Sodium-Dependent Movement of Covalently Bound FMN Residue(s) in Na(+)-Translocating NADH:Quinone Oxidoreductase

Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) is a component of respiratory electron-transport chain of various bacteria generating redox-driven transmembrane electrochemical Na(+) potential. We found that the change in Na(+) concentration in the reaction medium has no effect on the thermodynamic properties of prosthetic groups of Na(+)-NQR from Vibrio harveyi, as was revealed by the anaerobic equilibrium redox titration of the enzyme's EPR spectra. On the other hand, the change in Na(+) concentration strongly alters the EPR spectral properties of the radical pair formed by the two anionic semiquinones of FMN residues bound to the NqrB and NqrC subunits (FMN(NqrB) and FMN(NqrC)). Using data obtained by pulse X- and Q-band EPR as well as by pulse ENDOR and ELDOR spectroscopy, the interspin distance between FMN(NqrB) and FMN(NqrC) was found to be 15.3 ? in the absence and 20.4 ? in the presence of Na(+), respectively. Thus, the distance between the covalently bound FMN residues can vary by about 5 ? upon changes in Na(+) concentration. Using these results, we propose a scheme of the sodium potential generation by Na(+)-NQR based on the redox- and sodium-dependent conformational changes in the enzyme.     

Kinetics of the CRISPR-Cas9 effector complex assembly and the role of 3′-terminal segment of guide RNA

CRISPR-Cas9 is widely applied for genome engineering in various organisms. The assembly of single guide RNA (sgRNA) with the Cas9 protein may limit the Cas9/sgRNA effector complex function. We developed a FRET-based assay for detection of CRISPR–Cas9 complex binding to its targets and used this assay to investigate the kinetics of Cas9 assembly with a set of structurally distinct sgRNAs. We find that Cas9 and isolated sgRNAs form the effector complex efficiently and rapidly. Yet, the assembly process is sensitive to the presence of moderate concentrations of non-specific RNA competitors, which considerably delay the Cas9/sgRNA complex formation, while not significantly affecting already formed complexes. This observation suggests that the rate of sgRNA loading into Cas9 in cells can be determined by competition between sgRNA and intracellular RNA molecules for the binding to Cas9. Non-specific RNAs exerted particularly large inhibitory effects on formation of Cas9 complexes with sgRNAs bearing shortened 3′-terminal segments. This result implies that the 3′-terminal segment confers sgRNA the ability to withstand competition from non-specific RNA and at least in part may explain the fact that use of sgRNAs truncated for the 3′-terminal stem loops leads to reduced activity during genomic editing.     

Chiral HPLC Separation and Modeling of Four Stereomers of DL‐Leucine‐DL‐Tryptophan Dipeptide on Amylose Chiral Column

Chiral high‐performance liquid chromatography (HPLC) separation and modeling of four stereomers of DL‐leucine‐tryptophan DL‐dipeptide on AmyCoat‐RP column are described. The mobile phase applied was ammonium acetate (10 mM)‐methanol‐acetonitrile (50:5:45, v/v). The flow rate of the mobile phases was 0.8 mL/min with UV detection at 230 nm. The values of retention factors for LL‐, DD‐, DL‐, and LD‐ stereomers were 2.25, 3.60, 5.00, and 6.50, respectively. The values of separation and resolution factors were 1.60, 1.39, and 1.30 and 7.76, 8.05, and 7.19. The limits of detection and quantitation were ranging from 1.0–2.3 and 5.6–14.0 μg/mL. The simulation studies established the elution orders and the mechanism of chiral recognition. It was seen that π–π connections and hydrogen bondings were the main forces for enantiomeric resolution. The reported chiral HPLC method may be applied for the enantiomeric separation of DL‐leucine‐DL‐tryptophan in unknown matrices. Chirality 28:642–648, 2016. © 2016 Wiley Periodicals, Inc.     

Acetylation reduces SOX9 nuclear entry and ACAN gene transactivation in human chondrocytes

Changes in the content of aggrecan, an essential proteoglycan of articular cartilage, have been implicated in the pathophysiology of osteoarthritis (OA), a prevalent age‐related, degenerative joint disease. Here, we examined the effect of SOX9 acetylation on ACAN transactivation in the context of osteoarthritis. Primary chondrocytes freshly isolated from degenerated OA cartilage displayed lower levels of ACAN mRNA and higher levels of acetylated SOX9 compared with cells from intact regions of OA cartilage. Degenerated OA cartilage presented chondrocyte clusters bearing diffused immunostaining for SOX9 compared with intact cartilage regions. Primary human chondrocytes freshly isolated from OA knee joints were cultured in monolayer or in three‐dimensional alginate microbeads (3D). SOX9 was hypo‐acetylated in 3D cultures and displayed enhanced binding to a ?10 kb ACAN enhancer, a result consistent with higher ACAN mRNA levels than in monolayer cultures. It also co‐immunoprecipitated with SIRT1, a major deacetylase responsible for SOX9 deacetylation. Finally, immunofluorescence assays revealed increased nuclear localization of SOX9 in primary chondrocytes treated with the NAD SIRT1 cofactor, than in cells treated with a SIRT1 inhibitor. Inhibition of importin β by importazole maintained SOX9 in the cytoplasm, even in the presence of NAD. Based on these data, we conclude that deacetylation promotes SOX9 nuclear translocation and hence its ability to activate ACAN.     

The association between patellar alignment on magnetic resonance imaging and radiographic manifestations of knee osteoarthritis

The aim of our study was to evaluate the association between patellar alignment by using magnetic resonance imaging images and radiographic manifestations of patello-femoral osteoarthritis (OA). Subjects were recruited to participate in a natural history study of symptomatic knee OA. We examined the relation of patellar alignment in the sagittal plane (patellar length ratio (PLR)) and the transverse plane (sulcus angle (SA), lateral patellar tilt angle (LPTA), and bisect offset (BO)) to radiographic features of patello-femoral OA, namely joint space narrowing and patellar osteophytes, using a proportional odds logistic regression model while adjusting for age, sex, and bone mass index (BMI). The study sample consisted of 126 males (average age 68.0 years, BMI 31.2) and 87 females (average age 64.7 years, BMI 31.6), 75% of whom had tibiofemoral OA (a Kellgren-Lawrence score of 2 or more). PLR showed a statistically significant association with joint space narrowing and osteophytosis in the lateral compartment. SA showed significant association with medial joint space narrowing and with lateral and medial patellar osteophytosis. LPTA and BO showed significant association with both radiographic indices of the lateral compartment. Clear linear trends were found in association between PLR, LPTA and BO, and with outcomes associated with lateral patello-femoral OA. SA, LPTA, and BO showed linear trends of association with medial joint space narrowing. Results of our study clearly suggest the association between indices of patellar alignment and such features of patello-femoral OA as osteophytosis and joint space narrowing. Additional studies will be required to establish the normal and abnormal ranges of patellar alignment indices and their longitudinal relation to patello-femoral OA.     

A fresh look at an antiviral helicase

In order to survive, all organisms must guard against viral infections. Recognition of viruses is accomplished via multiple sensors. Many mammalian proteins can recognize viral products, such as double-stranded RNA （dsRNA）, yet few of them are known to induce interferon, the central antiviral messenger. Since interferon is indispensable for successful antiviral defense [1], the interferon-inducing sensors have been of particular interest. However, a clear understanding of such sensors has been elusive, and the first well-established sensor family, the toll-like receptors （TLRs）, was described relatively recently [2]. Antiviral TLRs are positioned in the endosomes, where they report the appearance of viral genetic material （DNA, single-and double-stranded RNA）. However, the question of potential virus sensors in the cytoplasm was left open. Given the particular effectiveness ofintracellular dsRNA at inducing interferon, it was suspected that dsRNA-binding sensor molecules would be found in the cytoplasm.[第一段]     

Plant phenolics regulate neoplastic cell growth and survival: a quantitative structure-activity and biochemical analysis

The anti-tumour activities of many plant phenolics at high concentrations (>100 micromol/L) suggest their potential use as dietary supplements in cancer chemoprevention and cancer chemotherapy. However, it is not clear what impact phenolic compounds have at the physiological concentrations obtained through consumption of high phenolic diets on neoplastic cells. In the present study, 54 naturally occurring phenolics were evaluated at physiologically relevant concentrations for their capacity to alter PC12 cell viability in response to serum deprivation, the chemotherepeutic agent etoposide, and the apoptogen C2-ceramide. Surprisingly, novel mitogenic, cytoprotective, and antiapoptotic activities were detected. Quantitative structure-activity relationship modelling indicated that many of these activities could be predicted by compound lipophilicity, steric bulk, and (or) antioxidant capacity, with the exception of inhibition of ceramide-induced apoptosis. Where quantitative structure-activity relationship analysis was insufficient, biochemical assessment demonstrated that the benzoate orsellinic acid blocked downstream caspase-12 activation following ceramide challenge. These findings demonstrate substantive mitogenic, cytoprotective, and antiapoptotic biological activities of plant phenolics on neoplastic cells at physiologically relevant dietary concentrations that should be considered in chemopreventive and chemotherapeutic strategies.