Dissociation of RalA from synaptic membranes by Ca2+/calmodulin.

Ras-related small GTP-binding proteins execute many cellular functions, such as cell growth, differentiation, cytoskeletal reorganization, membrane trafficking, and membrane fusion. RalA belongs to the superfamily of Ras-related small GTP-binding proteins. Synaptic vesicles (SV) contain small GTP-binding proteins, where RalA, Rab3A, and Rab5A are the major GTP-binding proteins. It has been postulated that a cycling of these proteins between membrane-bound and soluble states is required for regulating cellular functions. Calmodulin (CaM) was found to dissociate Rab3A from SV membranes by forming a 1:1 complex with Ca2+/CaM. RalA was also found to be a Ca2+/CaM-binding protein. Therefore, we examined if Ca2+/CaM can also cause the RalA to dissociate from SV membranes. In this study, we identified that Ca2+/CaM dissociates RalA as well as Rab3A from synaptic vesicles.     

Naphthazarin derivatives (II): formation of glutathione conjugate, inhibition of DNA topoisomerase-I and cytotoxicity.

6-(1-Hydroxyalkyl)-5,8-dimethoxy-1,4-naphthoquinones, expressing a higher reactivity in conjugation with glutathione, showed a greater potency in the inhibition of DNA topoisomerase-I and the cytotoxicity against L1210 cells than 2-(1-hydroxyalkyl)-DMNQ derivatives, implying the participation of electrophilic arylation in the bioactivities. In further study 6-(1-Hydroxyalkyl)-5,8-dimethoxy-1,4-naphthoquinones with an alkyl group of shorter chain length (C2-C6) exerted a greater bioactivities than those with longer chain length(>C6).     

Modification of the ADP-ribosyltransferase and NAD glycohydrolase activities of a mammalian transferase (ADP-ribosyltransferase 5) by auto-ADP-ribosylation.

Mono-ADP-ribosylation, a post-translational modification in which the ADP-ribose moiety of NAD is transferred to an acceptor protein, is catalyzed by a family of amino acid-specific ADP-ribosyltransferases. ADP-ribosyltransferase 5 (ART5), a murine transferase originally isolated from Yac-1 lymphoma cells, differed in properties from previously identified eukaryotic transferases in that it exhibited significant NAD glycohydrolase (NADase) activity. To investigate the mechanism of regulation of transferase and NADase activities, ART5 was synthesized as a FLAG fusion protein in Escherichia coli. Agmatine was used as the ADP-ribose acceptor to quantify transferase activity. ART5 was found to be primarily an NADase at 10 microM NAD, whereas at higher NAD concentrations (1 mM), after some delay, transferase activity increased, whereas NADase activity fell. This change in catalytic activity was correlated with auto-ADP-ribosylation and occurred in a time- and NAD concentration-dependent manner. Based on the change in mobility of auto-ADP-ribosylated ART5 by SDS-polyacrylamide gel electrophoresis, the modification appeared to be stoichiometric and resulted in the addition of at least two ADP-ribose moieties. Auto-ADP-ribosylated ART5 isolated after incubation with NAD was primarily a transferase. These findings suggest that auto-ADP-ribosylation of ART5 was stoichiometric, resulted in at least two modifications and converted ART5 from an NADase to a transferase, and could be one mechanism for regulating enzyme activity.     

Extracellular secretion of levansucrase from Zymomonas mobilis in Escherichia coli

Secretion of levansucrase from Zymomonas mobilis in Escherichiacoli by glycine supplement was investigated. A significant amount of levansucrase (about 25% of total activity) was found in intact whole-cells. Cell fractionation experiments showed that levansucrase was found both in the periplasmic space and in the cytoplasmic fraction of E. coli. None or only trace amounts of levansucrase was detected in the extracellular culture broth at 24 h of cultivation and it accrued with the increasing concentration of glycine in the culture medium and duration of the culture period. Optimal glycine concentration for the maximum secretion of levansucrase was in the range of 0.8-1%, in which approximately 20-50% of levansucrase was released into the extracellular fraction at 24 h of cultivation, although glycine retarded the bacterial growth.     

Cyclic nucleotide selectivity of protein kinase G isozymes

The intrinsic activity of the C‐terminal catalytic (C) domain of cyclic guanosine monophosphate (cGMP)‐dependent protein kinases (PKG) is inhibited by interactions with the N‐terminal regulatory (R) domain. Selective binding of cGMP to cyclic nucleotide binding (CNB) domains within the R‐domain disrupts the inhibitory R–C interaction, leading to the release and activation of the C‐domain. Affinity measurements of mammalian and plasmodium PKG CNB domains reveal different degrees of cyclic nucleotide affinity and selectivity; the CNB domains adjacent to the C‐domain are more cGMP selective and therefore critical for cGMP‐dependent activation. Crystal structures of isolated CNB domains in the presence and absence of cyclic nucleotides reveal isozyme‐specific contacts that explain cyclic nucleotide selectivity and conformational changes that accompany CNB. Crystal structures of tandem CNB domains identify two types of CNB‐mediated dimeric contacts that indicate cGMP‐driven reorganization of domain–domain interfaces that include large conformational changes. Here, we review the available structural and functional information of PKG CNB domains that further advance our understanding of cGMP mediated regulation and activation of PKG isozymes.     

Immune Checkpoint Blockade Augments Changes Within Oncolytic Virus-induced Cancer MHC-I Peptidome,Creating Novel Antitumor CD8 T Cell Reactivities

The combination cancer immunotherapies with oncolytic virus (OV) and immune checkpoint blockade (ICB) reinstate otherwise dysfunctional antitumor CD8 T cell responses. One major mechanism that aids such reinstatement of antitumor CD8 T cells involves the availability of new class I major histocompatibility complex (MHC-I)-bound tumor epitopes following therapeutic intervention. Thus, therapy-induced changes within the MHC-I peptidome hold the key to understanding the clinical implications for therapy-reinstated CD8 T cell responses. Here, using mass spectrometry–based immuno-affinity methods and tumor-bearing animals treated with OV and ICB (alone or in combination), we captured the therapy-induced alterations within the tumor MHC-I peptidome, which were then tested for their CD8 T cell response-stimulating activity. We found that the oncolytic reovirus monotherapy drives up- as well as downexpression of tumor MHC-I peptides in a cancer type and oncolysis susceptibility dependent manner. Interestingly, the combination of reovirus + ICB results in higher numbers of differentially expressed MHC-I-associated peptides (DEMHCPs) relative to either monotherapies. Most importantly, OV+ICB-driven DEMHCPs contain biologically active epitopes that stimulate interferon-gamma responses in cognate CD8 T cells, which may mediate clinically desired antitumor attack and cancer immunoediting. These findings highlight that the therapy-induced changes to the MHC-I peptidome contribute toward the reinstated antitumor CD8 T cell attack established following OV + ICB combination cancer immunotherapy.     

Graph Independent Component Analysis Reveals Repertoires of Intrinsic Network Components in the Human Brain

Does each cognitive task elicit a new cognitive network each time in the brain? Recent data suggest that pre-existing repertoires of a much smaller number of canonical network components are selectively and dynamically used to compute new cognitive tasks. To this end, we propose a novel method (graph-ICA) that seeks to extract these canonical network components from a limited number of resting state spontaneous networks. Graph-ICA decomposes a weighted mixture of source edge-sharing subnetworks with different weighted edges by applying an independent component analysis on cross-sectional brain networks represented as graphs. We evaluated the plausibility in our simulation study and identified 49 intrinsic subnetworks by applying it in the resting state fMRI data. Using the derived subnetwork repertories, we decomposed brain networks during specific tasks including motor activity, working memory exercises, and verb generation, and identified subnetworks associated with performance on these tasks. We also analyzed sex differences in utilization of subnetworks, which was useful in characterizing group networks. These results suggest that this method can effectively be utilized to identify task-specific as well as sex-specific functional subnetworks. Moreover, graph-ICA can provide more direct information on the edge weights among brain regions working together as a network, which cannot be directly obtained through voxel-level spatial ICA.     

Vitamins,stress and growth: the availability of antioxidants in early life influences the expression of cryptic genetic variation

Environmental inputs during early development can shape the expression of phenotypes, which has long‐lasting consequences in physiology and life history of an organism. Here, we study whether experimentally manipulated availability of dietary antioxidants, vitamins C and E, influences the expression of genetic variance for antioxidant defence, endocrine signal and body mass in yellow‐legged gull chicks using quantitative genetic models based on full siblings. Our experimental study in a natural population reveals that the expression of genetic variance in total antioxidant capacity in plasma increased in chicks supplemented with vitamins C and E despite the negligible effects on the average phenotype. This suggests that individuals differ in their ability to capture and transport dietary antioxidants or to respond to these extra resources, and importantly, this ability has a genetic basis. Corticosterone level in plasma and body mass were negatively correlated at the phenotypic level. Significant genetic variance of corticosterone level appeared only in control chicks nonsupplemented with vitamins, suggesting that the genetic variation of endocrine system, which transmits environmental cues to adaptively control chick development, appeared in stressful conditions (i.e. poor antioxidant availability). Therefore, environmental inputs may shape evolutionary trajectories of antioxidant capacity and endocrine system by affecting the expression of cryptic genetic variation.