Expression, isolation and characterization of a mutated human plasminogen kringle 3 with a functional lysine binding site

Each kringle of human plasminogen (HPg) except kringle 3 (K3) exhibits affinity for omega-aminocarboxylic acids. Assuming that the K3 domain contains a preformed but nonfunctional lysine binding site (LBS), Lys311 was altered by site-directed mutagenesis into Asp311 in accordance with the consensus sequence of the LBS. Cys297 involved in the interkringle disulfide bridge was mutated into Ser297 to minimize dimerization and aggregation. The mutated K3 TYQ[K3HPg/C297S/K311D]DS (r-K3mut) was expressed in Escherichia coli, isolated on an Ni2(+)-nitrilotriacetic acid-agarose column, refolded and purified on a lysine Bio-Gel column. Fluorescence titration indicates affinity of r-K3mut for omega-aminocarboxylic acids with the following association constants (Kass, mM-1): 5-aminopentanoic acid: 1.3; 6-aminohexanoic acid: 4.2; 7-aminoheptanoic acid: 0.5; trans-(aminomethyl)cyclohexanecarboxylic acid: 12.7; p-benzylaminesulfonic acid: 11.8. r-K3mut exhibits an affinity similar to native and mutated (R220G, E221D) K2. The results indicate the presence of a preformed but nonfunctional LBS in native K3 of HPg. We were able to demonstrate for the first time that an appropriate mutation in the LBS of a kringle produced a weak but distinct affinity for omega-aminocarboxylic acids.     

Gene–ethanol interactions underlying fetal alcohol spectrum disorders

Fetal alcohol spectrum disorders (FASD) is an umbrella term that describes a diverse set of ethanol-induced defects. The phenotypic variation is generated by numerous factors, including timing and dosage of ethanol exposure as well as genetic background. We are beginning to learn about how the concentration, duration, and timing of ethanol exposure mediate variability within ethanol teratogenesis. However, little is known about the genetic susceptibilities in FASD. Studies of FASD animal models are beginning to implicate a number of susceptibility genes that are involved in various pathways. Here we review the current literature that focuses on the genetic predispositions in FASD.     

Optical nano-imaging of gate-tunable graphene plasmons

The ability to manipulate optical fields and the energy flow of light is central to modern information and communication technologies, as well as quantum information processing schemes. However, because photons do not possess charge, a way of controlling them efficiently by electrical means has so far proved elusive. A promising way to achieve electric control of light could be through plasmon polaritons—coupled excitations of photons and charge carriers—in graphene. In this two-dimensional sheet of carbon atoms, it is expected that plasmon polaritons and their associated optical fields can readily be tuned electrically by varying the graphene carrier density. Although evidence of optical graphene plasmon resonances has recently been obtained spectroscopically, no experiments so far have directly resolved propagating plasmons in real space. Here we launch and detect propagating optical plasmons in tapered graphene nanostructures using near-field scattering microscopy with infrared excitation light. We provide real-space images of plasmon fields, and find that the extracted plasmon wavelength is very short—more than 40 times smaller than the wavelength of illumination. We exploit this strong optical field confinement to turn a graphene nanostructure into a tunable resonant plasmonic cavity with extremely small mode volume. The cavity resonance is controlled in situ by gating the graphene, and in particular, complete switching on and off of the plasmon modes is demonstrated, thus paving the way towards graphene-based optical transistors. This successful alliance between nanoelectronics and nano-optics enables the development of active subwavelength-scale optics and a plethora of nano-optoelectronic devices and functionalities, such as tunable metamaterials, nanoscale optical processing, and strongly enhanced light–matter interactions for quantum devices and biosensing applications.     

Genome-wide association analysis identifies three psoriasis susceptibility loci

We carried out a meta-analysis of two recent psoriasis genome-wide association studies with a combined discovery sample of 1,831 affected individuals (cases) and 2,546 controls. One hundred and two loci selected based on P value rankings were followed up in a three-stage replication study including 4,064 cases and 4,685 controls from Michigan, Toronto, Newfoundland and Germany. In the combined meta-analysis, we identified three new susceptibility loci, including one at NOS2 (rs4795067, combined P = 4 × 10?11), one at FBXL19 (rs10782001, combined P = 9 × 10?1?) and one near PSMA6-NFKBIA (rs12586317, combined P = 2 × 10??). All three loci were also associated with psoriatic arthritis (rs4795067, combined P = 1 × 10??; rs10782001, combined P = 4 × 10??; and rs12586317, combined P = 6 × 1??) and purely cutaneous psoriasis (rs4795067, combined P = 1 × 10??; rs10782001, combined P = 2 × 10??; and rs12586317, combined P = 1 × 10??). We also replicated a recently identified association signal near RNF114 (rs495337, combined P = 2 × 10??).     

Reduction of disulphide bonds unmasks potent antimicrobial activity of human β-defensin 1

Human epithelia are permanently challenged by bacteria and fungi, including commensal and pathogenic microbiota. In the gut, the fraction of strict anaerobes increases from proximal to distal, reaching 99% of bacterial species in the colon. At colonic mucosa, oxygen partial pressure is below 25% of airborne oxygen content, moreover microbial metabolism causes reduction to a low redox potential of -200?mV to -300?mV in the colon. Defensins, characterized by three intramolecular disulphide-bridges, are key effector molecules of innate immunity that protect the host from infectious microbes and shape the composition of microbiota at mucosal surfaces. Human β-defensin 1 (hBD-1) is one of the most prominent peptides of its class but despite ubiquitous expression by all human epithelia, comparison with other defensins suggested only minor antibiotic killing activity. Whereas much is known about the activity of antimicrobial peptides in aerobic environments, data about reducing environments are limited. Herein we show that after reduction of disulphide-bridges hBD-1 becomes a potent antimicrobial peptide against the opportunistic pathogenic fungus Candida albicans and against anaerobic, Gram-positive commensals of Bifidobacterium and Lactobacillus species. Reduced hBD-1 differs structurally from oxidized hBD-1 and free cysteines in the carboxy terminus seem important for the bactericidal effect. In vitro, the thioredoxin (TRX) system is able to reduce hBD-1 and TRX co-localizes with reduced hBD-1 in human epithelia. Hence our study indicates that reduced hBD-1 shields the healthy epithelium against colonisation by commensal bacteria and opportunistic fungi. Accordingly, an intimate interplay between redox-regulation and innate immune defence seems crucial for an effective barrier protecting human epithelia.     

The plasmin–antiplasmin system: structural and functional aspects

The plasmin–antiplasmin system plays a key role in blood coagulation and fibrinolysis. Plasmin and α₂-antiplasmin are primarily responsible for a controlled and regulated dissolution of the fibrin polymers into soluble fragments. However, besides plasmin(ogen) and α₂-antiplasmin the system contains a series of specific activators and inhibitors. The main physiological activators of plasminogen are tissue-type plasminogen activator, which is mainly involved in the dissolution of the fibrin polymers by plasmin, and urokinase-type plasminogen activator, which is primarily responsible for the generation of plasmin activity in the intercellular space. Both activators are multidomain serine proteases. Besides the main physiological inhibitor α₂-antiplasmin, the plasmin–antiplasmin system is also regulated by the general protease inhibitor α₂-macroglobulin, a member of the protease inhibitor I39 family. The activity of the plasminogen activators is primarily regulated by the plasminogen activator inhibitors 1 and 2, members of the serine protease inhibitor superfamily.