Genetic control of growth,biomass allocation,and survival under drought stress in Pinus radiata D. Don seedlings

Drought is one of the critical factors limiting plant growth, and knowledge about genetic adaptation to drought is necessary to develop strategies for successful reforestation on drought-prone sites. In this study, genetic variation was investigated in 98 full- and half-sib radiata pine (Pinus radiata D. Don) families, representing Chilean coastal and interior populations, subjected to two water regimes: well-watered and drought-stressed. Assessed traits, in 5-month-old seedlings, included height (H), diameter (D), height-to-diameter ratio (HDR), dry biomass of needles (NDW), stem (SDW), roots (RDW), and total (TDW), root-to-shoot ratio (RSR), and survival (SUR). After 115 days of treatment, growth, biomass, and survival were nearly two times higher under the well-watered regime than under the stressed one. Families differed significantly in most traits, with individual tree heritabilities ranging from 0.14 for SUR to 0.63 for D in the well-watered treatment. Families from the interior showed the highest heritability for D, SDW, RDW, and TDW when grown in the water-stress treatment. The genetic correlations between treatments were moderately strong, which suggests the presence of a genotype by watering regime interaction. Most traits were strongly correlated (genetic correlations often exceeded 0.40). Compared to the first generation families from coastal sites, the third generation families from the interior sites showed an increase in SUR and RSR. Thus, potential exists to screen families at the seedling stage for drought hardiness and to identify parents from the interior sites with potential to produce a more drought-resistant breed with satisfactory growth rates and yields in dry environments.     

Steroids linked with amide bond-extended cholesterol

New type of linear cholesterol-like molecules based on cholesterol extended by attachment of etienic acid derivatives was designed and oligosteroids with two to four units were synthesized. Amide bond was used for inter steroid connections and 1-hydroxybenzotriazole active ester method was adapted for their formations. Use of disteroids as larger building blocks was applied.     

Generation of Hydroxyl Radical in Isolated Pea Root Cell Wall, and the Role of Cell Wall-Bound Peroxidase, Mn-SOD and Phenolics in Their Production

The hydroxyl radical produced in the apoplast has been demonstratedto facilitate cell wall loosening during cell elongation. Cellwall-bound peroxidases (PODs) have been implicated in hydroxylradical formation. For this mechanism, the apoplast or cellwalls should contain the electron donors for (i) H₂O₂ formationfrom dioxygen; and (ii) the POD-catalyzed reduction of H₂O₂to the hydroxyl radical. The aim of the work was to identifythe electron donors in these reactions. In this report, hydroxylradical (·OH) generation in the cell wall isolated frompea roots was detected in the absence of any exogenous reductants,suggesting that the plant cell wall possesses the capacity togenerate ·OH in situ. Distinct POD and Mn-superoxidedismutase (Mn-SOD) isoforms different from other cellular isoformswere shown by native gel electropho-resis to be preferably boundto the cell walls. Electron paramagnetic resonance (EPR) spectroscopyof cell wall isolates containing the spin-trapping reagent,5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO),was used for detection of and differentiation between ·OHand the superoxide radical (O₂^–·). The data obtainedusing POD inhibitors confirmed that tightly bound cell wallPODs are involved in DEPMPO/OH adduct formation. A decreasein DEPMPO/OH adduct formation in the presence of H₂O₂ scavengersdemonstrated that this hydroxyl radical was derived from H₂O₂.During the generation of ·OH, the concentration of quinhydronestructures (as detected by EPR spectroscopy) increased, suggestingthat the H₂O₂ required for the formation of ·OH in isolatedcell walls is produced during the reduction of O₂ by hydroxycinnamicacids. Cell wall isolates in which the proteins have been denaturated(including the endogenous POD and SOD) did not produce ·OH.Addition of exogenous H₂O₂ again induced the production of ·OH,and these were shown to originate from the Fenton reaction withtightly bound metal ions. However, the appearance of the DEPMPO/OOHadduct could also be observed, due to the production of O₂^–·when endogenous SOD has been inactivated. Also, O₂^–·was converted to ·OH in an in vitro horseradish peroxidase(HRP)/H₂O₂ system to which exogenous SOD has been added. Takentogether with the discovery of the cell wall-bound Mn-SOD isoform,these results support the role of such a cell wall-bound SODin the formation of ·OH jointly with the cell wall-boundPOD. According to the above findings, it seems that the hydroxycinnamicacids from the cell wall, acting as reductants, contribute tothe formation of H₂O₂ in the presence of O₂ in an autocatalyticmanner, and that POD and Mn-SOD coupled together generate ·OHfrom such H₂O₂.     

Cell-cycle-dependent variations in FTIR micro-spectra of single proliferating HeLa cells: principal component and artificial neural network analysis

We have previously reported spectral differences for cells at different stages of the eukaryotic cell division cycle. These differences are due to the drastic biochemical and morphological changes that occur as a consequence of cell proliferation. We correlate these changes in FTIR absorption and Raman spectra of individual cells with their biochemical age (or phase in the cell cycle), determined by immunohistochemical staining to detect the appearance (and subsequent disappearance) of cell-cycle-specific cyclins, and/or the occurrence of DNA synthesis. Once spectra were correlated with their cells' staining patterns, we used methods of multivariate statistics to analyze the changes in cellular spectra as a function of cell cycle phase.     

A computational approach toward label-free protein quantification using predicted peptide detectability

We propose here a new concept of peptide detectability which could be an important factor in explaining the relationship between a protein's quantity and the peptides identified from it in a high-throughput proteomics experiment. We define peptide detectability as the probability of observing a peptide in a standard sample analyzed by a standard proteomics routine and argue that it is an intrinsic property of the peptide sequence and neighboring regions in the parent protein. To test this hypothesis we first used publicly available data and data from our own synthetic samples in which quantities of model proteins were controlled. We then applied machine learning approaches to demonstrate that peptide detectability can be predicted from its sequence and the neighboring regions in the parent protein with satisfactory accuracy. The utility of this approach for protein quantification is demonstrated by peptides with higher detectability generally being identified at lower concentrations over those with lower detectability in the synthetic protein mixtures. These results establish a direct link between protein concentration and peptide detectability. We show that for each protein there exists a level of peptide detectability above which peptides are detected and below which peptides are not detected in an experiment. We call this level the minimum acceptable detectability for identified peptides (MDIP) which can be calibrated to predict protein concentration. Triplicate analysis of a biological sample showed that these MDIP values are consistent among the three data sets.     

Methylglyoxal Activates Nociceptors through Transient Receptor Potential Channel A1 (TRPA1): A POSSIBLE MECHANISM OF METABOLIC NEUROPATHIES

Neuropathic pain can develop as an agonizing sequela of diabetes mellitus and chronic uremia. A chemical link between both conditions of altered metabolism is the highly reactive compound methylglyoxal (MG), which accumulates in all cells, in particular neurons, and leaks into plasma as an index of the severity of the disorder. The electrophilic structure of this cytotoxic ketoaldehyde suggests TRPA1, a receptor channel deeply involved in inflammatory and neuropathic pain, as a molecular target. We demonstrate that extracellularly applied MG accesses specific intracellular binding sites of TRPA1, activating inward currents and calcium influx in transfected cells and sensory neurons, slowing conduction velocity in unmyelinated peripheral nerve fibers, and stimulating release of proinflammatory neuropeptides from and action potential firing in cutaneous nociceptors. Using a model peptide of the N terminus of human TRPA1, we demonstrate the formation of disulfide bonds based on MG-induced modification of cysteines as a novel mechanism. In conclusion, MG is proposed to be a candidate metabolite that causes neuropathic pain in metabolic disorders and thus is a promising target for medicinal chemistry.     

Facilitation of long-term potentiation by muscarinic M(1) receptors is mediated by inhibition of SK channels

Muscarinic receptor activation facilitates the induction of synaptic plasticity and enhances cognitive function. However, the specific muscarinic receptor subtype involved and the critical intracellular signaling pathways engaged have remained controversial. Here, we show that the recently discovered highly selective allosteric M(1) receptor agonist 77-LH-28-1 facilitates long-term potentiation (LTP) induced by theta burst stimulation at Schaffer collateral synapses in the hippocampus. Similarly, release of acetylcholine by stimulation of cholinergic fibers facilitates LTP via activation of M(1) receptors. N-methyl-D-aspartate receptor (NMDAR) opening during theta burst stimulation was enhanced by M(1) receptor activation, indicating this is the mechanism for LTP facilitation. M(1) receptors were found to enhance NMDAR activation by inhibiting SK channels that otherwise act to hyperpolarize postsynaptic spines and inhibit NMDAR opening. Thus, we describe a mechanism where M(1) receptor activation inhibits SK channels, allowing enhanced NMDAR activity and leading to a facilitation of LTP induction in the hippocampus.     

Versatile and Simple Approach to Determine Astrocyte Territories in Mouse Neocortex and Hippocampus

Background

Besides their neuronal support functions, astrocytes are active partners in neuronal information processing. The typical territorial structure of astrocytes (the volume of neuropil occupied by a single astrocyte) is pivotal for many aspects of glia–neuron interactions.

Methods

Individual astrocyte territorial volumes are measured by Golgi impregnation, and astrocyte densities are determined by S100β immunolabeling. These data are compared with results from conventionally applied methods such as dye filling and determination of the density of astrocyte networks by biocytin loading. Finally, we implemented our new approach to investigate age-related changes in astrocyte territories in the cortex and hippocampus of 5- and 21-month-old mice.

Results

The data obtained by our simplified approach based on Golgi impregnation were compared to previously published dye filling experiments, and yielded remarkably comparable results regarding astrocyte territorial volumes. Moreover, we found that almost all coupled astrocytes (as indicated by biocytin loading) were immunopositive for S100β. A first application of this new experimental approach gives insight in age-dependent changes in astrocyte territorial volumes. They increased with age, while cell densities remained stable. In 5-month-old mice, the overlap factor was close to 1, revealing little or no interdigitation of astrocyte territories. However, in 21-month-old mice, the overlap factor was more than 2, suggesting that processes of adjacent astrocytes interdigitate.

Conclusion

Here we verified the usability of a simple, versatile method for assessing astrocyte territories and the overlap factor between adjacent territories. Second, we found that there is an age-related increase in territorial volumes of astrocytes that leads to loss of the strict organization in non-overlapping territories. Future studies should elucidate the physiological relevance of this adaptive reaction of astrocytes in the aging brain and the methods presented in this study might be a powerful tool to do so.