Mass‐Spectrometric Identification of Anandamide and 2‐Arachidonoylglycerol in Nematodes

The purpose of the study was to see if nematodes (Caenorhabditis elegans, Caenorhabditis briggsae, and Pelodera strongyloides) produce endocannabinoids; i.e., anandamide (AEA) and 2‐arachidonoylglycerol (2‐AG). In this study, AEA and 2‐AG were identified as endogenous products from nematodes by using electrospray‐ionization ion‐trap MS/MS (ESI‐IT‐MS) experiments operated in the positive‐ionization mode. Endocannabinoids were identified by product ion scan and concentrations were measured by triple quadrupole mass spectrometry in the multiple reaction monitoring mode (MRM). Both AEA and 2‐AG were identified in all of the nematode samples, even though these species lack known cannabinoid receptors. Neither AEA nor 2‐AG were detected in the fat‐3 mutant of C. elegans, which lacks the necessary enzyme to produce arachidonic acid, the fatty acid precursor of these endocannabinoids.     

Single-molecule micromanipulation studies of methylated DNA

Cytosine methylated at the five-carbon position is the most widely studied reversible DNA modification. Prior findings indicate that methylation can alter mechanical properties. However, those findings were qualitative and sometimes contradictory, leaving many aspects unclear. By applying single-molecule magnetic force spectroscopy techniques allowing for direct manipulation and dynamic observation of DNA mechanics and mechanically driven strand separation, we investigated how CpG and non-CpG cytosine methylation affects DNA micromechanical properties. We quantitatively characterized DNA stiffness using persistence length measurements from force-extension curves in the nanoscale length regime and demonstrated that cytosine methylation results in longer contour length and increased DNA flexibility (i.e., decreased persistence length). In addition, we observed the preferential formation of plectonemes over unwound single-stranded “bubbles” of DNA under physiologically relevant stretching forces and supercoiling densities. The flexibility and high structural stability of methylated DNA is likely to have significant consequences on the recruitment of proteins recognizing cytosine methylation and DNA packaging.     

The proximal region of a noncatalytic eukaryotic seryl-tRNA synthetase extension is required for protein stability in vitro and in vivo

Eukaryotic cytosolic seryl-tRNA synthetases (SerRS) have idiosyncratic C-terminal extensions not present in prokaryotic counterparts. The extensions of two eukaryotic SerRSs were subjected to mutagenesis and partial truncation. Only minor parts of the yeast or maize SerRS extensions, adjacent to the catalytic core (7 of 20 and 8 of 26 amino acids, respectively), were found to be indispensable for protein stability. Truncated proteins with substantially shortened extensions displayed unaltered catalytic properties and could complement a Saccharomyces cerevisiae strain with a disrupted SerRS gene, if these proximal regions were left intact. Although the yeast C-terminal SerRS extension is required for Pex21p binding, the maize counterpart with an appended yeast SerRS extension remained incapable of Pex21p binding, implying that additional regions of yeast SerRS may also contribute to the interaction with the peroxin. The proximal region of the eukaryotic SerRS C-terminal extension is indispensable for protein stability, while the remaining part of the extension remains available for other functions, such as species-specific protein:protein interactions.     

Biocatalyst assessment of recombinant whole-cells expressing the Baeyer-Villiger monooxygenase from Xanthobacter sp. ZL5

An Escherichia coli-based expression system for the Baeyer-Villiger monooxygenase (BVMO) from Xanthobacter sp. ZL5 was screened for whole-cell-mediated biotransformations. Biooxidation studies included kinetic resolutions and regiodivergent conversions of structurally diverse cycloketones. An extended phylogenetic analysis of the BVMOs currently available as recombinant systems with experimentally determined Baeyer-Villigerase activity showed that the enzyme originating from Xanthobacter sp. ZL5 clusters together with the sequences of bacterial CHMO-type BVMOs. The regio- and enantiopreferences experimentally observed for this enzyme are clearly similar to the biocatalytic performance of cyclohexanone monooxygenase from Acinetobacter as prototype for this group of BVMOs and support our previously reported family grouping.     

Establishing the stochastic nature of intracellular calcium oscillations from experimental data

Calcium has been established as a key messenger in both intra- and intercellular signaling. Experimentally observed intracellular calcium responses to different agonists show a variety of behaviors from simple spiking to complex oscillatory regimes. Here we study typical experimental traces of calcium oscillations in hepatocytes obtained in response to phenylephrine and ATP. The traces were analyzed with methods of nonlinear time series analysis in order to determine the stochastic/deterministic nature of the intracellular calcium oscillations. Despite the fact that the oscillations appear, visually, to be deterministic yet perturbed by noise, our analyses provide strong evidence that the measured calcium traces in hepatocytes are prevalently of stochastic nature. In particular, bursting calcium oscillations are temporally correlated Gaussian series distorted by a monotonic, instantaneous, time-independent function, whilst the spiking behavior appears to have a dynamical nonlinear component whereby the overall determinism level is still low. The biological importance of this finding is discussed in relation to the mechanisms incorporated in mathematical models as well as the role of stochasticity and determinism at cellular and tissue levels which resemble typical statistical and thermodynamic effects in physics.     

Lateral drift correction in time-laps images by the particle-tracking algorithm

In long-term time-laps imaging of living cells, a significant lateral drift of the fluorescently labeled structures is often observed due to many reasons including superfusion of solution, temperature gradients, bolus addition of pharmacological agents and cell motility. We have detected lateral drift in long-term time-laps confocal imaging by tracking fluorescent puncta, which represent single exocytotic vesicles expressing synaptopHluorin (spH), a pH sensitive green fluorescence protein. Following the initial increase in fluorescence intensity due to alkalinization of vesicle lumen, the spH fluorescent puncta dimmed, which may be attributed to the resealing of the fusion pore and subsequent slow reacidification of the vesicle, or alternatively the dimming may be due to a significant lateral drift of the vesicle out of the region of interest (ROI). We identified and compensated the lateral drift by tracking particles present in the confocal images, without any additional mechanical and/or optical hardware components. The peak of the Gaussian two-dimensional (2D) curve fitted to the fluorescent particle intensity profile was recorded as the X and Y coordinates of the vesicle in each frame. The resulting coordinates of vesicle positions were averaged and rounded to the nearest pixel value, which was used to correct the drift in the time-laps images. In drift corrected time-laps images, the vesicle remained enclosed by the ROI, and the time dependent changes of spH fluorescence intensity averaged from the ROI remained at a constant level, revealing that endocytosis with subsequent slow reacidification of vesicles was an unlikely event.     

Role of sarcolemmal ATP-sensitive potassium channel in oxidative stress-induced apoptosis: mitochondrial connection

From time of their discovery, sarcolemmal ATP-sensitive K+ (sarcK ATP) channels were thought to have an important protective role in the heart during stress whereby channel opening protects the heart from stress-induced Ca2+ overload and resulting damage. In contrast, some recent studies indicate that sarcK ATP channel closing can lead to cardiac protection. Also, the role of the sarcK ATP channel in apoptotic cell death is unclear. In the present study, the effects of channel inhibition on apoptosis and the specific interaction between the sarcK ATP channel and mitochondria were investigated. Apoptotic cell death of cultured HL-1 and neonatal cardiomyocytes following exposure to oxidative stress was significantly increased in the presence of sarcK ATP channel inhibitor HMR-1098 as evidenced by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling and caspase-3,7 assays. This was paralleled by an increased release of cytochrome c from mitochondria to cytosol, suggesting activation of the mitochondrial death pathway. sarcK ATP channel inhibition during stress had no effect on Bcl-2, Bad, and phospho-Bad, indicating that the increase in apoptosis cannot be attributed to these modulators of the apoptotic pathway. However, monitoring of mitochondrial Ca2+ with rhod-2 fluorescent indicator revealed that mitochondrial Ca2+ accumulation during stress is potentiated in the presence of HMR-1098. In conclusion, this study provides novel evidence that opening of sarcK ATP channels, through a specific Ca2+-related interaction with mitochondria, plays an important role in preventing cardiomyocyte apoptosis and mitochondrial damage during stress.     

VEGF promotes vascular sympathetic innervation

The sympathetic nervous system, via postganglionic innervation of blood vessels and the heart, is an important determinant of cardiovascular function. The mechanisms underlying sympathetic innervation of targets are not fully understood. This study tests the hypothesis that target-derived vascular endothelial growth factor (VEGF) promotes sympathetic innervation of blood vessels. Western blot and immunohistochemical analyses indicate that VEGF is produced by vascular cells in arteries and that VEGF receptors are expressed on sympathetic nerve fibers innervating arteries. In vitro, exogenously added VEGF and VEGF produced by vascular smooth muscle cells (VSMCs) in sympathetic neurovascular cocultures inhibited semaphorin 3A (Sema3A)-induced collapse of sympathetic growth cones. In the absence of Sema3A, VEGF and VSMCs also increased growth cone area. These effects were mediated via VEGF receptor 1. In vivo, the neutralization of VEGF inhibited the reinnervation of denervated femoral arteries. These data demonstrate that target-derived VEGF plays a previously unrecognized role in promoting the growth of sympathetic axons.     

Anisotropic diffusion of fluorescently labeled ATP in rat cardiomyocytes determined by raster image correlation spectroscopy

A series of experimental data points to the existence of profound diffusion restrictions of ADP/ATP in rat cardiomyocytes. This assumption is required to explain the measurements of kinetics of respiration, sarcoplasmic reticulum loading with calcium, and kinetics of ATP-sensitive potassium channels. To be able to analyze and estimate the role of intracellular diffusion restrictions on bioenergetics, the intracellular diffusion coefficients of metabolites have to be determined. The aim of this work was to develop a practical method for determining diffusion coefficients in anisotropic medium and to estimate the overall diffusion coefficients of fluorescently labeled ATP in rat cardiomyocytes. For that, we have extended raster image correlation spectroscopy (RICS) protocols to be able to discriminate the anisotropy in the diffusion coefficient tensor. Using this extended protocol, we estimated diffusion coefficients of ATP labeled with the fluorescent conjugate Alexa Fluor 647 (Alexa-ATP). In the analysis, we assumed that the diffusion tensor can be described by two values: diffusion coefficient along the myofibril and that across it. The average diffusion coefficients found for Alexa-ATP were as follows: 83 +/- 14 microm(2)/s in the longitudinal and 52 +/- 16 microm(2)/s in the transverse directions (n = 8, mean +/- SD). Those values are approximately 2 (longitudinal) and approximately 3.5 (transverse) times smaller than the diffusion coefficient value estimated for the surrounding solution. Such uneven reduction of average diffusion coefficient leads to anisotropic diffusion in rat cardiomyocytes. Although the source for such anisotropy is uncertain, we speculate that it may be induced by the ordered pattern of intracellular structures in rat cardiomyocytes.