Activation of AMP-activated Protein Kinase Regulates Hippocampal Neuronal pH by Recruiting Na+/H+ Exchanger NHE5 to the Cell Surface

Strict regulation of intra- and extracellular pH is an important determinant of nervous system function as many voltage-, ligand-, and H⁺-gated cationic channels are exquisitely sensitive to transient fluctuations in pH elicited by neural activity and pathophysiologic events such as hypoxia-ischemia and seizures. Multiple Na⁺/H⁺ exchangers (NHEs) are implicated in maintenance of neural pH homeostasis. However, aside from the ubiquitous NHE1 isoform, their relative contributions are poorly understood. NHE5 is of particular interest as it is preferentially expressed in brain relative to other tissues. In hippocampal neurons, NHE5 regulates steady-state cytoplasmic pH, but intriguingly the bulk of the transporter is stored in intracellular vesicles. Here, we show that NHE5 is a direct target for phosphorylation by the AMP-activated protein kinase (AMPK), a key sensor and regulator of cellular energy homeostasis in response to metabolic stresses. In NHE5-transfected non-neuronal cells, activation of AMPK by the AMP mimetic AICAR or by antimycin A, which blocks aerobic respiration and causes acidification, increased cell surface accumulation and activity of NHE5, and elevated intracellular pH. These effects were effectively blocked by the AMPK antagonist compound C, the NHE inhibitor HOE694, and mutation of a predicted AMPK recognition motif in the NHE5 C terminus. This regulatory pathway was also functional in primary hippocampal neurons, where AMPK activation of NHE5 protected the cells from sustained antimycin A-induced acidification. These data reveal a unique role for AMPK and NHE5 in regulating the pH homeostasis of hippocampal neurons during metabolic stress.     

Lipoprotein subclasses and endogenous sex hormones in women at midlife

The objective of this work was to evaluate the associations between levels of endogenous sex hormones in women at midlife and lipoprotein subclasses. One hundred and twenty women (68 late peri-/postmenopausal and 52 pre-/early perimenopausal) from the Study of Women’s Health Across the Nation (Pittsburgh site) were included. Lipoprotein subclasses were quantified using NMR spectroscopy. Participants (57.5% White and 42.5% Black) were 50.4 ± 1.9 years old. Adjusting for age, race, cycle day of blood draw, BMI, physical activity, and alcohol consumption, a negative correlation was found between estradiol (E2) and medium-small LDL particle (LDL-P) concentration (ρ = −0.19, P = 0.04). Further, E2 was positively correlated with HDL particle (HDL-P) size (ρ = 0.22, P = 0.02). For sex hormone binding globulin (SHBG), independent negative correlation was found with total small LDL-P concentration. SHBG was also positively correlated with LDL-P and HDL-P sizes (P < 0.05 for all). For free androgen index (FAI), positive correlations were found with concentrations of total VLDL particles, total LDL-Ps, and total small LDL-Ps. Additionally, FAI was negatively correlated with large HDL-P concentration, and HDL-P and LDL-P sizes (P < 0.05 for all). Lower levels of E2 and SHBG, and higher levels of FAI were associated with a more atherogenic profile of lipoprotein subclasses. Sex hormone levels at midlife may increase women’s risk of coronary heart disease.     

Mechanical and Electrokinetic Effects of Polyamines/Phospholipid Interactions in Model Membranes

The mechanical and electrical properties of phospholipids layers influenced by interaction with polyamines were determined by measuring surface pressure and compression modulus of monolayers and zeta potential of liposomes. The saturated derivative of phosphatidic acid (DPPA) formed layers of the organization varying with compression degree. Contact of DPPA layers with polyamines present in the subphase resulted in changing their mechanical properties and the conditions in which the layer reorganization appears. The parameters corresponding to the layer reorganization depended on the size and charge of polyamines’ molecules. The values of: area per DPPA molecule, surface pressure at the point of layer structure reorganization, and surface pressure at the point of collapse characterizing of DPPA layers in the studied systems were determined. It was found that polyamines influenced to a much lesser extent the mechanical properties of monolayers formed from unsaturated derivative of phosphatidic acid slightly increasing its mechanical resistance in the range of higher molecular packing. The results of electrokinetic measurements revealed that surface charge of phosphatidic acid liposomes was effectively neutralized in the presence of polyamines. A similar effect was observed for phosphatidyl glycerol and for negatively charged polystyrene latex particles used as a reference. The influence of polyamines on the mechanical properties of DPPA layers was interpreted assuming a possibility of penetration of the lipid layer by polyamines’ molecules. Comparison of action of putrescine and calcium ions and effects of polyamines on phosphatidyl glycerol provided additional justification for the proposed interpretation of the observed effects.     

The Population Structure of Vibrio cholerae from the Chandigarh Region of Northern India

Background

Cholera infection continues to be a threat to global public health. The current cholera pandemic associated with Vibrio cholerae El Tor has now been ongoing for over half a century.

Methodology/Principal Findings

Thirty-eight V. cholerae El Tor isolates associated with a cholera outbreak in 2009 from the Chandigarh region of India were characterised by a combination of microbiology, molecular typing and whole-genome sequencing. The genomic analysis indicated that two clones of V. cholera circulated in the region and caused disease during this time. These clones fell into two distinct sub-clades that map independently onto wave 3 of the phylogenetic tree of seventh pandemic V. cholerae El Tor. Sequence analyses of the cholera toxin gene, the Vibrio seventh Pandemic Island II (VSPII) and SXT element correlated with this phylogenetic position of the two clades on the El Tor tree. The clade 2 isolates, characterized by a drug-resistant profile and the expression of a distinct cholera toxin, are closely related to the recent V. cholerae isolated elsewhere, including Haiti, but fell on a distinct branch of the tree, showing they were independent outbreaks. Multi-Locus Sequence Typing (MLST) distinguishes two sequence types among the 38 isolates, that did not correspond to the clades defined by whole-genome sequencing. Multi-Locus Variable-length tandem-nucleotide repeat Analysis (MLVA) identified 16 distinct clusters.

Conclusions/Significance

The use of whole-genome sequencing enabled the identification of two clones of V. cholerae that circulated during the 2009 Chandigarh outbreak. These clones harboured a similar structure of ICEVchHai1 but differed mainly in the structure of CTX phage and VSPII. The limited capacity of MLST and MLVA to discriminate between the clones that circulated in the 2009 Chandigarh outbreak highlights the value of whole-genome sequencing as a route to the identification of further genetic markers to subtype V. cholerae isolates.     

Searching for new resistance sources to tomato yellow leaf curl virus within a highly variable wild Lycopersicon genetic pool

The high variability found among Tomato yellow leaf curl virus (TYLCV) isolates from different geographical areas makes progress in breeding for TYLCV resistance slow. By using Agrobacterium-mediated inoculation, we have identified several new resistant sources to TYLCV within a extraordinarily variable wild Lycopersicon gene pool, collected in semidesert areas of Ecuador and Peru changed into wet by “El Nińo”. This screening assay revealed a high susceptibility within L. esculentum and L. pennellii, but different levels of resistance within L. pimpinellifolium and L. hirsutum. Resistance level was related to the collection place, being concentrated in accessions collected in Northern Peru (Piura province). Agroinoculation allowed the selection of 4 Lycopersicon pimpinellifolium and 2 Lycopersicon hirsutum accessions with higher level of resistance than accessions of these species previously reported, avoiding interference due to vector resistance mechanisms reported in both species. These new resistance sources will be included in pyramiding strategies aimed at obtaining durable resistance to TYLCV.     

Maurotoxin and the Kv1.1 channel: voltage-dependent binding upon enantiomerization of the scorpion toxin disulfide bridge Cys31-Cys34.

Maurotoxin (MTX) is a 34-amino acid polypeptide cross-linked by four disulfide bridges that has been isolated from the venom of the scorpion Scorpio maurus palmatus and characterized. Maurotoxin competed with radiolabeled apamin and kaliotoxin for binding to rat brain synaptosomes and blocked K+ currents from Kv1 channel subtypes expressed in Xenopus oocytes. Structural characterization of the synthetic toxin identified half-cystine pairings at Cys3-Cys24, Cys9-Cys29, Cys13-Cys19 and Cys31-Cys34 This disulfide bridge pattern is unique among known scorpion toxins, particularly the existence of a C-terminal '14-membered disulfide ring' (i.e. cyclic domain 31-34), We therefore studied structure-activity relationships by investigating the structure and pharmacological properties of synthetic MTX peptides either modified at the C-terminus ?i.e. MTX(1-29), [Abu31,34]-MTX and [Cys31,34, Tyr32]D-MTX) or mimicking the cyclic C-terminal domain [i.e. MTX(31-34)]. Unexpectedly, the absence of a disulfide bridge Cys31-Cys34 in [Abu 31,34]-MTX and MTX(1-29) resulted in MTX-unrelated half-cystine pairings of the three remaining disulfide bridges for the two analogs, which is likely to be responsible for their inactivity against Kv1 channel subtypes. Cyclic MTX(31-34) was also biologically inactive. [Cys31,34, Tyr32]D-MTX, which had a 'native', MTX-related, disulfide bridge organization, but a D-residue-induced reorientation of the C-terminal disulfide bridge, was potent at blocking the Kv1.1 channel. This peptide-induced Kv1.1 blockage was voltage-dependent (a property not observed for MTX), maximal in the low depolarization range and associated with on-rate changes in ligand binding. Thus, the cyclic C-terminal domain of MTX seems to be crucial for recognition of Kv1.3, and to a lesser extent, Kv1.2 channels and it may contribute to the stabilization and strength of the interaction between the toxin and the Kv1.1 channel.     

High-performance liquid chromatographic determination of cotinine in urine in isocratic mode

A simple procedure for the determination of cotinine, major metabolite of nicotine in urine, is described. The assay involved a liquid–liquid extraction with dichloromethane in alkaline environment. The extract was dried at ambient temperature under a gentle stream of nitrogen. The residue was dissolved in 300 μl of mobile phase and 30 μl aliquot was injected via an automatic sampler into the liquid chromatograph and eluted with the mobile phase (10–9%, v/v methanol and acetonitrile, respectively in potassium dihydrogenphosphate buffer adjusted to pH 3.4) at a flow rate of 1 ml/min on a C₈ Symmetry cartridge column (5 μm, 150 mm×3.9 mm, Waters) at 25°C. The eluate was detected at 260 nm. Internal standard was 2-phenylimidazole. Sensitive and specific, this technique was performed to test urine of diabetic patients (smokers and non-smokers) admitted in an endocrinology service. Urinary cotinine seems to be a better marker of smoking status than thiocyanates.     

Effect of Dissemination of 2,4-Dichlorophenoxyacetic Acid (2,4-D) Degradation Plasmids on 2,4-D Degradation and on Bacterial Community Structure in Two Different Soil Horizons

Transfer of the 2,4-dichlorophenoxyacetic acid (2,4-D) degradation plasmids pEMT1 and pJP4 from an introduced donor strain, Pseudomonas putida UWC3, to the indigenous bacteria of two different horizons (A horizon, depth of 0 to 30 cm; B horizon, depth of 30 to 60 cm) of a 2,4-D-contaminated soil was investigated as a means of bioaugmentation. When the soil was amended with nutrients, plasmid transfer and enhanced degradation of 2,4-D were observed. These findings were most striking in the B horizon, where the indigenous bacteria were unable to degrade any of the 2,4-D (100 mg/kg of soil) during at least 22 days but where inoculation with either of the two plasmid donors resulted in complete 2,4-D degradation within 14 days. In contrast, in soils not amended with nutrients, inoculation of donors in the A horizon and subsequent formation of transconjugants (10⁵ CFU/g of soil) could not increase the 2,4-D degradation rate compared to that of the noninoculated soil. However, donor inoculation in the nonamended B-horizon soil resulted in complete degradation of 2,4-D within 19 days, while no degradation at all was observed in noninoculated soil during 89 days. With plasmid pEMT1, this enhanced degradation seemed to be due only to transconjugants (10⁵ CFU/g of soil), since the donor was already undetectable when degradation started. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes showed that inoculation of the donors was followed by a shift in the microbial community structure of the nonamended B-horizon soils. The new 16S rRNA gene fragments in the DGGE profile corresponded with the 16S rRNA genes of 2,4-D-degrading transconjugant colonies isolated on agar plates. This result indicates that the observed change in the community was due to proliferation of transconjugants formed in soil. Overall, this work clearly demonstrates that bioaugmentation can constitute an effective strategy for cleanup of soils which are poor in nutrients and microbial activity, such as those of the B horizon.     

Relationship between Cell Wall Susceptibility to Cellulases and Pectinases of Fusarium oxysporum and Susceptibility of Date Palm Cultivars

Fusarium oxysporum f. sp. albedinis, the bayoud disease agent of date palm, grows on a mineral medium containing the cell walls of date palm roots as a sole carbohydrate source. The growth and development of pathogen under these conditions was related to the production of extracellular cell wall-degrading enzymes (CWDE): cellulases, polygalacturonases, polygalacturonate transeliminases, and pectinmethylesterases. The mycelial growth and the sporulation of pathogen were higher in the presence of cell walls of susceptible cultivars (BFG, JHL, BSK) than in the presence of those of resistant cultivars (IKL, SLY, BSTN). After 8 d of fungal culture, the activity of CWDE was equal whatever is the origin of the cell walls (resistant or susceptible cultivars). After 16 d of culture, the activity of these CWDE was higher when the parasite was cultivated on the cell walls of the susceptible cultivars than on those of the resistant cultivars. A positive correlation was observed between CWDE activities and the growth and the sporulation of F. oxysporum after 16 d of culture. These results clearly show a relation between the susceptibility/resistance of the cell walls of the roots of the date palm to the parasitic CWDE and the susceptibility/resistance of the cultivars.