The effect of repeated acute mental stress on habituation and recovery responses in hemoconcentration and blood cells in healthy men

Acute mental stress elicits hemoconcentration and polycytosis. We investigated whether haematological response to repeated acute mental stress would habituate and be sustained 45 min and 105 min after stress. Twenty-four men underwent a 13-min stressor three times, one week apart; hematological variables were measured at week one and three. Hematocrit, hemoglobin, leukocytes, lymphocytes, erythrocytes, and thrombocytes all increased from rest to immediately post-stress (p's<.001). After 105 min of recovery, leukocytes and platelets both were higher, and hematocrit, hemoglobin, lymphocytes, and erythrocytes were all lower than at rest (p's<.001 to <.05). At all time points, hematocrit (p=.005) and erythrocytes (p=.006) were lower at week three than at week one. In contrast to an attenuation in systolic blood pressure increase from rest to immediately post-stress (p<.001), and in cortisol recovery from immediately post-stress to 45 min post-stress (p<.001), the magnitude of change in hemoconcentration and cell counts in stress and recovery experienced no habituation. Adjustment for stress-induced plasma volume shift altered findings: Elevated leukocytes post-stress persisted at 105 min (p<.001); any changes in lymphocytes became insignificant; erythrocytes decreased from rest to post-stress (p<.001) to increase again during recovery (p's<.05); platelets increased linearly between rest and 105 min of recovery (p=.005). We conclude that the magnitude of changes in hemoconcentration and blood cells during acute mental stress and recovery failed to habituate to stress repeats and, in part, sustained up to 105 min. Plasma volume shift accompanying stress affects the time course of stress polycytosis.     

Antioxidant properties of cystic fibrosis sputum

Oxidative stress is a likely contributor to the pathogenesis of cystic fibrosis (CF) lung disease. However, hydrogen peroxide (H(2)O(2)), a physiological oxidant, is not elevated in CF exhalates. H(2)O(2) may be neutralized by antioxidants in CF airway secretions. The H(2)O(2)-detoxifying capacity of CF airway secretions, obtained via sputum induction, was studied in an in vitro H(2)O(2) cytotoxicity model. 16HBE14o- cells were exposed to H(2)O(2) in culture medium containing either 0 or 10% fetal bovine serum (FBS) or 10% CF sputum supernatant (extracted without use of dithiothreitol). The efficiency of H(2)O(2) neutralization was estimated by measuring intracellular oxidant levels (dihydrorhodamine 123) after 2 h and cell viability (propidium iodide) after 24 h of H(2)O(2) exposure. Furthermore, the presence of reduced thiols (DTNB assay) and reduced glutathione (recycling assay) in CF sputum samples was evaluated. CF sputum extracts completely prevented intracellular oxidant accumulation seen in cells incubated with H(2)O(2) in both control media (i.e., 0 or 10% FBS). Furthermore, CF sputum abolished cell death in 16HBE14o- cells exposed to up to 1 mM H(2)O(2). In contrast, there was 100% cytotoxicity in cells exposed to 600 microM H(2)O(2) in both control media. The H(2)O(2)-detoxifying potential of CF sputum was sustained after catalase and heme peroxidases were inactivated by sodium azide, which does not affect glutathione peroxidase. In addition, reduced protein thiols were found in abundance in CF sputum. In conclusion, CF sputum is capable to neutralize H(2)O(2) and abundant reduced thiols and/or glutathione peroxidase are fully sufficient to detoxify H(2)O(2).     

Automated acquisition of cryo-electron micrographs for single particle reconstruction on an FEI Tecnai electron microscope

AutoEM is a software package developed by Zhang et al. [J. Struct. Biol. 1356, 251] for semi-automated acquisition of cryo-electron micrographs from Tecnai series electron microscopes and is used frequently at the lowest level of automation. We report here on the new progress that we have made based on their preliminary work. A fourth low-dose state is created where the system can pre-select all the good holes in a grid square from a single CCD image taken at low magnification, making the system operative at much higher levels of automation. An additional control interface enables the operator to monitor the status of the program and the quality of the data, interact with the program, and direct the execution process according to intermediate results. When data acquisition is in progress, all useful information is automatically saved in certain text files which are easily accessible by a database. More detailed improvements and general advantages are illustrated and discussed. We have started to use the program to perform routine data collection. A number of applications show that the performance of the program is satisfactory and the quality of the micrographs and their power spectra acquired by the program is comparable to those manually collected under the same conditions.     

Candida albicans MTLalpha tup1Delta mutants can reversibly switch to mating-competent, filamentous growth forms

Candida albicans strains that are homozygous at the mating type locus (MTLa or MTLalpha) can spontaneously switch from the normal round-to-oval yeast cell morphology to an elongated, so-called opaque cell form that can mate with opaque cells of the opposite mating type. In response to environmental signals, C. albicans also undergoes a transition from yeast to filamentous growth, which is negatively regulated by the general repressor Tup1p. Therefore, C. albicans mutants in which the TUP1 gene is inactivated grow constitutively in the filamentous form. We found that tup1Delta mutants of the MTLalpha strain WO-1 are still able to undergo phenotypic switching. Although the mutants had lost the capacity to grow in the normal yeast (white) or opaque forms, they could still reversibly switch between four different cell and colony phenotypes (designated as fuzzy, frizzy, wrinkled and smooth) at a frequency of about 10(-3) to 10(-4). Deletion of TUP1 resulted in deregulated expression of phase-specific genes. While the white-specific WH11 gene was constitutively expressed in all four cell types, the opaque-specific SAP1 gene remained repressed and the opaque-specific OP4 gene was weakly induced in all phase variants. In spite of the loss of white- and opaque-specific cell morphology and gene expression, the tup1Delta mutants retained an important characteristic of their wild-type parent, the ability to switch to a mating-competent form. The three filamentous phase variants (fuzzy, frizzy and wrinkled) all were able to mate and produce recombinant progeny with opaque cells of an MTLa strain at frequencies that were somewhat lower than those of normal opaque cells, whereas the smooth phase variant was unable to do so. Therefore, although deletion of TUP1 in C. albicans MTLalpha cells affects cellular morphology and gene expression patterns, the mutants can still reversibly switch between mating-competent and -incompetent cell types and mate with a partner of the opposite mating type.     

CBCAnalyzer: inferring phylogenies based on compensatory base changes in RNA secondary structures

The CBCAnalyzer (CBC=compensatory base change) is a custom written software toolbox consisting of three parts, CTTransform, CBCDetect, and CBCTree. CTTransform reads several ct-file formats, and generates a so called "bracket-dot-bracket" format that typically is used as input for other tools such as RNAforester, RNAmovie or MARNA. The latter one creates a multiple alignment based on primary sequences and secondary structures that now can be used as input for CBCDetect. CBCDetect counts CBCs in all against all of the aligned sequences. This is important in detecting species that are discriminated by their sexual incompatibility. The count (distance) matrix obtained by CBCDetect is used as input for CBCTree that reconstructs a phylogram by using the algorithm of BIONJ. In this note we describe the features of the toolbox as well as application examples. The toolbox provides a graphical user interface. It is written in C++ and freely available at: http://cbcanalyzer.bioapps.biozentrum.uni-wuerzburg.de.     

Correlative GC-TOF-MS-based metabolite profiling and LC-MS-based protein profiling reveal time-related systemic regulation of metabolite–protein networks and improve pattern recognition for multiple biomarker selection

A novel approach is presented combining quantitative metabolite and protein data and multivariate statistics for the analysis of time-related regulatory effects of plant metabolism at a systems level. For the analysis of metabolites, gas chromatography coupled to a time-of-flight mass analyzer (GC-TOF-MS) was used. Proteins were identified and quantified using a novel procedure based on shotgun sequencing as described recently (Weckwerth etal., 2004b, Proteomics 4, 78–83). For comparison, leaves of Arabidopsis thaliana wild type plants and starchless mutant plants deficient in phosphoglucomutase activity (PGM) were sampled at intervals throughout the day/night cycle. Using principal and independent components analysis, each dataset (metabolites and proteins) displayed discrete characteristics. Compared to the analysis of only metabolites or only proteins, independent components analysis (ICA) of the integrated metabolite/protein dataset resulted in an improved ability to distinguish between WT and PGM plants (first independent component) and, in parallel, to see diurnal variations in both plants (second independent component). Interestingly, levels of photorespiratory intermediates such as glycerate and glycine best characterized phases of diurnal rhythm, and were not influenced by high sugar accumulation in PGM plants. In contrast to WT plants, PGM plants showed an inversely regulated cluster of N-rich amino acid metabolites and carbohydrates, indicating a shift in C/N partitioning. This observation corresponds to altered utilization of urea cycle intermediates in PGM plants suggesting enhanced protein degradation and carbon utilization due to growth inhibition. Among the proteins chloroplastidic GAPDH (At3g26650) was the best discriminator between WT and PGM plants in contrast to the cytosolic isoform (At1g13440) according to the primary effect of mutation located in the chloroplast. The described method is applicable to all kinds of biological systems and enables the unbiased identification of biomarkers embedded in correlative metabolite–protein networks.     

A two-dimensional electrophoretic map of human mitochondrial proteins from immortalized lymphoblastoid cell lines: a prerequisite to study mitochondrial disorders in patients

Mitochondrial diseases may be caused by numerous mutations that alter proteins of the respiratory chain and of other metabolic pathways in the mitochondrium. For clinicians this disease group poses a considerable diagnostic challenge due to ambiguous genotype-phenotype relationships. Until now, only 30% of the mitochondriopathies can be diagnosed at the molecular level. We therefore need a new diagnostic tool that offers a wide view on the mitochondrial proteins. Here, we present a method to generate a high-resolution, large-gel two-dimensional gel electrophoretic (2-DE) map of a purified fraction of mitochondrial proteins from Epstein-Barr virus-immortalized lymphoblastoid cell line (LCL). LCLs can be easily obtained from patients and control subjects in a routine clinical setting. They often express the biochemical phenotype and can be cultured to high cell numbers, sufficient to gain enough purified material for 2-DE. In total we identified 166 mitochondrial proteins. Thirteen proteins were earlier not known to be of mitochondrial origin. Thirty-nine proteins were associated with human diseases ranging from respiratory chain enzyme deficiencies to disorders of beta-oxidation and amino acid metabolism. This 2-DE map is intended to be the first step to diagnose mitochondrial diseases at the proteomic level.     

P-deficiency increases the O2 uptake per N2 reduced in alfalfa

Nodulated alfalfa (Medicago sativa L. cv. Saranac) plants were grown in hydroponics at P-sufficient and P-deficient supply levels. After 5 weeks of growth, dry matter accumulation, nodulation, total N and P accumulation, as well as 15N2 uptake, were measured. Moreover, the response of nodule O2-uptake to raising external pO2 was determined in an open-flow measurement system and nodule permeability was calculated. Plants in the P-deficient supply treatment had a lower P concentration in all organs. In both treatments the highest P concentration was found in nodules. In the P-deficient supply treatment plants formed less dry matter, had a lower shoot/root ratio, less nodulation, decreased total N accumulation, and lower 15N2 uptake per dry matter nodule. Nodules in the P-deficient treatment were, on average, smaller and had a higher O2 uptake per N2 reduced, coinciding with increased nodule permeability and conductance. Thus increased oxygen uptake appears to be a mechanism to adjust nodule metabolism to P deficiency in indeterminate N2-fixing nodules such as in alfalfa, as has previously been shown for determinate nodule forms.     

A cell-based cGMP assay useful for ultra-high-throughput screening and identification of modulators of the nitric oxide/cGMP pathway

We have established a rapid, homogeneous, cell-based, and highly sensitive assay for guanosine 3'-5'-cyclic monophosphate (cGMP) that is suitable for fully automated ultra-high-throughput screening. In this assay system, cGMP production is monitored in living cells via Ca2+ influx through the olfactory cyclic nucleotide-gated cation channel CNGA2, acting as the intracellular cGMP sensor. A stably transfected Chinese hamster ovary (CHO) cell line was generated recombinantly expressing soluble guanylate cyclase, CNGA2, and aequorin as a luminescence indicator for the intracellular calcium concentration. This cell line was used to screen more than 900,000 compounds in an automated ultra-high-throughput screening assay using 1536-well microtiter plates. In this way, we have been able to identify BAY 58-2667, a member of a new class of amino dicarboxylic acids that directly activate soluble guanylate cyclase. The assay system allows the real-time cGMP detection within living cells and makes it possible to screen for activators and inhibitors of enzymes involved in the nitric oxide/cGMP pathway.     

Structural characterization of the interaction of the delta and alpha subunits of the Escherichia coli F1F0-ATP synthase by NMR spectroscopy

A critical point of interaction between F(1) and F(0) in the bacterial F(1)F(0)-ATP synthase is formed by the alpha and delta subunits. Previous work has shown that the N-terminal domain (residues 3-105) of the delta subunit forms a 6 alpha-helix bundle [Wilkens, S., Dunn, S. D., Chandler, J., Dahlquist, F. W., and Capaldi, R. A. (1997) Nat. Struct. Biol. 4, 198-201] and that the majority of the binding energy between delta and F(1) is provided by the interaction between the N-terminal 22 residues of the alpha- and N-terminal domain of the delta subunit [Weber, J., Muharemagic, A., Wilke-Mounts, S., and Senior, A. E. (2003) J. Biol. Chem. 278, 13623-13626]. We have now analyzed a 1:1 complex of the delta-subunit N-terminal domain and a peptide comprising the N-terminal 22 residues of the alpha subunit by heteronuclear protein NMR spectroscopy. A comparison of the chemical-shift values of delta-subunit residues with and without alpha N-terminal peptide bound indicates that the binding interface on the N-terminal domain of the delta subunit is formed by alpha helices I and V. NOE cross-peak patterns in 2D (12)C/(12)C-filtered NOESY spectra of the (13)C-labeled delta-subunit N-terminal domain in complex with unlabeled peptide verify that residues 8-18 in the alpha-subunit N-terminal peptide are folded as an alpha helix when bound to delta N-terminal domain. On the basis of intermolecular contacts observed in (12)C/(13)C-filtered NOESY experiments, we describe structural details of the interaction of the delta-subunit N-terminal domain with the alpha-subunit N-terminal alpha helix.