Intracellular pH regulation by Na(+)/H(+) exchange requires phosphatidylinositol 4,5-bisphosphate

The carrier-mediated, electroneutral exchange of Na(+) for H(+) across the plasma membrane does not directly consume metabolic energy. Nevertheless, acute depletion of cellular ATP markedly decreases transport. We analyzed the possible involvement of polyphosphoinositides in the metabolic regulation of NHE1, the ubiquitous isoform of the Na(+)/H(+) exchanger. Depletion of ATP was accompanied by a marked reduction of plasmalemmal phosphatidylinositol 4,5-bisphosphate (PIP(2)) content. Moreover, sequestration or hydrolysis of plasmalemmal PIP(2), in the absence of ATP depletion, was associated with profound inhibition of NHE1 activity. Examination of the primary structure of the COOH-terminal domain of NHE1 revealed two potential PIP(2)-binding motifs. Fusion proteins encoding these motifs bound PIP(2) in vitro. When transfected into antiport-deficient cells, mutant forms of NHE1 lacking the putative PIP(2)-binding domains had greatly reduced transport capability, implying that association with PIP(2) is required for optimal activity. These findings suggest that NHE1 activity is modulated by phosphoinositides and that the inhibitory effect of ATP depletion may be attributable, at least in part, to the accompanying net dephosphorylation of PIP(2).     

Phase Variation in Xenorhabdus nematophilus

Xenorhabdus nematophilus is a symbiotic bacterium that inhabits the intestine of entomopathogenic nematodes. The bacterium-nematode symbiotic pair is pathogenic for larval-stage insects. The phase I cell type is the form of the bacterium normally associated with the nematode. A variant cell type, referred to as phase II, can form spontaneously under stationary-phase conditions. Phase II cells do not elaborate products normally associated with the phase I cell type. To better define phase variation in X. nematophilus, several strains (19061, AN6, F1, N2-4) of this bacterium were analyzed for new phenotypic traits. An analysis of pathogenicity in Manduca sexta larvae revealed that the phase II form of AN6 (AN6/II) was significantly less virulent than the phase I form (AN6/I). The variant form of N2-4 was also avirulent. On the other hand, F1/II and 19061/II were as virulent as the respective phase I cells. Strain 19061/II was found to be motile, and AN6/II regained motility when the bacteria were grown in low-osmolarity medium. In contrast, F1/II remained nonmotile. The phase II cells did not produce the outer membrane protein, OpnB, that is normally induced during the stationary phase. Both phase I and phase II cells were able to support nematode growth and development. These findings indicate that while certain phenotypic traits are common to all phase II cells, other characteristics, such as virulence and motility, are variable and can be influenced by environmental conditions.     

Visualization of Phosphoinositides That Bind Pleckstrin Homology Domains: Calcium- and Agonist-induced Dynamic Changes and Relationship to Myo-[3H]inositol-labeled Phosphoinositide Pools

Phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P₂) pools that bind pleckstrin homology (PH) domains were visualized by cellular expression of a phospholipase C (PLC)δ PH domain–green fluorescent protein fusion construct and analysis of confocal images in living cells. Plasma membrane localization of the fluorescent probe required the presence of three basic residues within the PLCδ PH domain known to form critical contacts with PtdIns(4,5)P₂. Activation of endogenous PLCs by ionophores or by receptor stimulation produced rapid redistribution of the fluorescent signal from the membrane to cytosol, which was reversed after Ca²⁺ chelation. In both ionomycin- and agonist-stimulated cells, fluorescent probe distribution closely correlated with changes in absolute mass of PtdIns(4,5)P₂. Inhibition of PtdIns(4,5)P₂ synthesis by quercetin or phenylarsine oxide prevented the relocalization of the fluorescent probe to the membranes after Ca²⁺ chelation in ionomycin-treated cells or during agonist stimulation. In contrast, the synthesis of the PtdIns(4,5)P₂ imaged by the PH domain was not sensitive to concentrations of wortmannin that had been found inhibitory of the synthesis of myo-[³H]inositol– labeled PtdIns(4,5)P₂. Identification and dynamic imaging of phosphoinositides that interact with PH domains will further our understanding of the regulation of such proteins by inositol phospholipids.     

Supression of hemin-mediated oxidation of low-density lipoprotein and subsequent endothelial reactions by hydrogen sulfide (H2S)

Heme-mediated oxidative modification of low-density lipoprotein (LDL) plays a crucial role in early atherogenesis. It has been shown that hydrogen sulfide (H₂S) produced by vascular smooth muscle cells is present in plasma at a concentration of about 50 µmol/L. H₂S is a strong reductant which can react with reactive oxygen species like superoxide anion and hydrogen peroxide. The current study investigated the effect of H₂S on hemin-mediated oxidation of LDL and oxidized LDL (oxLDL)-induced endothelial reactions. H₂S dose dependently delayed the accumulation of lipid peroxidation products—conjugated dienes, lipid hydroperoxides (LOOH), and thiobarbituric acid reactive substances—during hemin-mediated oxidation. Moreover, H₂S decreased the LOOH content of both oxidized LDL and lipid extracts derived from soft atherosclerotic plaque, which was accompanied by reduced cytotoxicity. OxLDL-mediated induction of the oxidative stress responsive gene, heme oxygenase-1, was also abolished by H₂S. Finally we have shown that H₂S can directly protect endothelium against hydrogen peroxide and oxLDL-mediated endothelial cytotoxicity. These results demonstrate novel functions of H₂S in preventing hemin-mediated oxidative modification of LDL, and consequent deleterious effects, suggesting a possible antiatherogenic action of H₂S.     

New Insights into the Methodology of L-Arginine-Induced Acute Pancreatitis

Animal models are ideal to study the pathomechanism and therapy of acute pancreatitis (AP). The use of L-arginine-induced AP model is nowadays becoming increasingly popular in mice. However, carefully looking through the literature, marked differences in disease severity could be observed. In fact, while setting up the L-arginine (2×4 g/kg i.p.)-induced AP model in BALB/c mice, we found a relatively low rate (around 15%) of pancreatic necrosis, whereas others have detected much higher rates (up to 55%). We suspected that this may be due to differences between mouse strains. We administered various concentrations (5–30%, pH = 7.4) and doses (2×4, 3×3, or 4×2.5 g/kg) of L-arginine-HCl in BALB/c, FVB/n and C57BL/6 mice. The potential gender-specific effect of L-arginine was investigated in C57BL/6 mice. The fate of mice in response to the i.p. injections of L arginine followed one of three courses. Some mice (1) developed severe AP or (2) remained AP-free by 72 h, whereas others (3) had to be euthanized (to avoid their death, which was caused by the high dose of L-arginine and not AP) within 12 h., In FVB/n and C57BL/6 mice, the pancreatic necrosis rate (about 50%) was significantly higher than that observed in BALB/c mice using 2×4 g/kg 10% L–arginine, but euthanasia was necessary in a large proportion of animals, The i.p. injection of lower L-arginine concentrations (e.g. 5–8%) in case of the 2×4 g/kg dose, or other L-arginine doses (3×3 or 4×2.5 g/kg, 10%) were better for inducing AP. We could not detect any significant differences between the AP severity of male and female mice. Taken together, when setting up the L-arginine-induced AP model, there are several important factors that are worth consideration such as the dose and concentration of the administered L arginine-HCl solution and also the strain of mice.     

Roux-en-Y Gastric Bypass Alters Brain Activity in Regions that Underlie Reward and Taste Perception

Background

Roux-en-Y gastric bypass (RYGB) surgery is a very effective bariatric procedure to achieve significant and sustained weight loss, yet little is known about the procedure’s impact on the brain. This study examined the effects of RYGB on the brain’s response to the anticipation of highly palatable versus regular food.

Methods

High fat diet-induced obese rats underwent RYGB or sham operation and were then tested for conditioned place preference (CPP) for the bacon-paired chamber, relative to the chow-paired chamber. After CPP, animals were placed in either chamber without the food stimulus, and brain-glucose metabolism (BGluM) was measured using positron emission tomography (μPET).

Results

Bacon CPP was only observed in RYGB rats that had stable weight loss following surgery. BGluM assessment revealed that RYGB selectively activated regions of the right and midline cerebellum (Lob 8) involved in subjective processes related to reward or expectation. Also, bacon anticipation led to significant activation in the medial parabrachial nuclei (important in gustatory processing) and dorsomedial tegmental area (key to reward, motivation, cognition and addiction) in RYGB rats; and activation in the retrosplenial cortex (default mode network), and the primary visual cortex in control rats.

Conclusions

RYGB alters brain activity in areas involved in reward expectation and sensory (taste) processing when anticipating a palatable fatty food. Thus, RYGB may lead to changes in brain activity in regions that process reward and taste-related behaviors. Specific cerebellar regions with altered metabolism following RYGB may help identify novel therapeutic targets for treatment of obesity.     

Measurement of Inositol 1,4,5-Trisphosphate in Living Cells Using an Improved Set of Resonance Energy Transfer-Based Biosensors

Improved versions of inositol-1,4,5-trisphosphate (InsP₃) sensors were created to follow intracellular InsP₃ changes in single living cells and in cell populations. Similar to previous InsP₃ sensors the new sensors are based on the ligand binding domain of the human type-I InsP₃ receptor (InsP₃R-LBD), but contain a mutation of either R265K or R269K to lower their InsP₃ binding affinity. Tagging the InsP₃R-LBD with N-terminal Cerulean and C-terminal Venus allowed measurement of InsP₃ in single-cell FRET experiments. Replacing Cerulean with a Luciferase enzyme allowed experiments in multi-cell format by measuring the change in the BRET signal upon stimulation. These sensors faithfully followed the agonist-induced increase in InsP₃ concentration in HEK 293T cells expressing the Gq-coupled AT1 angiotensin receptor detecting a response to agonist concentration as low as 10 pmol/L. Compared to the wild type InsP₃ sensor, the mutant sensors showed an improved off-rate, enabling a more rapid and complete return of the signal to the resting value of InsP₃ after termination of M3 muscarinic receptor stimulation by atropine. For parallel measurements of intracellular InsP₃ and Ca²⁺ levels in BRET experiments, the Cameleon D3 Ca²⁺ sensor was modified by replacing its CFP with luciferase. In these experiments depletion of plasma membrane PtdIns(4,5)P₂ resulted in the fall of InsP₃ level, followed by the decrease of the Ca²⁺-signal evoked by the stimulation of the AT1 receptor. In contrast, when type-III PI 4-kinases were inhibited with a high concentration of wortmannin or a more specific inhibitor, A1, the decrease of the Ca²⁺-signal preceded the fall of InsP₃ level indicating an InsP₃-, independent, direct regulation of capacitative Ca²⁺ influx by plasma membrane inositol lipids. Taken together, our results indicate that the improved InsP₃ sensor can be used to monitor both the increase and decrease of InsP₃ levels in live cells suitable for high-throughput BRET applications.     

The protein kinase C cascade regulates recruitment of matrix metalloprotease 9 to podosomes and its release and activation

Podosomes are transient cell surface structures essential for degradation of extracellular matrix during cell invasion. Protein kinase C (PKC) is involved in the regulation of podosome formation; however, the roles of individual PKC isoforms in podosome formation and proteolytic function are largely unknown. Recently, we reported that PDBu, a PKC activator, induced podosome formation in normal human bronchial epithelial cells. Here, we demonstrate that phorbol-12,13-dibutyrate (PDBu)-induced podosome formation is mainly mediated through redistribution of conventional PKCs, especially PKCα, from the cytosol to the podosomes. Interestingly, although blocking atypical PKCζ did not affect PDBu-induced podosome formation, it significantly reduced matrix degradation at podosomes. Inhibition of PKCζ reduced recruitment of matrix metalloprotease 9 (MMP-9) to podosomes and its release and activation. Downregulation of MMP-9 by small interfering RNA (siRNA) or neutralization antibody also significantly reduced matrix degradation. The regulatory effects of PKCζ on matrix degradation and recruitment of MMP-9 to podosomes were PKCζ kinase activity dependent. PDBu-induced recruitment of PKCζ and MMP-9 to podosomes was blocked by inhibition of novel PKC with rottlerin or PKCδ siRNA. Our data suggest that multiple PKC isozymes form a signaling cascade that controls podosome formation and dynamics and MMP-9 recruitment, release, and activation in a coordinated fashion.     

Studies on solvatochromic properties of aminophenylstyryl-quinolinum dye, LDS 798, and its application in studying submicron lipid based structure

The styryl group of dyes has been used in cellular studies for over 20 years because of their solvatochromic and/or electrochromic properties. Here we report characterization of solubility and solvatochromic properties of a near infra-red styryl dye, styryl 11 or LDS 798. We have extended our studies to small unilamellar vesicles and lipid based nanoparticles and found that solvatochromic properties of this dye used in tandem with fluorescence correlation spectroscopy can be used to efficiently determine the diffusion coefficient and hence the size of the submicron lipid based particles. This technique has the potential to provide essential information about liposomal and vesicular structures and their movement in vitro and in situ.     

A rapid non-destructive DNA extraction method for insects and other arthropods

Preparation of arthropods for morphological identification often damages or destroys DNA within the specimen. Conversely, DNA extraction methods often destroy the external physical characteristics essential for morphological identification. We have developed a rapid, simple and non-destructive DNA extraction technique for arthropod specimens. This technique was tested on four arthropod orders, using specimens that were fresh, preserved by air drying, stored in ethanol, or collected with sticky or propylene glycol traps. The technique could be completed in 20 min for Coleoptera, Diptera and Hemiptera, and 2 min for the subclass Acarina, without significant distortion, discolouration, or other damage to the specimens.