CAT SIS NETWORK ACTIVATION SIS ID(2012) |LINK|

Figure 4. Theoretical models of biological mechanisms of DNA release in neutrophils. The three models are depicted and illustrate their potential triggers, biochemical pathways, and biological consequences. (A) NETosis is initiated by stimuli that potently activate NADPH oxidase and ROS production, but decreases PAD4 activation and likely mtROS production. NETosis is antimicrobial, potentially anti-inflammatory, and has limited protein citrullination. (B) Leukotoxic hypercitrullination is triggered by stimuli that generate prominent and sustained increases in cytosolic calcium, such as ionophores and proteins that damage the cellular membrane (e.g., pore-forming proteins). PAD hyperactivation and protein hypercitrullination are characteristic of this process, but not ROS production by NADPH oxidase. While mtROS may have some role in the release of DNA by calcium ionophores in neutrophils (32), the relevance of this pathway in neutrophil damage induced by pore-forming proteins is unknown. Antimicrobial functions and the immunogenicity of self-antigens are affected as a result of cellular hypercitrullination. (C,D) To compensate a constitutive defect in mitophagy, neutrophils normally extrude mitochondrial components, including mtDNA, as a mechanism for mitochondria clearance. (C) The process of mtDNA expulsion (MDEX) may be enhanced by inflammatory signals such LPS or C5a following neutrophil priming with GM-CSF and requires NADPH oxidase activity. (D) IFN-I and RNP-ICs alter the normal disposal of mitochondria in neutrophils and promote the expulsion of ox-mtDNA (OMEX). This process is dependent on mtROS, but not NADPH oxidase, and may be predisposed by factors that decrease NADPH oxidase activity. Different to mtDNA, ox-mtDNA is pro-inflammatory and highly interferogenic. LDGs from patients with SLE have spontaneous OMEX. The role of citrullination in (C,D) is unknown. The question marks (?) reflect pathways/functions that require further experimental confirmation.

The JDLink Satellite Modem enables JDLink usage in remote locations where a cellular network infrastructure is not available. When the satellite modem is in use, users can utilize the features provided with JDLink Select and JDLink Ultimate.

Here, we describe the substrate specificity of SisLac, providing extensive kinetic studies performed with various phosphotriesters, esters, N-acyl-homoserine lactones (AHLs) and other lactones as substrates. Moreover, we solved the X-ray structure of SisLac and structural comparisons with the closely related SsoPox structure highlighted differences in the surface salt bridge network and the dimerization interface. SisLac and SsoPox being close homologues (91% sequence identity), we undertook a mutational study to decipher these structural differences and their putative consequences on the stability and the catalytic properties of these proteins.

The plasmids preparations were performed in Escherichia coli strain DH5α (Invitrogen). Protein production was performed in E. coli BL21(DE3)-pGro7/GroEL strain (TaKaRa) using plasmids pET22b-StrepTevSisLac and pET22b-SsoPox (provided by GeneArt; Germany). Site directed mutagenesis was performed in 50 µL using Pfu polymerase (Invitrogen) on 100 ng of plasmid encoding corresponding genes and primers referenced in Table S1. The PCR cycle was performed using hybridization temperature of 55°C, elongation time of 12 min during 30 cycles and final elongation of 20 min. The template plasmid was eliminated by Fast Digest DpnI (Fermentas) digestion of 30 min at 37°C followed by inactivation step of 20 min at 80°C. Plasmids were concentrated by classical alcoholic precipitation and then electroporated (Gene-Pulser, Bio-Rad) into E. cloni 5alpha cells (Lucingen), a particularly competent strain of E. coli. Site directed mutagenesis was finally verified by sequencing.

SisLac sequence exhibits approximately the same amino acid content as SsoPox, containing 14.3 versus 16% of uncharged polar residues and 28.7 versus 28% of charged residues. This is no surprising since both enzymes possess high sequence identity (91%) (see sequence alignment, Fig. 2). As described for SsoPox, the charged residues are mainly located at the protein surface, forming complex electrostatic networks [47] that includes 28 salt bridges implicating 46 residues. This charge network mainly differs by the substitution K14E in SisLac that suppress a salt bridge network between E12-K14-D15 of SsoPox, and consequently creates a local concentration of 3 negative charges within 4 consecutive residues (Fig. 5A).

The pH dependence of SisLac was investigated and yields a bell-shaped curve with a pH optimum at pH 9, a consistent behavior with previously characterized PLLs [6], [7] and PTEs [48]. This pH dependence profile is also in agreement with the commonly accepted hydrolysis mechanism where a water molecule activated by the bi-metallic active site serves as nucleophile [13]. Additionally, the metal dependence was assayed and the metal nature was found to modulate the catalytic activities, as previously described in PTEs [19] and PLLs [34]. Amongst the tested metals, SisLac shows preference for cobalt cations for both lactonase and paraoxonase activities (supplementary information & Fig. S6), as previously reported for the paraoxonase activity of SsoPox [6] and the lactonase activity of DrOPH [34], MCP [4] and GkL [9]. The metal dependence of SisLac may be related to the relative pKa of considered metal with H2O, since the pKa of Co2+/H2O is lower than that of Zn2+/H2O and Mn2+/H2O (8.9 versus 9.0, and 10.6, respectively [20]), thus Co2+ would better contribute to the activation of the nucleophile. In addition, Co2+ is more electronegative than Zn2+ and Mn2+ (1.88 versus 1.65 and 1.55, respectively [19]), thus being more efficient for stabilizing the developing negative charge on the transition state.

Interactions between the estrogen signaling pathway and a wide variety of other intracellular signaling molecules affect breast cancer cell sensitivity to endocrine therapy.8 Ligand-activated ERα functions as a transcription factor that interacts with a large number of coregulator proteins and other transcription factors.9, 10 Activated ERα also initiates rapid intracellular signaling through interactions with growth factor signaling molecules at the plasma membrane.4, 11, 12, 13 Interactions of ERα with other signaling molecules affect functions of growth factor-activated protein kinases.14, 15, 16, 17, 18, 19, 20 The interferon-γ and the HER2/ERBB2-mitogen-activated protein kinase (MAPK) signaling pathways have been reported to play a pivotal role in antiestrogen resistance.21, 22, 23 These observations suggest that a highly complex network comprising many intracellular signaling molecules is involved in the development of resistance to endocrine therapy.

Because our previous study showed that expression of BIK mRNA is dependent on TP53,26 TP53 is a second-generation node (Figure 1b). TP53 also plays critical roles in DAPK1-induced cell death,30, 31 and in human breast cancers the epigenetic suppression of DAPK1 expression and mutational inactivation of TP53 occur in a mutually exclusive manner,43 supporting the notion that DAPK inactivation in breast cancers might contribute to attenuation of TP53-dependent drug actions.

In 1972, new rules regarding cable television became effective. These rules required cable television operators to obtain a certificate of compliance from the Commission prior to operating a cable television system or adding a television broadcast signal. The rules applicable to cable operators fell into several broad subject areas -- franchise standards, signal carriage, network program nonduplication and syndicated program exclusivity, nonbroadcast or cablecasting services, cross-ownership, equal employment opportunity, and technical standards. Cable television operators who originated programming were subject to equal time, sponsorship identification and other provisions similar to rules applicable to broadcasters. Cable operators were also required to maintain certain records and to file annual reports with the Commission concerning general statistics, employment, and finances.

Subject to the Commission's network nonduplication, syndicated exclusivity and sports broadcasting rules, cable systems must carry the entirety of the program schedule of every local television station carried pursuant to the mandatory carriage provisions or the retransmission consent provisions of the 1992 Cable Act. A broadcaster and a cable operator may negotiate for partial carriage of the signal where the station is not eligible for must carry rights, either because of the station's failure to meet the requisite definitions or because the cable system is outside the station's market. In those situations where the carriage in the entirety rule applies, the primary video and accompanying audio of all television broadcast stations must be carried in full, without alteration or deletion of their content. Ancillary services such as closed captioning and program-related material in the vertical blanking interval must be carried. However, other information contained in the vertical blanking interval need not be carried. 2b1af7f3a8