Superoxide production at phagosomal cup/phagosome through beta I protein kinase C during Fc gamma R-mediated phagocytosis in microglia

Protein kinase C (PKC) plays a prominent role in immune signaling. To elucidate the signal transduction in a respiratory burst and isoform-specific function of PKC during FcgammaR-mediated phagocytosis, we used live, digital fluorescence imaging of mouse microglial cells expressing GFP-tagged molecules. betaI PKC, epsilonPKC, and diacylglycerol kinase (DGK) beta dynamically and transiently accumulated around IgG-opsonized beads (BIgG). Moreover, the accumulation of p47(phox), an essential cytosolic component of NADPH oxidase and a substrate for betaI PKC, at the phagosomal cup/phagosome was apparent during BIgG ingestion. Superoxide (O(2)(-)) production was profoundly inhibited by G?6976, a cPKC inhibitor, and dramatically increased by the DGK inhibitor, R59949. Ultrastructural analysis revealed that BIgG induced O(2)(-) production at the phagosome but not at the intracellular granules. We conclude that activation/accumulation of betaI PKC is involved in O(2)(-) production, and that O(2)(-) production is primarily initiated at the phagosomal cup/phagosome. This study also suggests that DGKbeta plays a prominent role in regulation of O(2)(-) production during FcgammaR-mediated phagocytosis.     

HIV-1 Tat Activates Neuronal Ryanodine Receptors with Rapid Induction of the Unfolded Protein Response and Mitochondrial Hyperpolarization

Neurologic disease caused by human immunodeficiency virus type 1 (HIV-1) is ultimately refractory to highly active antiretroviral therapy (HAART) because of failure of complete virus eradication in the central nervous system (CNS), and disruption of normal neural signaling events by virally induced chronic neuroinflammation. We have previously reported that HIV-1 Tat can induce mitochondrial hyperpolarization in cortical neurons, thus compromising the ability of the neuron to buffer calcium and sustain energy production for normal synaptic communication. In this report, we demonstrate that Tat induces rapid loss of ER calcium mediated by the ryanodine receptor (RyR), followed by the unfolded protein response (UPR) and pathologic dilatation of the ER in cortical neurons in vitro. RyR antagonism attenuated both Tat-mediated mitochondrial hyperpolarization and UPR induction. Delivery of Tat to murine CNS in vivo also leads to long-lasting pathologic ER dilatation and mitochondrial morphologic abnormalities. Finally, we performed ultrastructural studies that demonstrated mitochondria with abnormal morphology and dilated endoplasmic reticulum (ER) in brain tissue of patients with HIV-1 inflammation and neurodegeneration. Collectively, these data suggest that abnormal RyR signaling mediates the neuronal UPR with failure of mitochondrial energy metabolism, and is a critical locus for the neuropathogenesis of HIV-1 in the CNS.     

Mechanism of Na‐Ion Storage in Hard Carbon Anodes Revealed by Heteroatom Doping

Hard carbon is the leading candidate anode for commercialization of Na‐ion batteries. Hard carbon has a unique local atomic structure, which is composed of nanodomains of layered rumpled sheets that have short‐range local order resembling graphene within each layer, but complete disorder along the c‐axis between layers. A primary challenge holding back the development of Na‐ion batteries is that a complete understanding of the structure–capacity correlations of Na‐ion storage in hard carbon has remained elusive. This article presents two key discoveries: first, the characteristics of hard carbons structure can be modified systematically by heteroatom doping, and second, that these structural changes greatly affect Na‐ion storage properties, which reveals the mechanisms for Na storage in hard carbon. Specifically, via P or S doping, the interlayer spacing is dilated, which extends the low‐voltage plateau capacity, while increasing the defect concentrations with P or B doping leads to higher sloping sodiation capacity. The combined experimental studies and first principles calculations reveal that it is the Na‐ion‐defect binding that corresponds to the sloping capacity, while the Na intercalation between graphenic layers causes the low‐potential plateau capacity. The understanding suggests a new design principle of hard carbon anode: more reversibly binding defects and dilated turbostratic domains, given that the specific surface area is maintained low.     

Serum Metabolite Biomarkers Discriminate Healthy Smokers from COPD Smokers

COPD (chronic obstructive pulmonary disease) is defined by a fixed expiratory airflow obstruction associated with disordered airways and alveolar destruction. COPD is caused by cigarette smoking and is the third greatest cause of mortality in the US. Forced expiratory volume in 1 second (FEV1) is the only validated clinical marker of COPD, but it correlates poorly with clinical features and is not sensitive enough to predict the early onset of disease. Using LC/MS global untargeted metabolite profiling of serum samples from a well-defined cohort of healthy smokers (n = 37), COPD smokers (n = 41) and non-smokers (n = 37), we sought to discover serum metabolic markers with known and/or unknown molecular identities that are associated with early-onset COPD. A total of 1,181 distinct molecular ions were detected in 95% of sera from all study subjects and 23 were found to be differentially-expressed in COPD-smokers vs. healthy-smokers. These 23 putative biomarkers were differentially-correlated with lung function parameters and used to generate a COPD prediction model possessing 87.8% sensitivity and 86.5% specificity. In an independent validation set, this model correctly predicted COPD in 8/10 individuals. These serum biomarkers included myoinositol, glycerophopshoinositol, fumarate, cysteinesulfonic acid, a modified version of fibrinogen peptide B (mFBP), and three doubly-charged peptides with undefined sequence that significantly and positively correlate with mFBP levels. Together, elevated levels of serum mFBP and additional disease-associated biomarkers point to a role for chronic inflammation, thrombosis, and oxidative stress in remodeling of the COPD airways. Serum metabolite biomarkers offer a promising and accessible window for recognition of early-stage COPD.     

Sucrose Phosphate Synthase Activity Rises in Correlation with High-Rate Cellulose Synthesis in Three Heterotrophic Systems

Based on work with cotton fibers, a particulate form of sucrose (Suc) synthase was proposed to support secondary wall cellulose synthesis by degrading Suc to fructose and UDP-glucose. The model proposed that UDP-glucose was then channeled to cellulose synthase in the plasma membrane, and it implies that Suc availability in cellulose sink cells would affect the rate of cellulose synthesis. Therefore, if cellulose sink cells could synthesize Suc and/or had the capacity to recycle the fructose released by Suc synthase back to Suc, cellulose synthesis might be supported. The capacity of cellulose sink cells to synthesize Suc was tested by analyzing the Suc phosphate synthase (SPS) activity of three heterotrophic systems with cellulose-rich secondary walls. SPS is a primary regulator of the Suc synthesis rate in leaves and some Suc-storing, heterotrophic organs, but its activity has not been previously correlated with cellulose synthesis. Two systems analyzed, cultured mesophyll cells of Zinnia elegans L. var. Envy and etiolated hypocotyls of kidney beans (Phaseolus vulgaris), contained differentiating tracheary elements. Cotton (Gossypium hirsutum L. cv Acala SJ-1) fibers were also analyzed during primary and secondary wall synthesis. SPS activity rose in all three systems during periods of maximum cellulose deposition within secondary walls. The Z. elegans culture system was manipulated to establish a tight linkage between the timing of tracheary element differentiation and rising SPS activity and to show that SPS activity did not depend on the availability of starch for degradation. The significance of these findings in regard to directing metabolic flux toward cellulose will be discussed.     

Potent inhibitors of farnesyltransferase and geranylgeranyltransferase-I

Compound 1 has been shown to be a dual prenylation inhibitor with FPTase (IC50=2 nM) and GGPTase-I (IC50=95 nM). Analogues of 1, which replaced the cyanophenyl group with various biaryls, led to the discovery of highly potent dual FPTase/GGPTase-I inhibitors. 4-trifluoromethylphenyl, trifluoropentynyl, and trifluoropentyl were identified as good p-cyano replacements.     

Probing domain swapping for the neuronal SNARE complex with electron paramagnetic resonance

Highly conserved soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins control membrane fusion at synapses. The target plasma membrane-associated SNARE proteins and the vesicle-associated SNARE protein assemble into a parallel four-helix bundle. Using a novel EPR approach, it is found that the SNARE four-helix bundles are interconnected via domain swapping that is achieved by substituting one of the two SNAP-25 helices with the identical helix from the second four-helical bundle. Domain swapping is likely to play a role in the multimerization of the SNARE complex that is required for successful membrane fusion. The new EPR application employed here should be useful to study other polymerizing proteins.     

Investigation of Four Classes of Non-nodulating White Sweetclover (Melilotus alba annua Desr.) Mutants and Their Responses to Arbuscular-Mycorrhizal Fungi

The nitrogen-fixing symbiosis between Rhizobiaceae and legumes is one of the best-studied interactions established between prokaryotes and eukaryotes. The plant develops root nodules in which the bacteria are housed, and atmospheric nitrogen is fixed into ammonia by the rhizobia and made available to the plant in exchange for carbon compounds. It has been hypothesized that this symbiosis evolved from the more ancient arbuscular mycorrhizal (AM) symbiosis, in which the fungus associates with roots and aids the plant in the absorption of mineral nutrients, particularly phosphate. Support comes from several fronts: 1) legume mutants where Nod(-) and Myc(-) co-segregate, and 2) the fact that various early nodulin (ENOD) genes are expressed in legume AM. Both strongly argue for the idea that the signal transduction pathways between the two symbioses are conserved. We have analyzed the responses of four classes of non-nodulating Melilotus alba (white sweetclover) mutants to Glomus intraradices (the mycorrhizal symbiont) to investigate how Nod(-) mutations affect the establishment of this symbiosis. We also re-examined the root hair responses of the non-nodulating mutants to Sinorhizobium meliloti (the nitrogen-fixing symbiont). Of the four classes, several sweetclover sym mutants are both Nod(-) and Myc(-). In an attempt to decipher the relationship between nodulation and mycorrhiza formation, we also performed co-inoculation experiments with mutant rhizobia and Glomus intraradices on Medicago sativa, a close relative of M. alba. Even though sulfated Nod factor was supplied by some of the bacterial mutants, the fungus did not complement symbiotically defective rhizobia for nodulation.