Top-Down Lipidomics Reveals Ether Lipid Deficiency in Blood Plasma of Hypertensive Patients

Background

Dyslipoproteinemia, obesity and insulin resistance are integrative constituents of the metabolic syndrome and are major risk factors for hypertension. The objective of this study was to determine whether hypertension specifically affects the plasma lipidome independently and differently from the effects induced by obesity and insulin resistance.

Methodology/Principal Findings

We screened the plasma lipidome of 19 men with hypertension and 51 normotensive male controls by top-down shotgun profiling on a LTQ Orbitrap hybrid mass spectrometer. The analysis encompassed 95 lipid species of 10 major lipid classes. Obesity resulted in generally higher lipid load in blood plasma, while the content of tri- and diacylglycerols increased dramatically. Insulin resistance, defined by HOMA-IR >3.5 and controlled for BMI, had little effect on the plasma lipidome. Importantly, we observed that in blood plasma of hypertensive individuals the overall content of ether lipids decreased. Ether phosphatidylcholines and ether phosphatidylethanolamines, that comprise arachidonic (20∶4) and docosapentaenoic (22∶5) fatty acid moieties, were specifically diminished. The content of free cholesterol also decreased, although conventional clinical lipid homeostasis indices remained unaffected.

Conclusions/Significance

Top-down shotgun lipidomics demonstrated that hypertension is accompanied by specific reduction of the content of ether lipids and free cholesterol that occurred independently of lipidomic alterations induced by obesity and insulin resistance. These results may form the basis for novel preventive and dietary strategies alleviating the severity of hypertension.     

Seed treatment technology: An attractive delivery system for controlling root parasitic weed Striga with mycoherbicide

Coating sorghum seeds with Fusarium oxysporum (Foxy 2) for control of the root parasitic weed Striga, appears to be an attractive option for minimizing the inoculum amount, establishing the biocontrol agent in the potential infection zone of the host plants, and offering a simple, easy and economical delivery system. Our investigations resulted in the selection of appropriate seed coating materials and a suitable type and form of fungal inoculum. The coating materials tested were arabic gum (AG10%, 20%, 40%), carboxymethylcellulose (CMC1%, 2%) and pectin (LS 440, LM-5 CS) 1%, while the fungal inoculum included microconidia and fresh and dried chlamydospores produced using different substrates. Foxy 2 survived the seed treatment processing and showed excellent viability on seeds for at least 8 months of storage after coating. In general, the performance of 40% arabic gum in combination with dried chlamydospores was the best among the other types of inoculum and coating material tested. Regardless of the type and form of inoculum and coating materials tested, Foxy 2 was able to colonize all roots, even root tips and hairs of the host (sorghum), thereby meeting important criteria of a promising candidate for controlling Striga when applied as a seed treatment. The efficacy of treated sorghum seed with Foxy 2 using different coating materials in reducing S. hermonthica infestation was evaluated in pot and root chamber trials. Foxy 2 markedly reduced Striga emergence and dry weight and increased the percentage of the diseased emerged Striga shoots. However, the efficacy of seed coating varied according to the type and form of fungal inoculum as well as coating material. Coating sorghum seed with dried chlamydospore inoculum homogenized into 20% arabic gum (as adhesive) showed the highest efficacy of 81 and 77% (i.e., percent reduction in healthy emerged Striga shoots compared to the control treatment) against Striga using either sterilized or non-sterilized soil, respectively. In root chamber bioassays, the application of Foxy 2 in combination with AG40% significantly caused disease in 77% of the germinated Striga seeds and in all tubercles after 25 days of sowing. These findings provide an optimized coating protocol as an attractive delivery system for bioherbicides for root parasitic weeds.     

Actions of gonadotropin-releasing hormone analogues in pituitary gonadotrophs and their modulation by ovarian steroids

Recently, GnRH antagonists (GnRHant) like cetrorelix and ganirelix have been introduced in protocols of controlled ovarian hyperstimulation for assisted reproductive techniques to prevent premature luteinizing hormone (LH) surges. Here we tested, whether the actions of cetrorelix and the GnRH agonist (GnRHag) triptorelin in gonadotrophs are dependent on the steroid milieu. Furthermore, we characterized the actions of cetrorelix and triptorelin on LH secretion and the total LH pool. Female rat pituitary cells were treated either with 0.1 nM triptorelin for 1, 2, 4 and 6 days or for 1, 3, 5 and 6 h or with 1, 10 or 100 nM cetrorelix for 1, 2, 3 and 5 h or for 10 min. Cells were stimulated for 3h with different concentrations of GnRH (10 pM-1 microM). For analysis of the total LH pool, which is composed of stored and released LH, cells were lysed with 0.1% Triton X-100 at -80 degrees C overnight. To test, whether the steroid milieu affects the actions of cetrorelix and triptorelin, cells were incubated for 52 h with 1 nM estradiol (E) alone or with combinations of 100 nM progesterone (P) for 4 or 52 h, respectively. Cells were then treated with 0.1 nM triptorelin for 9 h or 1 nM cetrorelix for 3 h and stimulated for 3 h with different concentrations of GnRH (10 pM-1 microM). The suppressive effect of triptorelin on LH secretion was fully accomplished after 3 h of treatment, for cetrorelix only 10 min were sufficient. The concentration of cetrorelix must be at least equimolar to GnRH to block LH secretion. Cetrorelix shifted the EC50s of the GnRH dose-response curve to the right. Triptorelin suppressed total LH significantly (from 137 to 36 ng/ml) after 1 h in a time-dependent manner. In contrast, only high concentrations of cetrorelix increased total LH. In steroid treated cells the suppressive effects of triptorelin were more distinct. One nanomolar cetrorelix suppressed GnRH-stimulated LH secretion of cells not treated with steroids from 10.1 to 3.5 ng/ml. In cells, additionally treated with estradiol alone or estradiol and short-term progesterone, LH levels were higher (from 3.5 to 5.4 or 4.5 ng/ml, respectively). In cells co-treated with estradiol and progesterone for 52 h LH secretion was only suppressed from 10.1 to 9.5 ng/ml. Steroid treatments diminished the suppressive effect of cetrorelix on LH secretion. In conclusion, the depletion of the total LH pool contributes to the desensitizing effect of triptorelin. The actions of cetrorelix and triptorelin are dependent on the steroid milieu.     

Integration specificity of phage phiC31 integrase in the human genome

The site-specific integrase from bacteriophage phiC31 functions in mammalian cells and is being applied for genetic engineering, including gene therapy. The phiC31 integrase catalyzes precise, unidirectional recombination between its 30-40-bp attP and attB recognition sites. In mammalian cells, the enzyme also mediates integration of plasmids bearing attB into native sequences that have partial sequence identity with attP, termed pseudo attP sites. Here, we analyzed the features of phiC31-mediated integration into pseudo attP sites in the human genome. Sequence analysis of 196 independent integration events derived from three cell lines revealed approximately 101 integration sites: 56% of the events were recurrent integrations distributed among 19 pseudo attP sequences. Bioinformatics analysis revealed a approximately 30-bp palindromic consensus sequence motif shared by all of the repeat occurrences and most of the single occurrence sites, verifying that phiC31-mediated integration into pseudo attP sites is significantly guided by DNA sequence recognition. The most favored unique sequence in these cell lines occurred at chromosome 19q13.31 and accounted for 7.5% of integration events. Other frequent integration sites were in three specific sequences in subfamilies of ERVL and L1 repetitive sequences, accounting for an additional 17.9% of integration events. Integrations could occur in either orientation at a pseudo attP site, were often accompanied by small deletions, and typically occurred in a single copy per cell. A number of aberrant events were also described, including large deletions and chromosome rearrangements. phiC31 integrase-mediated integration only slightly favored genes and did not favor promoter regions. Gene density and expression studies suggested chromatin context effects. An analysis of the safety of integration sites in terms of proximity to cancer genes suggested minimal cancer risk. We conclude that integration systems derived from phiC31 integrase have great potential utility.     

Drought Effects in Climate Change Manipulation Experiments: Quantifying the Influence of Ambient Weather Conditions and Rain-out Shelter Artifacts

Extreme drought events challenge ecosystem functioning. Ecological response to drought is studied worldwide in a growing number of field experiments by rain-out shelters. Yet, few meta-analyses face severe challenges in the comparability of studies. This is partly because build-up of drought stress in rain-out shelters is modified by ambient weather conditions. Rain-out shelters can further create confounding effects (radiation, temperature), which may influence plant responses. Yet, a quantification of ecophysiological effects within rain-out shelters under opposing ambient weather conditions and of microclimatological artifacts is missing. Here, we examined phytometers—standardized potted individuals of Plantago lanceolata—under rain-out shelter, rain-out shelter artifact control, and ambient control during opposing outside microclimatological conditions. Furthermore, we tested for artifacts of rain-out shelters on plant responses in a long-term semi-natural grassland experiment. Phytometer plants below the rain-out shelters showed lower stomatal conductance, maximum quantum efficiency, and leaf water potential during warm ambient conditions with high evaporative demand than during cold conditions with low evaporative demand. Plant performance was highly correlated with ambient temperature and vapor pressure deficit (VPD). Rain-out shelter artifacts on plant responses were nonsignificant. Rain-out shelters remain a viable tool for studying ecosystem responses to drought. However, drought manipulations using rain-out shelters are strongly modified by ambient weather conditions. Attributing the results from rain-out shelter studies to drought effects and comparability among studies and study years therefore requires the quantification of the realized drought stress, for example, by relating ecosystem responses to measured microclimatological parameters such as air temperature and VPD.     

The Na(+)-translocating F₁F₀-ATPase from the halophilic, alkalithermophile Natranaerobius thermophilus

Natranaerobius thermophilus is an unusual anaerobic extremophile, it is halophilic and alkalithermophilic; growing optimally at 3.3-3.9M Na(+), pH(50°C) 9.5 and 53°C. The ATPase of N. thermophilus was characterized at the biochemical level to ascertain its role in life under hypersaline, alkaline, thermal conditions. The partially purified enzyme (10-fold purification) displayed the typical subunit pattern for F-type ATPases, with a 5-subunit F(1) portion and 3-subunit-F(O) portion. ATP hydrolysis by the purified ATPase was stimulated almost 4-fold by low concentrations of Na(+) (5mM); hydrolysis activity was inhibited by higher Na(+) concentrations. Partially purified ATPase was alkaliphilic and thermophilic, showing maximal hydrolysis at 47°C and the alkaline pH(50°C) of 9.3. ATP hydrolysis was sensitive to the F-type ATPase inhibitor N,N'-dicylohexylcarbodiimide and exhibited inhibition by both free Mg(2+) and free ATP. ATP synthesis by inverted membrane vesicles proceeded slowly and was driven by a Na(+)-ion gradient that was sensitive to the Na(+)-ionophore monensin. Analysis of the atp operon showed the presence of the Na(+)-binding motif in the c subunit (Q(33), E(66), T(67), T(68), Y(71)), and a complete, untruncated ε subunit; suggesting that ATP hydrolysis by the enzyme is regulated. Based on these properties, the F(1)F(O)-ATPase of N. thermophilus is a Na(+)-translocating ATPase used primarily for expelling cytoplasmic Na(+) that accumulates inside cells of N. thermophilus during alkaline stress. In support of this theory are the presence of the c subunit Na(+)-binding motif and the low rates of ATP synthesis observed. The complete ε subunit is hypothesized to control excessive ATP hydrolysis and preserve intracellular Na(+) needed by electrogenic cation/proton antiporters crucial for cytoplasmic acidification in the obligately alkaliphilic N. thermophilus.