Effects of pH and Repellent Tactic Stimuli on Protein Methylation Levels in Escherichia coli

Intracellular pH (pH_int) and extracellular pH (pH_ext) of Escherichia coli were measured at 12-s time resolution by ³¹P-nuclear magnetic resonance: a sudden neutral-to-acid shift in pH_ext (e.g., from 7.0 to 5.6) caused a transient failure of homeostasis, with pH_int decreasing by about 0.4 unit in ca. 30 s and then returning to its original value (ca. 7.5) over a period of several minutes. Membrane proton conductance was estimated to be 20 pmol s⁻¹ cm⁻² pH unit⁻¹. Addition of the membrane-permeant weak acid benzoate at constant pH_ext also caused a lowering of pH_int; at high concentrations it generated an inverted transmembrane pH gradient (ΔpH). The buffering capacity of the cells was estimated by such experiments to be ca. 50 mM per pH unit. Effects of pH-related stimuli on the methyl-accepting chemotaxis proteins (MCPs) were examined: the steady-state methylation of MCP I was found to decrease when pH_int was lowered by weak acid addition or when pH_ext was lowered. The extent of demethylation in the latter case was too great to be explained by imperfect steady-state homeostasis; a small but reproducible undershoot in methylation level correlated with the observed short-term homeostatic failure. MCP II underwent smaller and more complex changes than MCP I, in response to pH-related stimuli. The methylation level of MCP I could not, by any condition tested, be driven below a limit of ca. 15% of the control level (unstimulated cells at pH_ext 7.0). The weak-acid concentration needed to reach that limit was dependent on pH_ext, as would be expected on the basis of ΔpH-driven concentrative effects. The potency ranking of weak acids was the same with respect to lowering pH_int, demethylating MCP I, and causing repellent behavioral responses. The data are consistent with a model whereby MCP I and hence tactic behavior are sensitive to both pH_int and pH_ext. Evidence is presented that pH_int may also have a direct (non-MCP-related) effect on motor function. Comparison of methyl-³H- and ³⁵S-labeled MCP I revealed that in both unstimulated and repellent-stimulated cells the major species did not carry methyl label, yet it had an electrophoretic mobility that indicated that it was more positively charged than the unmethylated form observed in methyltransferase mutants, and it was susceptible to base hydrolysis. This suggests that a substantial fraction of MCP I molecules is methylated or otherwise modified but neither exchanges methyl label nor undergoes reverse modification by repellent stimuli.     

GABAergic and cortical and subcortical glutamatergic axon terminals contain CB1 cannabinoid receptors in the ventromedial nucleus of the hypothalamus

Background

Type-1 cannabinoid receptors (CB₁R) are enriched in the hypothalamus, particularly in the ventromedial hypothalamic nucleus (VMH) that participates in homeostatic and behavioral functions including food intake. Although CB₁R activation modulates excitatory and inhibitory synaptic transmission in the brain, CB₁R contribution to the molecular architecture of the excitatory and inhibitory synaptic terminals in the VMH is not known. Therefore, the aim of this study was to investigate the precise subcellular distribution of CB₁R in the VMH to better understand the modulation exerted by the endocannabinoid system on the complex brain circuitries converging into this nucleus.

Methodology/Principal Findings

Light and electron microscopy techniques were used to analyze CB₁R distribution in the VMH of CB₁R-WT, CB₁R-KO and conditional mutant mice bearing a selective deletion of CB₁R in cortical glutamatergic (Glu-CB₁R-KO) or GABAergic neurons (GABA-CB₁R-KO). At light microscopy, CB₁R immunolabeling was observed in the VMH of CB₁R-WT and Glu-CB₁R-KO animals, being remarkably reduced in GABA-CB₁R-KO mice. In the electron microscope, CB₁R appeared in membranes of both glutamatergic and GABAergic terminals/preterminals. There was no significant difference in the percentage of CB₁R immunopositive profiles and CB₁R density in terminals making asymmetric or symmetric synapses in CB₁R-WT mice. Furthermore, the proportion of CB₁R immunopositive terminals/preterminals in CB₁R-WT and Glu-CB₁R-KO mice was reduced in GABA-CB₁R-KO mutants. CB₁R density was similar in all animal conditions. Finally, the percentage of CB₁R labeled boutons making asymmetric synapses slightly decreased in Glu-CB₁R-KO mutants relative to CB₁R-WT mice, indicating that CB₁R was distributed in cortical and subcortical excitatory synaptic terminals.

Conclusions/Significance

Our anatomical results support the idea that the VMH is a relevant hub candidate in the endocannabinoid-mediated modulation of the excitatory and inhibitory neurotransmission of cortical and subcortical pathways regulating essential hypothalamic functions for the individual''s survival such as the feeding behavior.     

The Role of Glycerol and Inorganic Ions in Osmoregulatory Responses of the Euryhaline Flagellate Chlamydomonas pulsatilla Wollenweber

The green euryhaline flagellate Chlamydomonas pulsatilla Wollenweber, isolated from a coastal marine environment, was grown exponentially over the salinity range of 10 to 200% artificial seawater (ASW). The cellular volume and aqueous space of the alga, measured by [¹⁴C] mannitol and ³H₂O tracer analyses of centrifuged cell pellets, ranged between 2.3 and 3.1 picoliters and between 1.5 and 2.1 picoliters, respectively. The nonaqueous space determined in those analyses (28-35%) was consistent with the cell composition of the alga. The glycerol content of the alga increased almost linearly with increasing salinity; its contribution to intracellular osmolality at 200% ASW was about 57%. The contribution of amino acids and soluble carbohydrates to the cell osmotic balance was small. Intracellular ion concentrations determined by analyzing centrifuged cell pellets of known [¹⁴C]mannitol space by atomic absorption spectrophotometry, and by neutron activation analyses of washed cells were similar. At 10% ASW, potassium and magnesium were the major cations, and chloride and phosphate were the major anions. The sodium and chloride content of the alga increased with increasing salinity; at 200% ASW the intracellular concentration of both sodium and chloride was about 400 millimolar. The intracellular osmolality (π_int) matched closely the external osmolality (π_ext) over the entire salinity range except at 10% ASW where π_int exceeded π_ext by 120 to 270 milliosmoles per kilogram H₂O.     

Bicarbonate uptake via an anion exchange protein is the main mechanism of inorganic carbon acquisition by the giant kelp Macrocystis pyrifera (Laminariales,Phaeophyceae) under variable pH

Macrocystis pyrifera is a widely distributed, highly productive, seaweed. It is known to use bicarbonate (HCO₃^?) from seawater in photosynthesis and the main mechanism of utilization is attributed to the external catalyzed dehydration of HCO₃^? by the surface‐bound enzyme carbonic anhydrase (CA_ext). Here, we examined other putative HCO₃^? uptake mechanisms in M. pyrifera under pH_T 9.00 (HCO₃^?: CO₂ = 940:1) and pH_T 7.65 (HCO₃^?: CO₂ = 51:1). Rates of photosynthesis, and internal CA (CA_int) and CA_ext activity were measured following the application of AZ which inhibits CA_ext, and DIDS which inhibits a different HCO₃^? uptake system, via an anion exchange (AE) protein. We found that the main mechanism of HCO₃^? uptake by M. pyrifera is via an AE protein, regardless of the HCO₃^?: CO₂ ratio, with CA_ext making little contribution. Inhibiting the AE protein led to a 55%–65% decrease in photosynthetic rates. Inhibiting both the AE protein and CA_ext at pH_T 9.00 led to 80%–100% inhibition of photosynthesis, whereas at pH_T 7.65, passive CO₂ diffusion supported 33% of photosynthesis. CA_int was active at pH_T 7.65 and 9.00, and activity was always higher than CA_ext, because of its role in dehydrating HCO₃^? to supply CO₂ to RuBisCO. Interestingly, the main mechanism of HCO₃^? uptake in M. pyrifera was different than that in other Laminariales studied (CA_ext‐catalyzed reaction) and we suggest that species‐specific knowledge of carbon uptake mechanisms is required in order to elucidate how seaweeds might respond to future changes in HCO₃^?:CO₂ due to ocean acidification.     

Interactions between Diet,Lifestyle and IL10, IL1B,and PTGS2/COX-2 Gene Polymorphisms in Relation to Risk of Colorectal Cancer in a Prospective Danish Case-Cohort Study

Background & Aims

Diet contributes to colorectal cancer development and may be potentially modified. We wanted to identify the biological mechanisms underlying colorectal carcinogenesis by assessment of diet-gene interactions.

Methods

The polymorphisms IL10 C-592A (rs1800872), C-rs3024505-T, IL1b C-3737T (rs4848306), G-1464C (rs1143623), T-31C (rs1143627) and PTGS2 (encoding COX-2) A-1195G (rs689466), G-765C (rs20417), and T8473C (rs5275) were assessed in relation to risk of colorectal cancer (CRC) and interaction with diet (red meat, fish, fibre, cereals, fruit and vegetables) and lifestyle (non-steroid-anti-inflammatory drug use and smoking status) was assessed in a nested case-cohort study of nine hundred and seventy CRC cases and 1789 randomly selected participants from a prospective study of 57,053 persons.

Results

IL1b C-3737T, G-1464C and PTGS2 T8473C variant genotypes were associated with risk of CRC compared to the homozygous wildtype genotype (IRR=0.81, 95%CI: 0.68-0.97, p=0.02, and IRR=1.22, 95%CI: 1.04-1.44, p=0.02, IRR=0.75, 95%CI: 0.57-0.99, p=0.04, respectively). Interactions were found between diet and IL10 rs3024505 (P-value for interaction (P_int); meat=0.04, fish=0.007, fibre=0.0008, vegetables=0.0005), C-592A (P_int; fibre=0.025), IL1b C-3737T (P_int; vegetables=0.030, NSAID use=0.040) and PTGS2 genotypes G-765C (P_int; meat=0.006, fibre=0.0003, fruit 0.004), and T8473C (P_int; meat 0.049, fruit=0.03) and A-1195G (P_int; meat 0.038, fibre 0.040, fruit=0.059, vegetables=0.025, and current smoking=0.046).

Conclusions

Genetically determined low COX-2 and high IL-1β activity were associated with increased risk of CRC in this northern Caucasian cohort. Furthermore, interactions were found between IL10, IL1b, and PTGS2 and diet and lifestyle factors in relation to CRC. The present study demonstrates that gene-environment interactions may identify genes and environmental factors involved in colorectal carcinogenesis.     

Hepatitis C Virus Induces the Cannabinoid Receptor 1

Background

Activation of hepatic CB₁ receptors (CB₁) is associated with steatosis and fibrosis in experimental forms of liver disease. However, CB₁ expression has not been assessed in patients with chronic hepatitis C (CHC), a disease associated with insulin resistance, steatosis and metabolic disturbance. We aimed to determine the importance and explore the associations of CB₁ expression in CHC.

Methods

CB₁ receptor mRNA was measured by real time quantitative PCR on extracted liver tissue from 88 patients with CHC (genotypes 1 and 3), 12 controls and 10 patients with chronic hepatitis B (CHB). The Huh7/JFH1 Hepatitis C virus (HCV) cell culture model was used to validate results.

Principal Findings

CB₁ was expressed in all patients with CHC and levels were 6-fold higher than in controls (P<0.001). CB₁ expression increased with fibrosis stage, with cirrhotics having up to a 2 fold up-regulation compared to those with low fibrosis stage (p<0.05). Even in mild CHC with no steatosis (F0-1), CB₁ levels remained substantially greater than in controls (p<0.001) and in those with mild CHB (F0-1; p<0.001). Huh7 cells infected with JFH-1 HCV showed an 8-fold upregulation of CB₁, and CB₁ expression directly correlated with the percentage of cells infected over time, suggesting that CB₁ is an HCV inducible gene. While HCV structural proteins appear essential for CB₁ induction, there was no core genotype specific difference in CB₁ expression. CB₁ significantly increased with steatosis grade, primarily driven by patients with genotype 3 CHC. In genotype 3 patients, CB₁ correlated with SREBP-1c and its downstream target FASN (SREBP-1c; R = 0.37, FASN; R = 0.39, p<0.05 for both).

Conclusions/Significance

CB₁ is up-regulated in CHC and is associated with increased steatosis in genotype 3. It is induced by the hepatitis C virus.     

Study on the biochemical basis of mefloquine resistant Plasmodium falciparum

Increase in drug detoxification and alteration of drug uptake and efflux of Plasmodium falciparum were investigated for their possible association with mefloquine (MQ) resistance in five different clones of P. falciparum from Thailand (T994b₃, K1CB₂, PR70CB₁, PR71CB₂ and TM₄CB8-2.2.3). Fifty percent inhibitory concentration (IC₅₀) values from these five clones varied between 30- and 50-fold. Regarding the detoxification mechanism, the ability of P. falciparum clones to biotransform MQ was shown in vitro by parasite microsomal protein prepared from parasite infected red blood cells protein (30 μg), NADPH (1 nM) and phosphate buffer pH 7.4, carried out at 37 °C with agitation. Radiolabelled unmetabolized MQ and possible metabolite(s) generated from the reaction was extracted into ethylacetate and separated by radiometric-HPLC after 1 h. All clones were capable of converting MQ into carboxymefloquine (CMQ), which is the main metabolite in human plasma. In addition, another unidentified metabolite eluted at 4.2 min on the chromatograph could be detected from the incubation reaction. This metabolite has never been detected in human liver microsomes before. There was no significant difference in the percentages of CMQ formed in the resistant (T994_b3, PR₇₀CB₁, PR₇₁CB₂) and sensitive (TM₄CB8-2.2.3, K1CB₂) clones. Another possible mechanism, i.e., alteration in the accumulation of MQ in the parasites was investigated in vitro using [¹⁴C]MQ as a tracer. The time courses of [¹⁴C]MQ uptake and efflux were generally characterized by two phases. A trend of increased efflux of [¹⁴C]MQ was observed in the resistant compared with sensitive clones.     

Endocannabinoids mediate acute fear adaptation via glutamatergic neurons independently of corticotropin-releasing hormone signaling

Recent evidence showed that the endocannabinoid system plays an important role in the behavioral adaptation of stress and fear responses. In this study, we chose a behavioral paradigm that includes criteria of both fear and stress responses to assess whether the involvement of endocannabinoids in these two processes rely on common mechanisms. To this end, we delivered a footshock and measured the fear response to a subsequently presented novel tone stimulus. First, we exposed different groups of cannabinoid receptor type 1 (CB₁)‐deficient mice (CB₁^?/?) and their wild‐type littermates (CB₁^+/+) to footshocks of different intensities. Only application of an intense footshock resulted in a sustained fear response to the tone in CB₁^?/?. Using the intense protocol, we next investigated whether endocannabinoids mediate their effects via an interplay with corticotropin‐releasing hormone (CRH) signaling. Pharmacological blockade of CB₁ receptors by rimonabant in mice deficient for the CRH receptor type 1 (CRHR1^?/?) or type 2 (CRHR2^?/?), and in respective wild‐type littermates, resulted in a sustained fear response in all genotypes. This suggests that CRH is not involved in the fear‐alleviating effects of CB₁. As CRHR1^?/? are known to be severely impaired in stress‐induced corticosterone secretion, our observation also implicates that corticosterone is dispensable for CB₁‐mediated acute fear adaptation. Instead, conditional mutants with a specific deletion of CB₁ in principal neurons of the forebrain (CaMK‐CB₁^?/?), or in cortical glutamatergic neurons (Glu‐CB₁^?/?), showed a similar phenotype as CB₁^?/?, thus indicating that endocannabinoid‐controlled glutamatergic transmission plays an essential role in acute fear adaptation.     

Synthesis and SAR of novel imidazoles as potent and selective cannabinoid CB2 receptor antagonists with high binding efficiencies

The synthesis and structure–activity relationship studies of imidazoles are described. The target compounds 6–20 represent a novel chemotype of potent and CB₂/CB₁ selective cannabinoid CB₂ receptor antagonists/inverse agonists with very high binding efficiencies in combination with favourable log P and calculated polar surface area values. Compound 12 exhibited the highest CB₂ receptor affinity (K_i = 1.03 nM) in this series, as well as the highest CB₂/CB₁ subtype selectivity (>9708-fold).     

Development of fluorinated CB2 receptor agonists for PET studies

A convergent strategy was followed to modify systematically carbazole based CB₂ receptor ligands. The length of the N-(fluoroalkyl) group (n in 7), the length of the alkanamide (m in 7) and the substitution pattern of the phenyl moiety (X and Y in 7) were varied systematically. The highest CB₂ affinity was found for the 2-fluoroethyl substituted carbazole derivative 20a (K_i = 5.8 nM) containing the propionamide and the 2-bromo-4-fluorophenyl moiety. According to docking studies 20a fits nicely into the binding pocket of the CB₂ receptor, but elongation of the fluoroethyl side chain leads to a different binding mode of the ligands. The high CB₂ affinity together with the high selectivity over the CB₂ subtype qualifies the fluoroethyl derivative 20a to be developed as a PET tracer.     

Intracellular S1P generation is essential for S1P-induced motility of human lung endothelial cells: role of sphingosine kinase 1 and S1P lyase

Background

Earlier we have shown that extracellular sphingosine-1-phosphate (S1P) induces migration of human pulmonary artery endothelial cells (HPAECs) through the activation of S1P₁ receptor, PKCε, and PLD2-PKCζ-Rac1 signaling cascade. As endothelial cells generate intracellular S1P, here we have investigated the role of sphingosine kinases (SphKs) and S1P lyase (S1PL), that regulate intracellular S1P accumulation, in HPAEC motility.

Methodology/Principal Findings

Inhibition of SphK activity with a SphK inhibitor 2-(p-Hydroxyanilino)-4-(p-Chlorophenyl) Thiazole or down-regulation of Sphk1, but not SphK2, with siRNA decreased S1P_int, and attenuated S1P_ext or serum-induced motility of HPAECs. On the contrary, inhibition of S1PL with 4-deoxypyridoxine or knockdown of S1PL with siRNA increased S1P_int and potentiated motility of HPAECs to S1P_ext or serum. S1P_ext mediates cell motility through activation of Rac1 and IQGAP1 signal transduction in HPAECs. Silencing of SphK1 by siRNA attenuated Rac1 and IQGAP1 translocation to the cell periphery; however, knockdown of S1PL with siRNA or 4-deoxypyridoxine augmented activated Rac1 and stimulated Rac1 and IQGAP1 translocation to cell periphery. The increased cell motility mediated by down-regulation was S1PL was pertussis toxin sensitive suggesting “inside-out” signaling of intracellularly generated S1P. Although S1P did not accumulate significantly in media under basal or S1PL knockdown conditions, addition of sodium vanadate increased S1P levels in the medium and inside the cells most likely by blocking phosphatases including lipid phosphate phosphatases (LPPs). Furthermore, addition of anti-S1P mAb to the incubation medium blocked S1P_ext or 4-deoxypyridoxine-dependent endothelial cell motility.

Conclusions/Significance

These results suggest S1P_ext mediated endothelial cell motility is dependent on intracellular S1P production, which is regulated, in part, by SphK1 and S1PL.     

Cellulose amendment promotes P solubilization by Penicillium aculeatum in non-sterilized soil

Successful application of microbial biofertilizers, such as phosphorus (P) solubilizing fungi to agroecosystems, is constrained from the lack of knowledge about their ecology; for example in terms of how they respond to an external input of carbon (C) to get established in the soil. In two soil incubation experiments we examined the performance of the P solubilizing fungus Penicillium aculeatum in non-sterile and semi-sterile (γ-irradiated) soil with different C and P sources. Results from the first experiment with C sources showed that starch and cellulose generally improved P solubilization by P. aculeatum measured as water extractable P (P_wep), though only significantly in non-sterile soil. This coincided with an increased population density of P. aculeatum measured with a hygromycin B resistant strain of this fungus. Soil respiration used to measure soil microbial activity was overall much higher in treatments with C compounds than without C in both non-sterile and semi-sterile soil. However, soil respiration was highest with cellulose in semi-sterile soil, especially in combination with P. aculeatum. Hence, for the second experiment with P sources (tricalcium phosphate (TCP) and sewage sludge ash) cellulose was used as a C source for P. aculeatum growth in all treatments. Main results showed that P. aculeatum in combination with cellulose soil amendment increased soil P_wep independent of soil sterilization and P source treatments. Soil resin P (P_res) and microbial P (P_mic), which represents stocks of potentially plant available P, were also affected from P. aculeatum inoculation. Increased soil P_res from TCP and sewage sludge ash was observed with P. aculeatum independent of soil type. On the other hand soil P_mic was higher after P. aculeatum inoculation only in semi-sterile soil. Population density of P. aculeatum measured with qPCR was maintained or increased in non-sterile and semi-sterile soil, respectively, compared to the original inoculum load of P. aculeatum. In conclusion, our results underline the importance of C source addition for P. aculeatum if used as a biofertilizer. For this, cellulose seems to be a promising option promoting P. aculeatum growth and P solubilization also in non-sterilized soil.     

Dimerization with Cannabinoid Receptors Allosterically Modulates Delta Opioid Receptor Activity during Neuropathic Pain

The diversity of receptor signaling is increased by receptor heteromerization leading to dynamic regulation of receptor function. While a number of studies have demonstrated that family A G-protein-coupled receptors are capable of forming heteromers in vitro, the role of these heteromers in normal physiology and disease has been poorly explored. In this study, direct interactions between CB₁ cannabinoid and delta opioid receptors in the brain were examined. Additionally, regulation of heteromer levels and signaling in a rodent model of neuropathic pain was explored. First we examined changes in the expression, function and interaction of these receptors in the cerebral cortex of rats with a peripheral nerve lesion that resulted in neuropathic pain. We found that, following the peripheral nerve lesion, the expression of both cannabinoid type 1 receptor (CB₁R) and the delta opioid receptor (DOR) are increased in select brain regions. Concomitantly, an increase in CB₁R activity and decrease in DOR activity was observed. We hypothesize that this decrease in DOR activity could be due to heteromeric interactions between these two receptors. Using a CB₁R-DOR heteromer-specific antibody, we found increased levels of CB₁R-DOR heteromer protein in the cortex of neuropathic animals. We subsequently examined the functionality of these heteromers by testing whether low, non-signaling doses of CB₁R ligands influenced DOR signaling in the cortex. We found that, in cortical membranes from animals that experienced neuropathic pain, non-signaling doses of CB₁R ligands significantly enhanced DOR activity. Moreover, this activity is selectively blocked by a heteromer-specific antibody. Together, these results demonstrate an important role for CB₁R-DOR heteromers in altered cortical function of DOR during neuropathic pain. Moreover, they suggest the possibility that a novel heteromer-directed therapeutic strategy for enhancing DOR activity, could potentially be employed to reduce anxiety associated with chronic pain.     

Δ-Tetrahydrocannabinol regulates the p53 post-translational modifiers Murine double minute 2 and the Small Ubiquitin MOdifier protein in the rat brain

The phytocannabinoid Δ⁹-Tetrahydrocannabinol (Δ⁹-THC), the main psychoactive cannabinoid in cannabis, activates a number of signalling cascades including p53. This study examines the role of Δ⁹-THC in regulating the p53 post-translational modifier proteins, Murine double minute (Mdm2) and Small Ubquitin-like MOdifier protein 1 (SUMO-1) in cortical neurons. Δ⁹-THC increased both Mdm2 and SUMO-1 protein expression and induced the deSUMOylation of p53 in a cannabinoid receptor type 1 (CB₁)-receptor dependent manner. We demonstrate that Δ⁹-THC decreased the SUMOylation of the CB₁ receptor. The data reveal a novel role for cannabinoid receptor activation in modulating the SUMO regulatory system.

Structured summary

MINT-7266621: Cb1 (uniprotkb:P20272) physically interacts (MI:0915) with SUMO-1 (uniprotkb:Q5I0H3) by anti bait coimmunoprecipitation(MI:0006)MINT-7266633: SUMO-1 (uniprotkb:Q5I0H3) and Cb1 (uniprotkb:P20272) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7266611: p53 (uniprotkb:P10361) physically interacts (MI:0915) with SUMO-1 (uniprotkb:Q5I0H3) by anti bait coimmunoprecipitation (MI:0006)     

Transmembrane Helical Domain of the Cannabinoid CB1 Receptor

Brain cannabinoid (CB₁) receptors are G-protein coupled receptors and belong to the rhodopsin-like subfamily. A homology model of the inactive state of the CB₁ receptor was constructed using the x-ray structure of β₂-adrenergic receptor (β₂AR) as the template. We used 105 ns duration molecular-dynamics simulations of the CB₁ receptor embedded in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer to gain some insight into the structure and function of the CB₁ receptor. As judged from the root mean-square deviations combined with the detailed structural analyses, the helical bundle of the CB₁ receptor appears to be fully converged in 50 ns of the simulation. The results reveal that the helical bundle structure of the CB₁ receptor maintains a topology quite similar to the x-ray structures of G-protein coupled receptors overall. It is also revealed that the CB₁ receptor is stabilized by the formation of extensive, water-mediated H-bond networks, aromatic stacking interactions, and receptor-lipid interactions within the helical core region. It is likely that these interactions, which are often specific to functional motifs, including the S(N)LAxAD, D(E)RY, CWxP, and NPxxY motifs, are the molecular constraints imposed on the inactive state of the CB₁ receptor. It appears that disruption of these specific interactions is necessary to release the molecular constraints to achieve a conformational change of the receptor suitable for G-protein activation.     

Bone microarchitecture in ankylosing spondylitis and the association with bone mineral density,fractures, and syndesmophytes

Introduction

Osteoporosis of the axial skeleton is a known complication of ankylosing spondylitis (AS), but bone loss affecting the peripheral skeleton is less studied. This study on volumetric bone mineral density (vBMD) and bone microarchitecture in AS was conducted to compare peripheral vBMD in AS patients with that in healthy controls, to study vBMD in axial compared with peripheral bone, and to explore the relation between vertebral fractures, spinal osteoproliferation, and peripheral bone microarchitecture and density.

Methods

High-resolution peripheral quantitative computed tomography (HRpQCT) of ultradistal radius and tibia and QCT and dual-energy x-ray absorptiometry (DXA) of lumbar spine were performed in 69 male AS patients (NY criteria). Spinal radiographs were assessed for vertebral fractures and syndesmophyte formation (mSASSS). The HRpQCT measurements were compared with the measurements of healthy controls.

Results

The AS patients had lower cortical vBMD in radius (P = 0.004) and lower trabecular vBMD in tibia (P = 0.033), than did the controls. Strong correlations were found between trabecular vBMD in lumbar spine, radius (r_S = 0.762; P < 0.001), and tibia (r_S = 0.712; P < 0.001).When compared with age-matched AS controls, patients with vertebral fractures had lower lumbar cortical vBMD (-22%; P = 0.019), lower cortical cross-sectional area in radius (-28.3%; P = 0.001) and tibia (-24.0%; P = 0.013), and thinner cortical bone in radius (-28.3%; P = 0.001) and tibia (-26.9%; P = 0.016).mSASSS correlated negatively with trabecular vBMD in lumbar spine (r_S = -0.620; P < 0.001), radius (r_S = -0.400; p = 0.001) and tibia (r_S = -0.475; p < 0.001) and also with trabecular thickness in radius (r_S = -0.528; P < 0.001) and tibia (r_S = -0.488; P < 0.001).Adjusted for age, syndesmophytes were significantly associated with decreasing trabecular vBMD, but increasing cortical vBMD in lumbar spine, but not with increasing cortical thickness or density in peripheral bone. Estimated lumbar vBMD by DXA correlated with trabecular vBMD measured by QCT (r_S = 0.636; P < 0.001).

Conclusions

Lumbar osteoporosis, syndesmophytes, and vertebral fractures were associated with both lower vBMD and deteriorated microarchitecture in peripheral bone. The results indicate that trabecular bone loss is general, whereas osteoproliferation is local in AS.     

The structural definition of adducts of stoichiometry MX:dpex (2:3)(∞), M = Cu, Ag, X = simple anion, dpex = Ph2E(CH2)xEPh2, E = P, As

Single crystal X-ray structural characterizations are recorded for a number of adducts of MX:dpex (2:3) stoichiometry (MX = simple univalent copper or silver salt; dpex = Ph₂E(CH₂)_xEPh₂ (E = P, As)). CuX:dppe (2:3) (X = Cl, Br, I, CN) are binuclear [(dppe-P,P′)CuX(P-dppe-P′)CuX(P,P′-dppe)], all centrosymmetric. AgX:dpex (2:3) (dpex = ‘dpae’ (Ph₂As(CH₂)₂AsPh₂), X = Br, F₃CCO₂ (= ‘tfa’), F₃CSO₃ (≡ ‘tfs’); dpex = ‘dpape’ (Ph₂As(CH₂)₂PPh₂), X = CN, SCN, OClO₃) are one-dimensional polymers ?-E′)₁AgX(E-dpex-E′)₂-AgX(E-dpex-E′)₁AgX?, P, As sites scrambled in the latter. AgNO₃:dpam (2:3) is also a one-dimensional polymer, ?AgO·NO·OAg(As-dpam-As)AgO·NO·OAg? (‘dpam’ ≡ Ph₂As(CH₂)₂AsPh₂). AgX:dpae (2:3) (X = I, CN, ClO₄, NO₃) and AgX:dpape (2:3) (X = Br, I, NO₃) are two-dimensional polymers with large 30-membered macrocyclic rings; similar webs are found for dppx ligands in AgOH:dppb (2:3) and AgNCO, Agtfa:dpph (2:3) with 42- and 54-membered rings. Complexes AgX:dpape (1:3) (X = Cl, Br) are defined as mono-nuclear [XAg(Ph₂P(CH₂)₂AsPh₂)₃] arrays, the unidentate ligands predominantly P-bound. Synthetic procedures for the adducts are reported, selected compounds being characterized both in solution (¹H, ³¹P NMR, ESI MS) and in the solid state (IR).     

Changes of cationic transport in <Emphasis Type="Italic">AtCAX5</Emphasis> transformant yeast by electromagnetic field environments

The electromagnetic field (EMF) is newly considered as an exogenous environmental stimulus that is closely related to ion transportation on the cellular membrane, maintaining the internal ionic homeostasis. Cation transports of Ca²⁺ and other metal ions, Cd²⁺, Zn²⁺, and Mn²⁺were studied in terms of the external Ca²⁺ stress, [Ca²⁺]_ext, and exposure to the physical EMF. A specific yeast strain K667 was used for controlling CAX5 (cation/H⁺ exchanger) expression. Culture samples were exposed to 60 Hz, 0.1 mT sinusoidal or square magnetics waves, and intracellular cations of each sample were measured and analyzed. AtCAX5 transformant yeast grew normally under the metallic stress. However, the growth of the control group was significantly inhibited under the same cation concentration; 60 Hz and 0.1 mT magnetic field enhanced intracellular cation concentrations significantly as exposure time increased both in the AtCAX5 transformed yeast and in the control group. However, the AtCAX5-transformed yeast showed higher concentration of the intracellular cations than the control group under the same exposure EMF. AtCAX5-transformed yeasts displayed an increment in [Ca²⁺]_int, [K⁺]_int, [Na⁺]_int, and [Zn²⁺]_int concentration under the presence of both sinusoidal and square-waved EMF stresses compared to the control group, which shows that AtCAX5 expressed in the vacuole play an important role in maintaining the homeostasis of intracellular cations. These findings could be utilized in the cultivation of the crops which were resistant to excessive exogenous ions or in the production of biomass containing a large proportion of ions for nutritional food or in the bioremediation process in metal-polluted environments.     

Molecular Imaging of Pancreatic Duct Adenocarcinoma Using a Type 2 Cannabinoid Receptor-Targeted Near-Infrared Fluorescent Probe

Imaging probes targeting type 2 cannabinoid receptor (CB₂R) overexpressed in pancreatic duct adenocarcinoma (PDAC) tissue have the potential to improve early detection and surgical outcome of PDAC. The aim of our study was to evaluate the molecular imaging potential of a CB₂R-targeted near-infrared (NIR) fluorescent probe (NIR760-XLP6) for PDAC. CB₂R overexpression was observed in both PDAC patient tissues and various pancreatic cancer cell lines. In vitro fluorescence imaging indicated specific binding of NIR760-XLP6 to CB₂R in human PDAC PANC-1 cells. In a xenograft mouse tumor model, NIR760-XLP6 showed remarkable 50- (ex vivo) and 3.2-fold (in vivo) tumor to normal contrast enhancement with minimal liver and kidney uptake. In a PDAC lymph node metastasis model, significant signal contrast was observed in bilateral axillary lymph nodes with PDAC metastasis after injection of the probe. In conclusion, NIR760-XLP6 exhibits promising characteristics for imaging PDAC, and CB₂R appears to be an attractive target for PDAC imaging.