can now prove that under the molecular clock assumption, the comb has a unique (local and global) ML point. (Such uniqueness was previously shown for the fork.).