Redox-Regulation of α-Globin in Vascular Physiology

Interest in the structure, function, and evolutionary relations of circulating and intracellular globins dates back more than 60 years to the first determination of the three-dimensional structure of these proteins. Non-erythrocytic globins have been implicated in circulatory control through reactions that couple nitric oxide (NO) signaling with cellular oxygen availability and redox status. Small artery endothelial cells (ECs) express free α-globin, which causes vasoconstriction by degrading NO. This reaction converts reduced (Fe2+) α-globin to the oxidized (Fe3+) form, which is unstable, cytotoxic, and unable to degrade NO. Therefore, (Fe3+) α-globin must be stabilized and recycled to (Fe2+) α-globin to reinitiate the catalytic cycle. The molecular chaperone α-hemoglobin-stabilizing protein (AHSP) binds (Fe3+) α-globin to inhibit its degradation and facilitate its reduction. The mechanisms that reduce (Fe3+) α-globin in ECs are unknown, although endothelial nitric oxide synthase (eNOS) and cytochrome b5 reductase (CyB5R3) with cytochrome b5 type A (CyB5a) can reduce (Fe3+) α-globin in solution. Here, we examine the expression and cellular localization of eNOS, CyB5a, and CyB5R3 in mouse arterial ECs and show that α-globin can be reduced by either of two independent redox systems, CyB5R3/CyB5a and eNOS. Together, our findings provide new insights into the regulation of blood vessel contractility.

Sulfonated poly(arylene ether nitrile)-based composite membranes enhanced with Ca2+ bridged carbon nanotube-graphene oxide networks

As the core component of proton exchange membrane fuel cell, proton exchange membranes (PEM) have attracted much attention of researchers. To trade-off the proton conduction, dimensional stability and anti-oxidation ability, graphene oxide (GO) and acidized multi-walled carbon nanotubes (MWCNT) using calcium ion as coordination bridge (GO-Ca2+-MWCNT) was synthesized, and then incorporating into sulfonated poly(arylene ether nitrile) (SPEN) to fabricate SPEN/GO-Ca2+-MWCNT organic-inorganic composite membranes by solution-casting method and explore the influence of varying loading on performances as PEM. It was found that the proton conductivity of the composite membranes was higher than that of SPEN, while maintaining better dimensional stability, excellent anti-oxidation ability and good mechanical properties. All of these were attributed to the formation of three-dimensional structure between GO and MWCNT bridged by Ca2+. Particularly, the SPEN/GO-Ca2+-MWCNT-1 composite membrane exhibited excellent tensile strength of 71.45 MPa, better thermal stability as well as high proton conductivity (0.054 S/cm at 30 ℃, and 0.193 S/cm at 90 ℃), above 10-2 S/cm, satisfying the requirement of fuel cells. All in all, the results indicate that the filler with three-dimensional network structure can effectively improve the performances of SPEN, and the prepared composite membranes show potential applications in many fields.

Genome-Wide Identification of JRL Genes in Moso Bamboo and Their Expression Profiles in Response to Multiple Hormones and Abiotic Stresses

Jacalin-related lectins (JRLs) are a new subfamily of plant lectins that has recently been recognized and plays an important role in plant growth, development, and abiotic stress response. Although moso bamboo (Phyllostachys edulis) is an economically and industrially important bamboo worldwide, there has been no systematic identification of JRLs in this species. Here, we identified 25 JRL genes in moso bamboo, and these genes are unequally distributed among 10 genome scaffolds. Phylogenetic analysis showed that the moso bamboo JRLs were clustered into four JRL subgroups: I, II, V, and VII. Numerous stress-responsive and hormone-regulated cis-elements were detected in the upstream promoter regions of the JRLs. Genome collinearity analyses showed that the JRL genes of moso bamboo are more closely related to those of Brachypodium distachyon than to those of Oryza sativa and Zea mays. Sixty-four percent of the PeJRL genes are present as segmental and tandem duplicates. qRT-PCR expression analysis showed that JRL genes in the same subgroup were significantly downregulated in response to salicylic acid (SA), abscisic acid (ABA), and methyl jasmonate (MeJA) treatments and significantly upregulated under low temperature, drought, and salt stress; they also exhibited tissue-specific expression patterns. Subcellular localization experiments revealed that PeJRL04 and PeJRL13 were localized to the cell membrane, nucleus, and cytoplasm. Three dimensional structure prediction and yeast two-hybrid assays were used to verify that PeJRL13 exists as a self-interacting homodimer in vivo. These findings provide an important reference for understanding the functions of specific moso bamboo JRL genes and for the effective selection of stress-related genes.

Structure of the Potential Virulence Factor from Francisella tularensis Shows Unexpected Presence of the SHS2 Motif and Similarity to Other Bacterial Virulence Factors

Pathogenic bacteria attack their host by secreting virulence factors that in various ways interrupt host defenses and damage their cells. Functions of many virulence factors, even from well-studied pathogens, are still unknown. Francisella tularensis is a class A pathogen and a causative agent of tularemia, a disease that is lethal without proper treatment. Here we report the three-dimensional structure and preliminary analysis of the potential virulence factor identified by the transcriptomic analysis of the F. tularensis disease models that is encoded by the FTT_1539 gene. The structure of the FTT_1539 protein contains two sets of three stranded antiparallel beta sheets, with a helix placed between the first and the second beta strand in each sheet. This structural motif, previously seen in virulence factors from other pathogens, was named the SHS2 motif and identified to play a role in protein-protein interactions and small molecule recognition. Sequence and structure analysis identified FTT_1539 as a member of a large family of secreted proteins from a broad range of pathogenic bacteria, such as Helicobacter pylori and Mycobacterium tuberculosis. While the specific function of the proteins from this class is still unknown, their similarity to the H. pylori Tip-α protein that induces TNF-a and other chemokines through NF-kB activation suggests the existence of a common pathogen-host interference mechanism shared by multiple human pathogens.

The Employee Spirituality Scale as a Measure of Employees’ Spirituality

The aim of the study was to assess the psychometric properties of the employee spirituality scale. The employee spirituality scale was found to be a reliable measure with good internal consistency. The internal consistency of this instrument, measured with Cronbach’s α coefficient, was 0.94. Factor analysis confirmed the two-dimensional structure of this measure, the dimensions being: relationship to a Higher Power (God) and attitude towards workmates and employer. A statistically significant positive correlation was found between employee spirituality and job satisfaction, as well as age, and a negative correlation was found with their intent to leave their organization. According to expectation, the relationship to a Higher Power (God) as a religious dimension of employee spirituality was strong, positively related to religious practices and attitude towards workmates and employer, and a secular dimension of employee spirituality did not correlate with religious measures. Gender did not differentiate participants in terms of employee spirituality. The presented results provide evidence that the employee spirituality scale has good psychometric properties and is therefore recommended for use by researchers studying employee spirituality in Polish organizations.

Validation of the Orgasm Rating Scale in Context of Sexual Relationships of Gay and Lesbian Adults

Currently, no validated instrument exists for assessing the subjective experience of orgasm in the gay population. The Orgasm Rating Scale (ORS), previously validated in the heterosexual population, comprises four dimensions: Affective, Sensory, Intimacy, and Rewards. This study validated it for sexual relationships in the gay population by obtaining its factorial invariance by sexual orientation and sex, its internal consistency reliability, and evidence of validity in its relationship with other variables. We assessed 1600 cisgender Spanish adults–heterosexuals, gays, and lesbians–divided into 4, sex-based groups of 400 each, according to the Kinsey scale scores. Participants reported recent experiences of orgasm in the context of sexual relationships and responded to the ORS and other scales assessing attitude toward sexual fantasies and sexual functioning. The ORS structure showed a strict multigroup-level invariance by sexual orientation and sex, confirming its four-dimensional structure. The subjective orgasm intensity was associated with a positive attitude toward sexual fantasies and sexual functioning. Scores obtained on the Affective, Intimacy, and Rewards dimensions confirmed the ability to discriminate between gay people with and without orgasmic difficulties. The ORS’s Spanish version presents good psychometric properties as a validated scale to evaluate the subjective experience of orgasm in the gay population.

Cloning and Characterization of a Novel Alginate Lyase from Paenibacillus sp. LJ-23

As a low molecular weight alginate, alginate oligosaccharides (AOS) exhibit improved water solubility, better bioavailability, and comprehensive health benefits. In addition, their biocompatibility, biodegradability, non-toxicity, non-immunogenicity, and gelling capability make them an excellent biomaterial with a dual curative effect when applied in a drug delivery system. In this paper, a novel alginate lyase, Algpt, was cloned and characterized from a marine bacterium, Paenibacillus sp. LJ-23. The purified enzyme was composed of 387 amino acid residues, and had a molecular weight of 42.8 kDa. The optimal pH of Algpt was 7.0 and the optimal temperature was 45 °C. The analysis of the conserved domain and the prediction of the three-dimensional structure indicated that Algpt was a novel alginate lyase. The dominant degradation products of Algpt on alginate were AOS dimer to octamer, depending on the incubation time, which demonstrated that Algpt degraded alginate in an endolytic manner. In addition, Algpt was a salt-independent and thermo-tolerant alginate lyase. Its high stability and wide adaptability endow Algpt with great application potential for the efficient preparation of AOS with different sizes and AOS-based products.

Proprioceptive sensitivity to imposed finger deflections

Hand proprioception - the sense of the posture and movements of the wrist and digits - is critical to dexterous manual behavior and to stereognosis, the ability to sense the three-dimensional structure of objects held in the hand. To better understand this sensory modality and its role in hand function, we sought to characterize the acuity with which the postures and movements of finger joints are sensed. To this end, we measured the ability of human subjects to discriminate changes in posture and speed around the three joints of the index finger. In these experiments, we isolated the sensory component by imposing the postures on an otherwise still hand, to complement other studies, in which subjects made judgments on actively achieved postures. We found that subjects could reliably sense 12-16% changes in joint angle and 18-32% changes in joint speed. Furthermore, the acuity for posture and speed was comparable across the three joints of the finger. Finally, task performance was unaffected by the presence of a vibratory stimulus, calling into question the role of cutaneous cues in hand proprioception.

Topoisomerase I (TOP1) dynamics: conformational transition from open to closed states

Eukaryotic topoisomerases I (TOP1) are ubiquitous enzymes removing DNA torsional stress. However, there is little data concerning the three-dimensional structure of TOP1 in the absence of DNA, nor how the DNA molecule can enter/exit its closed conformation. Here, we solved the structure of thermostable archaeal Caldiarchaeum subterraneum CsTOP1 in an apo-form. The enzyme displays an open conformation resulting from one substantial rotation between the capping (CAP) and the catalytic (CAT) modules. The junction between these two modules is a five-residue loop, the hinge, whose flexibility permits the opening/closing of the enzyme and the entry of DNA. We identified a highly conserved tyrosine near the hinge as mediating the transition from the open to closed conformation upon DNA binding. Directed mutagenesis confirmed the importance of the hinge flexibility, and linked the enzyme dynamics with sensitivity to camptothecin, a TOP1 inhibitor targeting the TOP1 enzyme catalytic site in the closed conformation.

Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate

NMR-assisted crystallography—the integrated application of solid-state NMR, X-ray crystallography, and first-principles computational chemistry—holds significant promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in enzyme active sites, including hydrogen atom locations and tautomeric equilibria, NMR crystallography offers insight into both structure and chemical dynamics. Here, this integrated approach is used to characterize the tryptophan synthase α-aminoacrylate intermediate, a defining species for pyridoxal-5′-phosphate–dependent enzymes that catalyze β-elimination and replacement reactions. For this intermediate, NMR-assisted crystallography is able to identify the protonation states of the ionizable sites on the cofactor, substrate, and catalytic side chains as well as the location and orientation of crystallographic waters within the active site. Most notable is the water molecule immediately adjacent to the substrate β-carbon, which serves as a hydrogen bond donor to the ε-amino group of the acid–base catalytic residue βLys87. From this analysis, a detailed three-dimensional picture of structure and reactivity emerges, highlighting the fate of the L-serine hydroxyl leaving group and the reaction pathway back to the preceding transition state. Reaction of the α-aminoacrylate intermediate with benzimidazole, an isostere of the natural substrate indole, shows benzimidazole bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. When modeled into the benzimidazole position, indole is positioned with C3 in contact with the α-aminoacrylate Cβ and aligned for nucleophilic attack. Here, the chemically detailed, three-dimensional structure from NMR-assisted crystallography is key to understanding why benzimidazole does not react, while indole does.