Acid-sensing ion channel (ASIC) 1a/2a heteromers have a flexible 2:1/1:2 stoichiometry

Acid-sensing ion channels (ASICs) are widely expressed proton-gated Na⁺ channels playing a role in tissue acidosis and pain. A trimeric composition of ASICs has been suggested by crystallization. Upon coexpression of ASIC1a and ASIC2a in Xenopus oocytes, we observed the formation of heteromers and their coexistence with homomers by electrophysiology, but could not determine whether heteromeric complexes have a fixed subunit stoichiometry or whether certain stoichiometries are preferred over others. We therefore imaged ASICs labeled with green and red fluorescent proteins on a single-molecule level, counted bleaching steps from GFP and colocalized them with red tandem tetrameric mCherry for many individual complexes. Combinatorial analysis suggests a model of random mixing of ASIC1a and ASIC2a subunits to yield both 2:1 and 1:2 ASIC1a:ASIC2a heteromers together with ASIC1a and ASIC2a homomers.Acid-sensing ion channels (ASICs) are proton-gated Na⁺ channels, which are probably ubiquitously expressed in neurons, yet surprisingly little is known about their physiological function in the brain (1). It is believed that ASICs localize to the postsynapse and carry an excitatory postsynaptic current (2). ASICs in central neurons are mainly composed of homomeric ASIC1a and heteromeric ASIC1a/2a (3–6) and ASIC1a/2b (7). Genetic ablation of ASIC1a has indeed led to the conclusion that ASICs contribute to long-term potentiation and memory formation (2), but a more recent study challenged these findings (8). In contrast to our incomplete understanding of the physiological functions of ASICs, there is a comparatively large body of evidence that activation of ASIC1a-containing channels in pathophysiological states that are associated with sustained acidosis contributes to neuronal or axonal degeneration (9, 10). In addition, blockade of the ASIC1a/2a heteromer causes central analgesia (11). Together, these findings render ASIC1a-containing channels attractive drug target candidates.The crystal structure of chicken ASIC1 revealed an acidic pocket at subunit interfaces, which has been proposed to be the ligand-binding site of ASICs (12). In agreement, it has recently been shown that a gating modifier toxin of ASIC1, psalmotoxin 1, which behaves like an agonist (13), binds at the acidic pocket at subunit interfaces (14–16). Therefore, knowing the subunit composition of the ASIC1a/2a heteromer is of major interest, in particular for pharmacologically targeting this subtype.Fluorescence resonance energy transfer analysis suggested that the ASIC1a/2a heteromer contains at least two ASIC1a and two ASIC2a subunits (17). In agreement, several studies reported that the related epithelial Na⁺ channel (ENaC) is composed of four (18–21) or nine subunits (22–24). In strong contrast, however, the crystal structure of chicken ASIC1 revealed a number of three subunits (12, 25), suggesting that all ASICs and their relatives like ENaC are trimers. The trimeric structure of ASIC1a has in the meanwhile been confirmed by atomic force microscopy imaging (26). The stoichiometry of heteromeric ASICs, however, remains completely unknown.In this study, we first used electrophysiology to characterize mixtures of ASIC1a and ASIC2a at different expression ratios in Xenopus laevis oocytes to demonstrate that at least one heteromeric channel forms. Because we were unable to decide on the existence of a second heteromeric species by electrophysiology, we used a single-molecule photobleaching approach that resolves stoichiometries of membrane proteins with high accuracy. We tagged ASIC1a and ASIC2a with green and red fluorescent reporter proteins, which did not change the electrophysiological characteristics of homo- and heteromeric ASICs, suggesting that fusion of a fluorescent reporter has no impact on the molecular composition of ASICs. Colocalization of red and green reporter tags on a single-molecule level and counting of green bleaching steps confirms the trimeric nature of functional ASICs and shows that ASIC1a and ASIC2a randomly assemble into a complex with a flexible stoichiometry of either 1:2 or 2:1.     

Epitaxial Growth of Diamond-Shaped Au1/2Ag1/2CN Nanocrystals on Graphene

Epitaxial synthesis of inorganic nanomaterials on pristine 2D materials is of interest in the development of nanostructured devices and nanocomposite materials, but is quite difficult because pristine surfaces of 2D materials are chemically inert. Previous studies found a few exceptions including AuCN, AgCN, CuCN, and Cu_0.5Au_0.5CN, which can be preferentially synthesized and epitaxially aligned onto various 2D materials. Here, we discover that Au_1/2Ag_1/2CN forms diamond-shaped nanocrystals epitaxially grown on pristine graphene surfaces. The nanocrystals synthesized by a simple drop-casting method are crystallographically aligned to lattice structures of the underlying graphene. Our experimental investigations on 3D structures and the synthesis conditions of the nanocrystals imply that the rhombic 2D geometries originate from different growth rates depending on orientations along and perpendicular to 1D molecular chains of Au_1/2Ag_1/2CN. We also perform in situ TEM observations showing that Au_1/2Ag_1/2CN nanocrystals are decomposed to Au and Ag alloy nanocrystals under electron beam irradiation. Our experimental results provide an additional example of 1D cyanide chain families that form ordered nanocrystals epitaxially aligned on 2D materials, and reveal basic physical characteristics of this rarely investigated nanomaterial.     

Netrin-1 induces angiogenesis via a DCC-dependent ERK1/2-eNOS feed-forward mechanism

Netrin-1 is critical for axonal pathfinding which shares similarities with formation of vascular network. Here we report that netrin-1 induction of angiogenesis is mediated by an increase in endothelial nitric oxide (NO*) production, which occurs via a DCC-dependent, ERK1/2-eNOS feed-forward mechanism. Exposure of mature aortic endothelial cells to netrin-1 resulted in a potent, dose-dependent increase in NO* production, detected by electron spin resonance. Scavenging NO* with 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) abolished netrin-1 stimulated angiogenesis. Netrin-1-stimulated NO* production or angiogenesis was inhibited by DCC antibody, DCC small interfering RNA (siRNA), specific inhibitors (PD98059, U0126), or siRNAs for MEK1/2. PTIO attenuated ERK1/2 phosphorylation, indicating a feed-forward mechanism. Netrin-1 induced a time-dependent phosphorylation of eNOS(s1179, s116) and a rapid dephosphorylation of eNOS(t497). Only eNOS(s1179) was sensitive to U0126 or PTIO. These data characterized a mechanism whereby netrin-1 promotes angiogenesis, which may broadly relate to cardiovascular, neuronal and cancer physiology.     

Uroporphyria in the Cyp1a2-/- mouse

Cytochrome P4501A2 (Cyp1a2) is important in the development of uroporphyria in mice, a model of porphyria cutanea tarda in humans. Heretofore, mice homozygous for the Cyp1a2−/− mutation do not develop uroporphyria with treatment regimens that result in uroporphyria in wild-type mice. Here we report uroporphyria development in Cyp1a2−/− mice additionally null for both alleles of the hemochromatosis (Hfe) gene and heterozygous for deletion of the uroporphyrinogen decarboxylase (Urod) gene (genotype: Cyp1a2−/−;Hfe−/−;Urod+/−), demonstrating that upon adding porphyria-predisposing genetic manipulations, Cyp1a2 is not essential. Cyp1a2−/−;Hfe−/−;Urod+/− mice were treated with various combinations of an iron-enriched diet, parenteral iron-dextran, drinking water containing δ-aminolevulinic acid and intraperitoneal Aroclor 1254 (a polychlorinated biphenyl mixture) and analyzed for uroporphyrin accumulation. Animals fed an iron-enriched diet alone did not develop uroporphyria but uroporphyria developed with all treatments that included iron supplementation and δ-aminolevulinic acid, even with a regimen without Aroclor 1254. Hepatic porphyrin levels correlated with low UROD activity and high levels of an inhibitor of UROD but marked variability in the magnitude of the porphyric response was present in all treatment groups. Gene expression profiling revealed no major differences between genetically identical triple cross mice exhibiting high and low magnitude porphyric responses from iron-enriched diet and iron-dextran supplementation, and δ-aminolevulinic acid. Even though the variation in porphyric response did not parallel the hepatic iron concentration, the results are compatible with the presence of a Cyp1a2-independent, iron-dependent pathway for the generation of uroporphomethene, the UROD inhibitor required for the expression of uroporphyria in mice and PCT in humans.     

Reparixin,a CXCR1/2 inhibitor in islet allotransplantation

Quality of life in Type 1 diabetic patients may be improved with islet transplantation, but lifelong immunosuppression is required to prevent rejection. Allo-immune response is a key player in graft dysfunction and although the adaptive immune response is well characterized, the effect of the innate immune reaction after transplantation is only recently becoming appreciated. In this study, we address how the innate response affects long-term outcomes in a murine islet allotransplant model. CTLA-4 Ig treatment is known to significantly prolong kidney subcapsular islet allograft survival and enhance glucose tolerance. The combination of CTLA-4 Ig with reparixin, which blocks against inflammatory neutrophil infiltration, yielded no long-term graft survival in an intrahepatic allotransplant model but had similar long-term graft survival in the kidney subcapsular model. Seven days after transplant, serum blood IFN-γ levels were significantly lower in the CTLA-4 Ig with reparixin treatment group compared to controls. IL-12p70 cytokine levels were increased with combination treatment, a positive modulation of the inflammatory response to the allograft. Furthermore, KC GRO, also known as CXCL1, was decreased in serum 7 d after transplant. Histologically, we found that immune cell infiltrate, CD4+ and CD8+ T cell populations along with both CXCR1+ and CXCR2+ cell populations were decreased within the CTLA-4 Ig and reparixin islet transplant graft. Overall these data provide insight into the down regulation of T-cell recruitment by CTLA-4 Ig and decreased neutrophil activation and recruitment with reparixin after long-term islet graft survival.     

Development of a monoclonal antibody capable of differentiating platelet PLA1/PLA1, PLA1/PLA2 and PLA2/PLA2 genotypes

A monoclonal antibody, LK-4, has been developed which distinguishes platelet PLA1/PLA1, PLA1/PLA2 and PLA2/PLA2 genotypes on platelet glycoprotein GPIIIa of Triton-solubilized platelet extracts. An ELISA assay has been developed which traps GPIIIa with Concanavalin A, enriching the platelet extract for the PLA antigens. A second monoclonal antibody, DEK-10, which reacts equally with GPIIIa of PLA1/PLA1 and PLA2/PLA2 platelet extracts is employed as an internal standard to correct for individual differences in GPIIIa content, GPIIIa extracted by Triton X-100 and GPIIIa trapped with Concanavalin A. This ELISA assay clearly differentiated 11 different PLA1/PLA1 subjects from eight PLA2/PLA2 women with a history of neonatal alloimmune thrombocytopenia as well as six unrelated obligate heterozygotes and should be useful in evaluating the PLA genotype of pregnant women and their families.     

p-ERK1/2 is a predictive factor of response to erythropoiesis-stimulating agents in low/int-1 myelodysplastic syndromes

Serum erythropoietin level less than 100U/L and a transfusion requirement of less than 2 units per month are the best predictive factors for response to treatment by erythropoiesis-stimulating agents in low/int-1 myelodysplastic syndromes. To investigate the factors influencing the response to erythropoiesis-stimulating agents, we enrolled 127 low/int-1 myelodysplastic syndrome patients at diagnosis in a biological study of erythropoiesis. The 54 non-responders had a significantly lower number of burst-forming unit-erythroid and colony-forming unit-erythroid than responders. Erythropoietin-dependent proliferation and survival, and phospho (p)-ERK1/2 expression in steady state and after erythropoietin stimulation were defective in cultured erythroblasts. By flow cytometry, p-ERK1/2 was significantly lower in bone marrow CD45⁻/CD71⁺/GPA⁻cells from non-responders compared to responders or controls. Receiver Operator Characteristic curve analysis showed that this flow cytometry test was a sensitive biomarker for predicting the response to erythropoiesis-stimulating agents.     

MEK1/2-ERK1/2 mediates alpha1-adrenergic receptor-stimulated hypertrophy in adult rat ventricular myocytes

We examined the relative roles of the mitogen-activated protein kinases (MAPK) in mediating the alpha1-adrenergic receptor (alpha1-AR) stimulated hypertrophic phenotype in adult rat ventricular myocytes (ARVM). Norepinephrine (NE; 1 microM) in the presence of the beta -AR antagonist propranolol (Pro; 2 microM) caused activation of Ras (>six-fold), MAPK/ERK kinase 1 and 2 (MEK1/2, >10-fold) and extracellular signal-regulated kinases 1 and 2 (ERK1/2, approximately 30-fold) within 5 min, as determined by kinase activity assays and Western blots using phospho-specific antibodies. Conversely, p38 and c-Jun amino-terminal kinases (JNK) were not activated by NE/Pro. Activated MEK1/2 signals remained detectable at 2 h, and activated ERK1/2 remained detectable at 48 h. The alpha1-AR selective inhibitor prazosin (100 nM) completely inhibited the NE/Pro-stimulated activation of Ras, MEK1/2 and ERK1/2. The MEK inhibitor PD98059 caused a concentration-dependent inhibition of NE/Pro-stimulated protein synthesis (as assessed by [3H]leucine incorporation and cellular protein accumulation) and ERK1/2 activation, with approximately 50% inhibition at a concentration between 10 and 50 microM, which is consistent with the known IC50 values of PD98059 for MEK1 (4 microM) and MEK2 (50 microM). Thus, these data show that alpha1-AR stimulated hypertrophy in ARVM is dependent on the MEK1/2-ERK1/2 signaling pathway.     

Netrin-1 prevents ischemia/reperfusion-induced myocardial infarction via a DCC/ERK1/2/eNOSs1177/NO/DCC feed-forward mechanism

We have recently shown that a novel endothelial mitogen netrin-1 potently stimulates nitric oxide (NO) production via a DCC-ERK1/2 dependent mechanism. In view of the well-established cardioprotective role of NO, the present study investigated whether netrin-1 is cardioprotective via NO signaling in the heart. Netrin-1 receptor DCC was abundantly expressed in the C57BL/6J mouse hearts. Perfusion of heart with netrin-1 (100 ng/mL) using a Langendorff system significantly increased NO production. Under ischemia/reperfusion (I/R), netrin-1 induced a substantial reduction in infarct size (21.8 ± 4.9% from 42.5 ± 3.6% in the controls), which was accompanied by an augmented production of NO. Pre-perfusion with DCC-antibody, U0126 (MEK1/2 inhibitor), L-NAME or PTIO (NO scavenger) attenuated protective effects of netrin-1 on infarct size and NO production, indicating upstream roles of DCC and ERK1/2 in NO production, as well as an essential role of NO in cardioprotection. Netrin-1 induced reduction in infarct size was significantly attenuated in DCC+/− mice, confirming an intermediate role of DCC. In additional experiments we found netrin-1 increased ERK1/2 and eNOS_s1177 phosphorylation, and DCC protein expression, which was diminished by I/R. Furthermore, netrin-1-induced DCC upregulation was NO and ERK1/2-dependent, implicating a feed-forward mechanism. DAF-AM staining revealed enhanced NO production in both cardiac endothelial cells (ECs) and myocytes. In primarily isolated cardiomyocytes, netrin-1 also increased NO production, DCC abundance and ERK1/2 phosphorylation. Of note, cardiac apoptosis was significantly attenuated by netrin-1, which was reversed by DCC-antibody, U0126, L-NAME or PTIO. In summary, our data clearly demonstrate that netrin-1 potently protects the heart from I/R injury by stimulating NO production from cardiac ECs and myocytes. This potent effect is mediated by a DCC/ERK1/2/eNOS_s1177/NO/DCC feed-forward mechanism in both cell types.     

CREG, a new regulator of ERK1/2 in cardiac hypertrophy

OBJECTIVES: The cellular repressor of E1A-stimulated genes (CREG), a mannose-6-phosphate-containing secreted glycoprotein, enhances differentiation and inhibits proliferation. In this study, our aim was to understand the role of CREG in cardiac hypertrophy. METHODS: Two models of cardiac hypertrophy were used: the in vivo pressure-overloaded rat cardiac hypertrophy and the in vitro stretched neonatal rat cardiomyocyte hypertrophy. CREG's function in cardiac cells was investigated after over-expression or antisense inhibition of CREG. RESULTS: We found reduced CREG expression in rat hearts after the in vivo overload, as shown by Northern blot analysis. CREG over-expression inhibited cardiac cell growth, as demonstrated by reduced protein content, cell area and ERK1/2 level in cultured neonatal rat cardiomyocytes, and by the reduced proliferation of cultured neonatal rat cardiac fibroblasts. Additionally, over-expression of CREG dampened the stretched cardiomyocyte hypertrophy through ERK1/2. On the other hand, the opposite effects were observed when CREG expression was decreased using antisense. This modulation of CREG expression resulted in no changes in the cardiomyocyte expressions of the hypertrophic or apoptotic signaling molecules such as protein kinase C (PKC) epsilon, PKC beta1, PKC alpha, PKC beta2, PKC delta, JNK1/2, p38, p53, Bax, Bcl2 and Fas. CONCLUSIONS: CREG was found to inhibit cardiac cell growth as a novel regulator of ERK1/2 and might participate in the development of cardiac hypertrophy under pressure overload. The insight that CREG inhibited the growth in rat hearts in vivo and in cardiac cells in vitro provides new clues for further investigation of the mechanism of heart remodeling.     

Cytoplasmic FMRP interacting protein 1/2 (CYFIP1/2) expression analysis in autism

Metabolic Brain Disease - Cytoplasmic FMRP interacting proteins 1 and 2 (CYFIP1/2) have been previously shown to be associated with central nervous system (CNS) disorders such as autism spectrum...     

Asymptomatic cor triatriatum in a 2 1/4 year old patient

We report on an asymptomatic 2 1/4 year old girl. Echocardiography revealed cor triatriatum with a membrane separating the left atrium. Transesophageal echocardiography demonstrated 3 large perforations of the intraatrial membrane. Using Doppler sonography, no pressure gradients across these orifices were identified. Obstruction of the intraatrial membrane and pulmonary hypertension were excluded by cardiac catheterization. We therefore decided against surgical resection of the membrane and to follow the patient noninvasively by echocardiography. Follow-up over 18 months revealed no development of an obstruction across the intraatrial membrane. Our case shows that immediate surgical correction is not necessary in all patients with cor triatriatum. Conservative management of these patients requires full evaluation of the hemodynamics and careful follow-up examinations by echocardiography.     

Recidivism among high risk youths: a 2 1/2-year follow-up of a cohort of juvenile detainees.

We report some results from a longitudinal study of juvenile detainees. Analyses were directed toward determining whether the youths' alcohol or other drug use and their emotional/psychological problems at entry into the detention center predicted subsequent arrests for new offenses during the 24 and 30 months following their initial interviews. Statistically significant relationships were found between the youths' demographic characteristics (age, race, gender), referral history, reason for placement in the detention center, cocaine use (as measured by urinalysis), and recidivism. The magnitude of these relationships increased with the length of the follow-up period.     

Enhanced Pyroelectric Performance of Lead-Free Zn-Doped Na1/2Bi1/2TiO3-BaTiO3 Ceramics

Lead-free Na_1/2Bi_1/2TiO₃-BaTiO₃ (NBT-BT) has gained revived interest due to its exceptionally good high power properties in comparison to commercial lead-based piezoelectrics. Recently, Zn-modified NBT-BT-based materials as solid solution and composites have been reported to exhibit enhanced depolarization temperatures and a high mechanical quality factor. In this work, the pyroelectric properties of Zn-doped NBT-6mole%BT and NBT-9mole%BT ceramics are investigated. The doped compositions of NBT-6BT and NBT-9BT feature a relatively stable pyroelectric property in a wide temperature range of ~37 K (300–330 K) and 80 K (300–380 K), respectively. A threefold increase in detector figure of merit is noted for 0.01 mole Zn-doped NBT-6mole% BT at room temperature in comparison to undoped NBT-6mole%BT and this increase is higher than those of major lead-free materials. A broad range of the temperature-independent behavior for the figures of merit was noted (303–380 K) for Zn-doped NBT-6mole% BT, which is 30 K higher than the undoped material. The large pyroelectric figures of merit and good temperature stability renders Zn-doped NBT-BT an ideal candidate for pyroelectric detector and energy harvesting applications.     

牛乳腺炎无乳链球菌PI-2a菌毛岛屿AP1-AP2-BP基因亚单位抗体的制备

目的 构建牛乳腺炎无乳链球菌菌毛岛屿PI-2a亚单位重组抗原AP₁-AP₂-BP,并制备其多亚单位抗体,为后期研制新型免疫疫苗和检测试剂提供实验基础。方法 利用延伸PCR技术构建了AP₁-AP₂-BP三联基因,并将该串联基因进行转化,诱导,表达,纯化,并将纯化蛋白制作为抗原,对家兔免疫,制备多亚单位抗体,并通过亲和层析的方式从免疫后的血清中获得纯化的IgG,完成多亚单位抗体的制备。结果 AP₁-AP₂-BP三基因串联重组工程菌株通过诱导、表达,对产物亲和层析等方法获得的纯化蛋白,其相对分子质量为65 kDa,其含量达到3.3 mg/mL,并具有较好的免疫原性。经间接ELISA可知,经过4周的免疫抗体的滴度已达到1∶5 600的水平。通过 Protein A280测量数据表明,纯化后的IgG的含量高达9.1 mg/mL。结论 牛乳腺炎无乳链球菌的菌毛岛屿AP₁-AP₂-BP三基因串联重组工程菌经诱导、表达后获得的纯化蛋白AP₁-AP₂-BP多亚单位抗原有较好的免疫原性,为制备相应多亚单位抗体的制备提供了良好的多价抗原,同时为将研制新型工程疫苗及检测试剂提供了科学研究基础。