New positive allosteric modulators of the metabotropic glutamate receptor 2 (mGluR2): identification and synthesis of N-propyl-8-chloro-6-substituted isoquinolones

A series of N-propyl-8-chloro-6-substituted isoquinolones was identified as positive allosteric modulators of metabotropic glutamate receptor 2 (mGluR2 PAM) via high throughput screening (HTS). The subsequent synthesis and initial SAR exploration that led to the identification of compound 28 is described.     

Elk Kelp, Pelagophycus porra, distribution limited due to susceptibility of microscopic stages to high light

Elk Kelp, Pelagophycus porra (Leman) Setchell, is commonly observed in deep (20-50 m) water along the outer edge of Giant Kelp, Macrocystis pyrifera (C. Agardh), forests in southern California, USA and northern Baja California, Mexico, but rarely occurs in shallower water or within the adjacent M. pyrifera beds. To investigate the factors that limit P. porra from establishing populations within these shallower habitats, juvenile P. porra sporophytes were transplanted from a deep (20 m) water P. porra zone to shallower (15 and 8 m) sites within and inside an adjacent M. pyrifera beds. Transplanted P. porra exhibited no differences in survival or growth among depths, although reproductive maturity was observed only at the two shallower sites. When P. porra propagules were experimentally introduced to areas under dense M. pyrifera canopies and areas cleared of the M. pyrifera canopies within the shallower M. pyrifera bed, P. porra still failed to recruit even though this procedure resulted in a four-fold increase in recruitment in the natural P. porra zone. Laboratory-based culturing of P. porra microscopic stages revealed that they grew and survived better under low light conditions characteristic of the natural P. porra zone (2-4 μmol photons m^− 2 s^− 1) than under higher light conditions characteristic of the M. pyrifera beds(18-20 μmol photons m^− 2 s^− 1). Pulse-amplitude modulated (PAM) fluorometry indicated that while naturally-occurring P. porra's adult sporophytes were able to photoacclimate to increasing irradiance as they grew from the benthos towards the surface, P. porra's microscopic stages were unable to photoacclimate to increased irradiances, and subsequently exhibited 100% mortality under these higher light conditions. Altogether, our study suggests that vulnerability of P. porra microscopic stages to higher irradiances appears to be the primary factor inhibiting P. porra from establishing populations in shallower water and stresses the importance of a multiple life-history approach when investigating species distributions.     

A CHLOROPHYLL FLUORESCENCE INDEX TO ESTIMATE SHORT‐TERM RATES OF PHOTOSYNTHESIS BY INTERTIDAL MICROPHYTOBENTHOS1

An index based on chl a fluorescence quenching analysis was tested as a predictor of photosynthetic rates of undisturbed intertidal microphytobenthic assemblages. The fluorescence index, P_fluo, was derived from the combination of different chl a fluorescence parameters chosen to represent the two main sources of short‐term variability in the community‐level microphytobenthic photosynthesis: 1) the quantum yield of photosynthesis of the microalgae present in the photic zone of the sediment, φP, and 2) the community‐level efficiency of photosynthetic light absorption, ?, determined by the microalgal concentration in the photic zone. Variations in φP were traced by the fluorescence index ΔF/F_m′ (the effective quantum yield of charge separation at PSII), whereas changes in ? were followed by the fluorescence parameter F_o (dark or minimum fluorescence level). Gross photosynthetic rate, P, and fluorescence parameters were measured nondestructively and simultaneously under in situ conditions, on the same samples, using oxygen microelectrodes and pulse amplitude modulation fluorometry, respectively. Despite the large and uncorrelated hourly variability in irradiance, photosynthetic rate, and fluorescence parameters included in P_fluo, highly significant correlations between P_fluo and P were found for all the sampling periods, encompassing hourly, biweekly, and seasonal time scales. The variability in P explained by P_fluo ranged from 84.3% to 91.4% when sampling periods were considered separately and reached 81.1% when all data were pooled. The results of the study show that despite its simplicity, the index P_fluo can be used to trace short‐term variations in the photosynthetic rate of undisturbed microphytobenthic assemblages undergoing rhythmic vertical migration.     

Photosynthetic rapid light curves (RLC) of Thalassia testudinum exhibit diurnal variation

The use of pulse-amplitude-modulated fluorometry (PAM) and rapid light curves (RLC) were evaluated for monitoring the physiological condition of the seagrass, Thalassia testudinum, at the landscape scale in Florida Bay, USA. PAM fluorometry provides rapid, non-invasive, and quantitative physiological information on the state of photosynthesis. Yet, previous studies of effective and maximum quantum yields have shown that problems arise when expanding measurements from the organismal scale to the landscape scale, mainly due to temporal and irradiance-induced changes in photophysiology. Here, the magnitude of diurnal and spatial variation of photosynthetic characteristics among 10 sample basins and between two sample years was investigated using RLCs. Because RLCs measure effective quantum yields over a range of changing actinic irradiances, we hypothesized that the response parameters might be less sensitive to diurnal light history effects. Our results indicate that the RLC parameters, alpha and ETR_max, significantly changed diurnally, as was previously found for both maximum and effective quantum yields, but the diurnal patterns were variable among the 10 basins. Both among-basin and between-year comparisons were confounded by diurnal variation and statistical analyses comparing morning, mid-day, and afternoon time periods were unable to definitively discern which time of day was best suited for assessing the relative photophysiological status of T. testudinum. However, pooling RLC data at the basin scale revealed among-basin differences and landscape scale trends that were consistent with basin-level morphometric variation in this seagrass. Thus, PAM fluorometry may be useful as a landscape scale monitoring tool within certain constraints. When using this approach over large spatial and temporal scales, diurnal variability must be considered.     

Structural model of the R state of Escherichia coli aspartate transcarbamoylase with substrates bound

The allosteric enzyme aspartate transcarbamoylase (ATCase) exists in two conformational states. The enzyme, in the absence of substrates is primarily in the low-activity T state, is converted to the high-activity R state upon substrate binding, and remains in the R state until substrates are exhausted. These conformational changes have made it difficult to obtain structural data on R-state active-site complexes. Here we report the R-state structure of ATCase with the substrate Asp and the substrate analog phosphonoactamide (PAM) bound. This R-state structure represents the stage in the catalytic mechanism immediately before the formation of the covalent bond between the nitrogen of the amino group of Asp and the carbonyl carbon of carbamoyl phosphate. The binding mode of the PAM is similar to the binding mode of the phosphonate moiety of N-(phosphonoacetyl)-l-aspartate (PALA), the carboxylates of Asp interact with the same residues that interact with the carboxylates of PALA, although the position and orientations are shifted. The amino group of Asp is 2.9 A away from the carbonyl oxygen of PAM, positioned correctly for the nucleophilic attack. Arg105 and Leu267 in the catalytic chain interact with PAM and Asp and help to position the substrates correctly for catalysis. This structure fills a key gap in the structural determination of each of the steps in the catalytic cycle. By combining these data with previously determined structures we can now visualize the allosteric transition through detailed atomic motions that underlie the molecular mechanism.     

Phospho-mimicry mutant of atToc33 affects early development of Arabidopsis thaliana

The precursor protein receptor at the chloroplast outer membrane atToc33 is a GTPase, which can be inactivated by phosphorylation in vitro, being arrested in the GDP loaded state. To assess the physiological function of phosphorylation, attoc33 knock out mutants were complemented with a mutated construct mimicking the constitutively phosphorylated state. Our data suggest that the reduced functionality of the mutant protein can be compensated by its upregulation. Chloroplast biogenesis and photosynthetic activity are impaired in the mutants during the early developmental stage, which is consistent with the requirement of atToc33 in young photosynthetic tissues.     

Parallel assessment of ROS formation and photosynthesis in leaves by fluorescence imaging

Stress-induced generation of reactive oxygen species (ROS) leads to lowering of the biochemical yield of photosynthesis in plant leaves. The detrimental effects of oxidative stress by paraquat are initiated by the generation of superoxide anion radicals in the vicinity of the thylakoid membrane. However, direct proof of ROS production has been elusive. In this study, we report first in vivo detection and imaging of the generated superoxide in illuminated tobacco leaves following paraquat infiltration. This was done using a newly developed imaging apparatus capable of detecting changes in the fluorescence of the ROS sensor 3-(N-dansyl)aminomethyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole. Under identical conditions, the effects on photosynthesis caused by the oxidative stress were assessed via chlorophyll fluorescence imaging and the saturation pulse method. In the future, the combination of these two imaging techniques may provide information on the spatial distribution and extent of stress induced ROS production in plant leaves, as well as on the protective ability of various free radical scavengers and antioxidants.     

Glycinebetaine alleviates the inhibitory effect of moderate heat stress on the repair of photosystem II during photoinhibition

Transformation with the bacterial gene codA for choline oxidase allows Synechococcus sp. PCC 7942 cells to accumulate glycinebetaine when choline is supplemented exogenously. First, we observed two types of protective effect of glycinebetaine against heat-induced inactivation of photosystem II (PSII) in darkness; the codA transgene shifted the temperature range of inactivation of the oxygen-evolving complex from 40-52 °C (with half inactivation at 46 °C) to 46-60 °C (with half inactivation at 54 °C) and that of the photochemical reaction center from 44-55 °C (with half inactivation at 51 °C) to 52-63 °C (with half inactivation at 58 °C). However, in light, PSII was more sensitive to heat stress; when moderate heat stress, such as 40 °C, was combined with light stress, PSII was rapidly inactivated, although these stresses, when applied separately, did not inactivate either the oxygen-evolving complex or the photochemical reaction center. Further our studies demonstrated that the moderate heat stress inhibited the repair of PSII during photoinhibition at the site of synthesis de novo of the D1 protein but did not accelerate the photodamage directly. The codA transgene and, thus, the accumulation of glycinebetaine alleviated such an inhibitory effect of moderate heat stress on the repair of PSII by accelerating the synthesis of the D1 protein. We propose a hypothetical scheme for the cyanobacterial photosynthesis that moderate heat stress inhibits the translation machinery and glycinebetaine protects it against the heat-induced inactivation.     

Deciphering the potential involvement of PXMP2 and PEX11B in hydrogen peroxide permeation across the peroxisomal membrane reveals a role for PEX11B in protein sorting

Peroxisomes have the intrinsic ability to produce and scavenge hydrogen peroxide (H₂O₂), a diffusible second messenger that controls diverse cellular processes by modulating protein activity through cysteine oxidation. Current evidence indicates that H₂O₂, a molecule whose physicochemical properties are similar to those of water, traverses cellular membranes through specific aquaporin channels, called peroxiporins. Until now, no peroxiporin-like proteins have been identified in the peroxisomal membrane, and it is widely assumed that small molecules such as H₂O₂ can freely permeate this membrane through PXMP2, a non-selective pore-forming protein with an upper molecular size limit of 300–600 Da. By employing the CRISPR-Cas9 technology in combination with a Flp-In T-REx 293 cell line that can be used to selectively generate H₂O₂ inside peroxisomes in a controlled manner, we provide evidence that PXMP2 is not essential for H₂O₂ permeation across the peroxisomal membrane, neither in control cells nor in cells lacking PEX11B, a peroxisomal membrane-shaping protein whose yeast homologue facilitates the permeation of molecules up to 400 Da. During the course of this study, we unexpectedly noted that inactivation of PEX11B leads to partial localization of both peroxisomal membrane and matrix proteins to mitochondria and a decrease in peroxisome density. These findings are discussed in terms of the formation of a functional peroxisomal matrix protein import machinery in the outer mitochondrial membrane.     

Analysis of gene expression for studying tumor progression: the case of glucocorticoid administration

Background

Glucocorticoids are commonly used as adjuvant treatment for side-effects and have anti-proliferative activity in several tumors but, on the other hand, their proliferative effect has been reported in several studies, some of them involving the spread of cancer. We shall attempt to reconcile these incongruities from the genomic and tissue-physiology perspectives with our findings.

Methods

An accurate phenotype analysis of microarray data can help to solve multiple paradoxes derived from tumor-progression models. We have developed a new strategy to facilitate the study of interdependences among the phenotypes defined by the sample clusters obtained by common clustering methods (HC, SOTA, SOM, PAM). These interdependences are obtained by the detection of non-linear expression-relationships where each fluctuation in the relationship implies a phenotype change and each relationship typology implies a specific phenotype interdependence. As a result, multiple phenotypic changes are identified together with the genes involved in the phenotype transitions. In this way, we study the phenotypic changes from microarray data that describe common phenotypes in cancer from different tissues, and we cross our results with biomedical databases to relate the glucocorticoid activity to the phenotypic changes.

Results

11,244 significant non-linear expression relationships, classified into 11 different typologies, have been detected from the data matrix analyzed. From them, 415 non-linear expression relationships were related to glucocorticoid activity. Studying them, we have found the possible reason for opposite effects of some stressor agents like dexamethasone on tumor progression and it has been confirmed by literature. This hidden reason has resulted in being linked with the type of tumor progression of the tissues. In the first type of tumor progression found, new cells can be stressed during proliferation and stressor agents increase tumor proliferation. In the second type, cell stress and tumor proliferation are antagonists so, therefore, stressor agents stop tumor proliferation in order to stress the cells. The non-linear expression relationships among DUSP6, FERMT2, FKBP5, EGFR, NEDD4L and CITED2 genes are used to synthesize these findings.