Calmodulin binding to the dehydrogenase domain of NADPH oxidase 5 alters its oligomeric state

Superoxide generated by NADPH Oxidase 5 (Nox5) is regulated by Ca2+ through the interaction of its self-contained Ca2+ binding domain and dehydrogenase domain (DH). Recently, calmodulin (CaM) has been reported to enhance the Ca2+ sensitivity of Nox5 by binding to the CaM-binding domain sequence (CMBD), in which the interaction between CaM and Nox5 is largely unclear. Here, we used the CMBD peptide and truncated DH constructs, and separately studied their interaction with CaM by fluorescence, calorimetry, and dynamic light scattering. Our results revealed that each half-domain of CaM binds one CMBD peptide with a binding constant near 106 M-1 and a binding enthalpy change of −3.81 kcal/mol, consistent with an extended 1:2 CaM:CMBD structure. However, the recombinant truncated DH proteins exist as oligomers, possibly trimer and tetramer. The oligomeric states are concentration and salt dependent. CaM binding appears to stabilize the DH dimer complexed with CaM. The thermodynamics of CaM binding to the DH is comparable to the peptide-based study except that the near unity binding stoichiometry and a large conformational change were observed. Our result suggests that the oligomeric states of Nox5, mediated by its DH domain and CaM, may be important for its superoxide-generating activity

3-Methyl-4-[(E)-3-thien­ylmethyl­idene­amino]-1H-1,2,4-triazole-5(4H)-thione

The asymmetric unit of the title compound, C8H8N4S2, contains two crystallographically independent mol­ecules. The thio­phene ring of one mol­ecule is disordered over two positions with refined site occupancies of 0.6375 (19) and 0.3625 (19). One mol­ecule is almost planar and the other one is twisted, the dihedral angles between the thio­phene and triazole rings being 7.28 (7) and 48.9 (2)° [48.5 (4)° for the minor component], respectively. An intra­molecular C—H⋯S hydrogen bond stabilizes the mol­ecular conformation of the planar molecule. In the crystal, the two mol­ecules are inter­connected by N—H⋯S hydrogen bonds into dimers, which are further consolidated into chains along the b axis by C—H⋯N hydrogen bonds. Weak C–H⋯π and π–π inter­actions [centroid–centroid distance = 3.5149 (7) Å] are also observed

4-Hy­droxy-3-[(4-hy­droxy-6,7-dimethyl-2-oxo-2H-chromen-3-yl)(4-oxo-4H-chromen-3-yl)meth­yl]-6,7-dimethyl-2H-chromen-2-one

In the title compound, C32H24O8, the mol­ecular structure is disordered over two positions with refined site occupancies of 0.8746 (10) and 0.1254 (10). The mean plane of the three chromeno rings make dihedral angles with each other of 65.12 (4), 62.91 (4) and 59.70 (4)° in the major occupancy component and 59.1 (3), 66.1 (3) and 58.8 (3)° in the minor component. Intra­molecular O—H⋯O hydrogen bonds stabil­ize the mol­ecular structure and the crystal structure is stabilized by weak C–H⋯π and π–π inter­actions [centroid–centroid distances 3.496 (6)–3.672 (7) Å]

Developing the Effective Method of Spectral Harmonic Energy Ratio to Analyze the Arterial Pulse Spectrum

In this article, we analyze the arterial pulse in the spectral domain. A parameter, the spectral harmonic energy ratio (SHER), is developed to assess the features of the overly decreased spectral energy in the fourth to sixth harmonic for palpitation patients. Compared with normal subjects, the statistical results reveal that the mean value of SHER in the patient group (57.7 ± 27.9) is significantly higher than that of the normal group (39.7 ± 20.9) (P-value = .0066 < .01). This means that the total energy in the fourth to sixth harmonic of palpitation patients is significantly less than it is in normal subjects. In other words, the spectral distribution of the arterial pulse gradually decreases for normal subjects while it decreases abruptly in higher-order harmonics (the fourth, fifth and sixth harmonics) for palpitation patients. Hence, SHER is an effective method to distinguish the two groups in the spectral domain. Also, we can thus know that a “gradual decrease” might mean a “balanced” state, whereas an “abrupt decrease” might mean an “unbalanced” state in blood circulation and pulse diagnosis. By SHER, we can determine the ratio of energy distribution in different harmonic bands, and this method gives us a novel viewpoint from which to comprehend and quantify the spectral harmonic distribution of circulation information conveyed by the arterial pulse. These concepts can be further applied to improve the clinical diagnosis not only in Western medicine but also in traditional Chinese medicine (TCM)

Removal of Acid Yellow 25 from Aqueous Solution by Chitin Prepared from Waste Snow Crab Legs

Acid Yellow 25 (AY25) is used in the textile industry for dyeing of natural and synthetic fibers, and is also used as a coloring agent in paints, inks, plastics, and leathers. Effluents from such industries are major sources of water pollution. Hence, it is important to find simple, efficient, and inexpensive ways to remove these dyes from wastewater. Here, we determined the suitability of chitin extracted from waste crab legs as an adsorbent for removing AY25 dye. The adsorption kinetics was modeled using pseudo-first order, pseudo-second order, and intraparticle diffusion equations to determine the rate controlling step. Results showed that the pseudo-second order adsorption mechanism is predominant, and the overall rate of the dye adsorption process is therefore controlled by an adsorption reaction. Adsorption isotherms were analyzed by utilizing the Langmuir, Freundlich, Dubinin-Radushkevich (D-R) and Temkin isotherm models at 23˚C, with data collected by using various initial dye concentrations with different chitin dosages. Our results show the highest correlation with the Langmuir model, consistent with the fact that chitin contains both a monolayer and homogeneous adsorption sites. Based on the D-R model, the adsorption of AY25 dye onto chitin is via chemisorption. Furthermore, we have concluded that the rate constants of both pseudo-second order adsorption and film diffusion are correlated to the initial dye concentrations and chitin dosages. In conclusion, chitin from waste crab legs is a very suitable adsorbent material that is capable of rapidly removing up to 95% of the initial concentration of AY25 dye at a pH of 2 and room temperature

The Arabidopsis Malectin-Like/LRR-RLK IOS1 is Critical for BAK1-Dependent and BAK1-Independent Pattern-Triggered Immunity

Plasma membrane-localized pattern recognition receptors (PRRs) such as FLAGELLIN SENSING2 (FLS2), EF-TU RECEPTOR (EFR) and CHITIN ELICITOR RECEPTOR KINASE 1 (CERK1) recognize microbe-associated molecular patterns (MAMPs) to activate pattern-triggered immunity (PTI). A reverse genetics approach on genes responsive to the priming agent beta-aminobutyric acid (BABA) revealed IMPAIRED OOMYCETE SUSCEPTIBILITY1 (IOS1) as a critical PTI player. Arabidopsis thaliana ios1 mutants were hyper-susceptible to Pseudomonas syringae bacteria. Accordingly, ios1 mutants showed defective PTI responses, notably delayed up-regulation of the PTI-marker gene FLG22-INDUCED RECEPTOR-LIKE KINASE1 (FRK1), reduced callose deposition and mitogen-activated protein kinase activation upon MAMP treatment. Moreover, Arabidopsis lines over-expressing IOS1 were more resistant to bacteria and showed a primed PTI response. In vitro pull-down, bimolecular fluorescence complementation, co-immunoprecipitation, and mass spectrometry analyses supported the existence of complexes between the membrane-localized IOS1 and BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1)-dependent PRRs FLS2 and EFR, as well as with the BAK1-independent PRR CERK1. IOS1 also associated with BAK1 in a ligand-independent manner, and positively regulated FLS2-BAK1 complex formation upon MAMP treatment. In addition, IOS1 was critical for chitin-mediated PTI. Finally, ios1 mutants were defective in BABA-induced resistance and priming. This work reveals IOS1 as a novel regulatory protein of FLS2-, EFR- and CERK1-mediated signaling pathways that primes PTI activation

The exchanged EF-hands in calmodulin and troponin C chimeras impair the Ca2+-induced hydrophobicity and alter the interaction with Orai1: a spectroscopic, thermodynamic and kinetic study

Background Calmodulin (CaM) plays an important role in Ca2+-dependent signal transduction. Ca2+ binding to CaM triggers a conformational change, forming a hydrophobic patch that is important for target protein recognition. CaM regulates a Ca2+-dependent inactivation process in store-operated Ca2+entry, by interacting Orai1. To understand the relationship between Ca2+-induced hydrophobicity and CaM/Orai interaction, chimera proteins constructed by exchanging EF-hands of CaM with those of Troponin C (TnC) are used as an informative probe to better understand the functionality of each EF-hand. Results ANS was used to assess the context of the induced hydrophobic surface on CaM and chimeras upon Ca2+ binding. The exchanged EF-hands from TnC to CaM resulted in reduced hydrophobicity compared with wild-type CaM. ANS lifetime measurements indicated that there are two types of ANS molecules with rather distinct fluorescence lifetimes, each specifically corresponding to one lobe of CaM or chimeras. Thermodynamic studies indicated the interaction between CaM and a 24-residue peptide corresponding to the CaM-binding domain of Orail1 (Orai-CMBD) is a 1:2 CaM/Orai-CMBD binding, in which each peptide binding yields a similar enthalpy change (ΔH = −5.02 ± 0.13 kcal/mol) and binding affinity (Ka = 8.92 ± 1.03 × 105 M−1). With the exchanged EF1 and EF2, the resulting chimeras noted as CaM(1TnC) and CaM(2TnC), displayed a two sequential binding mode with a one-order weaker binding affinity and lower ΔH than that of CaM, while CaM(3TnC) and CaM(4TnC) had similar binding thermodynamics as CaM. The dissociation rate constant for CaM/Orai-CMBD was determined to be 1.41 ± 0.08 s−1 by rapid kinetics. Stern-Volmer plots of Orai-CMBD Trp76 indicated that the residue is located in a very hydrophobic environment but becomes more solvent accessible when EF1 and EF2 were exchanged. Conclusions Using ANS dye to assess induced hydrophobicity showed that exchanging EFs for all Ca2+-bound chimeras impaired ANS fluorescence and/or binding affinity, consistent with general concepts about the inadequacy of hydrophobic exposure for chimeras. However, such ANS responses exhibited no correlation with the ability to interact with Orai-CMBD. Here, the model of 1:2 binding stoichiometry of CaM/Orai-CMBD established in solution supports the already published crystal structure

“Same same” but different? Exploring the impact of perceived organizational support at the school and teacher levels on teachers’ job engagement and organizational citizenship behavior

All countries in the world are currently trying to implement educational reform, which increases the additional workload of teachers. It is more important to discuss how to inspire teachers’ enthusiasm for educational reform from the perspective of organizational support (OS). Previous research on OS was limited to perceived organizational support (POS), but in recent years group-level OS has been considered the most promising. There is no study comparing POS and group-level OS in education, and therefore this study explored the relationships between OS, job engagement (JE) and organizational citizenship behavior (OCB) in an educational context. In particular, OS was examined at the individual-level (POS) and the aggregated group-level (school organizational support, SOS). Analysis was performed using structural equation modeling (SEM) at both single and multiple levels. SEM results showed direct and positive relationships of individual-level POS with both JE and OCB. Moreover, JE is directly and positively related to OCB and plays the partial mediating role of the indirect and positive impact of POS on OCB. Multilevel structural equation modeling (MSEM) analysis revealed direct and positive relationships of SOS with teachers’ JE, which was directly and positively related to their OCB. While SOS had no significant positive relationship with OCB, it did have a positive impact on OCB through the mediation of JE. Comparison between SEM and MSEM results revealed the change in effect of OS on OCB from significant to insignificant, thus implying full mediation effect of JE when SOS is considered