Addressing single molecules of a thin magnetic film

Electron spin noise-scanning tunneling microscopy (ESN-STM) represents the most promising technique for single spin centres addressing on surfaces. After a brief introduction on the results previously obtained by using this technique for the detection of both isolated molecules and small aggregates of BDPA, DPPH and TTM free radicals, we discuss here our improved results in the single-molecule addressing of thio-functionalized nitronyl nitroxide radicals (NNRs) chemically bound to gold. ESN-STM spectra of NNRs self-assembled monolayers (SAMs) on Au(1 1 1) surfaces are reported together with the crystal structure of the studied radical NN–Ph–CH₂SMe, considered up to now as the best aromatic NNR for the obtainment of thin magnetic films.     

Comparative analyses of a small molecule/enzyme interaction by multiple users of Biacore technology

To gauge the experimental variability associated with Biacore analysis, 36 different investigators analyzed a small molecule/enzyme interaction under similar conditions. Acetazolamide (222 g/mol) binding to carbonic anhydrase II (CAII; 30,000 Da) was chosen as a model system. Both reagents were stable and their interaction posed a challenge to measure because of the low molecular weight of the analyte and the fast association rate constant. Each investigator created three different density surfaces of CAII and analyzed an identical dilution series of acetazolamide (ranging from 4.1 to 1000 nM). The greatest variability in the results was observed during the enzyme immobilization step since each investigator provided their own surface activating reagents. Variability in the quality of the acetazolamide binding responses was likely a product of how well the investigators’ instruments had been maintained. To determine the reaction kinetics, the responses from the different density surfaces were fit globally to a 1:1 interaction model that included a term for mass transport. The averaged association and dissociation rate constants were 3.1 ± 1.6 × 10⁶ M⁻¹ s⁻¹ and 6.7 ± 2.5 × 10⁻² s⁻¹, respectively, which corresponded to an average equilibrium dissociation constant (K_D) of 2.6 ± 1.4 × 10⁻⁸ M. The results provide a benchmark of variability in interpreting binding constants from the biosensor and highlight keys areas that should be considered when analyzing small molecule interactions.     

Crystal structures of a type-1 ribosome inactivating protein from Momordica balsamina in the bound and unbound states

The ribosome inactivating proteins (RIPs) of type 1 are plant toxins that eliminate adenine base selectively from the single stranded loop of rRNA. We report six crystal structures, type 1 RIP from Momordica balsamina (A), three in complexed states with ribose (B), guanine (C) and adenine (D) and two structures of MbRIP-1 when crystallized with adenosine triphosphate (ATP) (E) and 2′-deoxyadenosine triphosphate (2′-dATP) (F). These were determined at 1.67 Å, 1.60 Å, 2.20 Å, 1.70 Å, 2.07 Å and 1.90 Å resolutions respectively. The structures contained, (A) unbound protein molecule, (B) one protein molecule and one ribose sugar, (C) one protein molecule and one guanine base, (D) one protein molecule and one adenine base, (E) one protein molecule and one ATP-product adenine molecule and (F) one protein molecule and one 2′-dATP-product adenine molecule. Three distinct conformations of the side chain of Tyr70 were observed with (i) χ¹ = − 66°and χ² = 165° in structures (A) and (B); (ii) χ¹ = − 95° and χ² = 70° in structures (C), (D) and (E); and (iii) χ¹ = − 163° and χ² = 87° in structure (F). The conformation of Tyr70 in (F) corresponds to the structure of a conformational intermediate. This is the first structure which demonstrates that the slow conversion of DNA substrates by RIPs can be trapped during crystallization.     

Dye-sensitized photocurrents and adsorption properties of merocyanine dye at atomically flat rutile (1 1 0) and (1 0 0) TiO2 surfaces

The crystal-face dependence of the dye-sensitized photocurrents and the adsorption properties of benzothiazole merocyanine (Mc[18,1]) dye molecules were investigated, using atomically flat (1 0 0) and (1 1 0) TiO₂ single crystal surfaces. From the estimation of the amount of the transferred charge from the TiO₂ surface to CO groups of dye molecules based on NEXAFS data, it was revealed that the interaction of the adsorbed molecules and the (1 1 0) surface was much stronger than that for the (1 0 0) surface. On the other hand, the absorbed photon to current conversion efficiency (APCE) value was almost the same for both surfaces. We suggested a possible explanation as follows: the energy difference between the LUMO of Mc[18,1] and the conduction band of TiO₂ was large enough to give a nearly 100% quantum efficiency of electron transfer from photoexcited dye to TiO₂, which made the difference in the interaction between dye molecules and TiO₂ not apparent. The incident photon to current conversion efficiency (IPCE) for the (1 0 0) surface was much larger than that for the (1 1 0) surface, which was explained by the fact that the amount of the adsorbed dye molecules on the (1 0 0) surface was larger than on the (1 1 0) surface, probably due to the larger surface density of five-coordinated Ti sites in the former surface.     

Nitroxidative chemistry interferes with fluorescent probe chemistry: Implications for nitric oxide detection using 2,3-diaminonaphthalene

Simultaneous production of nitric oxide (NO) and superoxide generates peroxynitrite and causes nitroxidative stress. The fluorometric method for NO detection is based on the formation of a fluorescent product from the reaction of a nonfluorescent probe molecule with NO-derived nitrosating species. Here, we present an example of how nitroxidative chemistry could interact with fluorescent probe chemistry. 2,3-Naphthotriazole (NAT) is the NO-derived fluorescent product of 2,3-diaminonaphthalene (DAN), a commonly used NO-detecting molecule. We show that NO/superoxide cogeneration, and particularly peroxynitrite, mediates the chemical decomposition of NAT. Moreover, the extent of NAT decomposition depends on the relative fluxes of NO and superoxide; the maximum effect being reached at almost equivalent generation rates for both radicals. The rate constant for the reaction of NAT with peroxynitrite was determined to be 2.2 × 10³ M⁻¹ s⁻¹. Further, various peroxynitrite scavengers were shown to effectively inhibit NO/superoxide- and peroxynitrite-mediated decomposition of NAT. Taken together, the present study suggests that the interference of a fluorometric NO assay can be originated from the interaction between the final fluorescent product and the formed reactive nitrogen and oxygen species.     

Galactomannan thin films as supports for the immobilization of Concanavalin A and/or dengue viruses

The immobilization of the glucose/mannose-binding lectin from Concanavalia ensiformis seeds (ConA) onto a monolayer made of a galactomannan extracted from Leucaena leucocephala seeds (GML), which was adsorbed onto - amino-terminated surfaces, was investigated by means of ellipsometry and atomic force microscopy. The mean thickness of GML monolayer, which polysaccharide consists of linear 1 → 4-linked β-d-mannopyranosil units partially substituted at C-6 by α-d-galactopyranosyl units, amounted to (1.5 ± 0.2) nm. ConA molecules adsorbed onto GML surfaces forming (2.0 ± 0.5) nm thick layers. However, in the presence of mannose the adsorption failed, indicating that ConA binding sites were blocked by mannose and were no longer available for mannose units present in the GML backbone. The GML film was also used as support for the adsorption of three serotypes of dengue virus particles (DENV-1, DENV-2 and DENV-3), where DENV-2 formed the thickest film (4 ± 2) nm. The adsorbed layer of DENV-2 onto ConA-covered GML surfaces presented mean thickness values similar to that determined for DENV-2 onto bare GML surfaces. The addition of free mannose units prevented DENV-2 adsorption onto ConA-covered GML films by ∼50%, suggesting competition between virus and mannose for ConA binding sites. This finding suggests that if ConA is also adsorbed to GML surface and its binding site is blocked by free mannose, virus particles are able to recognized GML mannose unities substituted by galactose. Interactions between polysaccharides thin films, proteins, and viruses are of great relevance since they can provide basis for the development of biotechnological devices. These results indicate that GML is a potential polysaccharide for biomaterials development, as those could involve interactions between ConA in immune system and viruses.     

Interleukin-1β regulates cell proliferation and activity of extracellular matrix remodelling enzymes in cultured primary pig heart cells

After myocardial infarction, elevated levels of interleukins (ILs) are found within the myocardial tissue and IL-1β is considered to play a major role in tissue remodelling events throughout the body. In the study presented, we have established a cell culture model of primary pig heart cells to evaluate the effects of different concentrations of IL-1β on cell proliferation as well as expression and activity of enzymes typically involved in tissue remodelling.Primary pig heart cell cultures were derived from three different animals and stimulated with recombinant pig IL-1β. RNA expression was detected by RT-PCR, protein levels were evaluated by Western blotting, activity of matrix metalloproteinases (MMPs) was quantified by gelatine zymography and cell proliferation was measured using colorimetric MTS assays.Pig heart cells express receptors for IL-1 and application of IL-1β resulted in a dose-dependent increase of cell proliferation (P < 0.05 vs. control; 100 ng/ml; 24 h). Gene expression of caspase-3 was increased by IL-1β (P < 0.05 vs. control; 100 ng/ml; 3 h), and pro-caspase-3 but not active caspase was detected in lysates of pig heart cells by Western blotting. MMP-2 gene expression as well as enzymatic activities of MMP-2 and MMP-9 were increased by IL-1β (P < 0.05 vs. control; 100 ng/ml; 3 h for gene expression, 48 and 72 h for enzymatic activities of MMP-2 and MMP-9, respectively).Our in vitro data suggest that IL-1β plays a major role in the events of tissue remodelling in the heart. Combined with our recently published in vivo data (Meybohm et al., PLoS One, 2009), the results presented here strongly suggest IL-1β as a key molecule guiding tissue remodelling events after myocardial infarction.     

Monitoring the redox and protonation dependent contributions of cardiolipin in electrochemically induced FTIR difference spectra of the cytochrome bc1 complex from yeast

Biochemical studies have shown that cardiolipin is essential for the integrity and activity of the cytochrome bc₁ complex and many other membrane proteins. Recently the direct involvement of a bound cardiolipin molecule (CL) for proton uptake at center N, the site of quinone reduction, was suggested on the basis of a crystallographic study. In the study presented here, we probe the low frequency infrared spectroscopy region as a technique suitable to detect the involvement of the lipids in redox induced reactions of the protein. First the individual infrared spectroscopic features of lipids, typically present in the yeast membrane, have been monitored for different pH values in micelles and vesicles. The pK_a values for cardiolipin molecule have been observed at 4.7 ± 0.3 and 7.9 ± 1.3, respectively. Lipid contributions in the electrochemically induced FTIR spectra of the bc₁ complex from yeast have been identified by comparing the spectra of the as isolated form, with samples where the lipids were digested by lipase-A₂. Overall, a noteworthy perturbation in the spectral region typical for the protein backbone can be reported. Interestingly, signals at 1159, 1113, 1039 and 980 cm^− 1 have shifted, indicating the perturbation of the protonation state of cardiolipin coupled to the reduction of the hemes. Additional shifts are found and are proposed to reflect lipids reorganizing due to a change in their direct environment upon the redox reaction of the hemes. In addition a small shift in the alpha band from 559 to 556 nm can be seen after lipid depletion, reflecting the interaction with heme b_H and heme c. Thus, our work highlights the role of lipids in enzyme reactivity and structure.     

Nano reengineering of horseradish peroxidase with dendritic macromolecules for stability enhancement

A simple bio-conjugation procedure to surround a single horseradish peroxidase (HRP) enzyme molecule with dendritic polyester macromolecules (polyester-32-hydroxyl-1-carboxyl bis-MPA dendron, generation 5) was proposed. The characterization of resultant nanoparticles entitled HRP dendrozyme, was performed by transmission electron microscopy, dynamic light scattering, gel permeation chromatography and Fourier transform infrared spectroscopy. The results showed that HRP nanoparticles were spherical in shape and have an average size of 14 ± 2 nm in diameter. Furthermore, bio-conformational characterization of HRP dendrozyme was performed by means of circular dichroism and fluorescence spectroscopy to evaluate the secondary and tertiary structure changes after enzyme modification. These investigations revealed that protein conformation had small changes (in secondary and tertiary structures) after bio-conjugation. We also reported here that dendritic modification did not significantly affect the kinetic parameters of free HRP. The stabilization of HRP with dendron macromolecules as single enzyme nanoparticles resulted in improvement of half-life over 70 days storage at 4 °C as well as its tolerance under different elevated temperatures up to 80 °C and in the presence of organic solvents for 15 min. These significant results promise extensive applications of HRP particularly in harsh environmental conditions.     

Ovalbumin labeling with p-hydroxymercurybenzoate: The effect of different denaturing agents and the kinetics of reaction

The aim of our study was to investigate how denaturing agents commonly used in protein analysis influence the labeling between a reactive molecule and proteins. For this reason, we investigated the labeling of ovalbumin (OVA) as a globular model protein with p-hydroxymercurybenzoate (pHMB) in its native state (phosphate buffer solution) and in different denaturing conditions (8 mol L⁻¹ urea, 3 mol L⁻¹ guanidinium thiocyanate, 6 mol L⁻¹ guanidinium chloride, 0.2% sodium dodecyl sulfate, and 20% methanol). In addition to chemical denaturation, thermal denaturation was also tested. The protein was pre-column simultaneously denatured and derivatized, and the pHMB-labeled denatured OVA complexes were analyzed by size exclusion chromatography (SEC) coupled online with chemical vapor generation–atomic fluorescence spectrometry (CVG–AFS). The number of –SH groups titrated greatly depends on the protein structure in solution. Indeed, we found that, depending on the adopted denaturing conditions, OVA gave different aggregate species that influence the complexation process. The results were compared with those obtained by a common alternative procedure for the titration of –SH groups that employs monobromobimane (mBBr) as tagging molecule and molecular fluorescence spectroscopy as detection technique.     

Spectral characteristic of fluorescence induction in a model cyanobacterium, Synechococcus sp. (PCC 7942)

We present here three-dimensional time-wavelength-intensity displays of changes in variable fluorescence, during the O(JI)PSMT transient, observed in cyanobacterium at room temperature. We were able to measure contributions of individual chromophores to fluorescence spectra at various times of fluorescence induction (FI). The method was applied to a freshwater cyanobacterium, Synechococcus sp. (PCC 7942). Analysis of our experimental results provides the following new conclusions: (i) the main chlorophyll (Chl) a emission band at ∼ 685 nm that originates in Photosystem (PS) II exhibits typical fast (OPS) and slow (SMT) FI kinetics with both orange (622 nm) and blue (464 nm) excitation. (ii) Similar kinetics are exhibited for its far-red emission satellite band centered at ∼ 745 nm, where the PS II contribution predominates. (iii) A significant OPS-SMT-type kinetics of C-phycocyanin emission at ∼ 650 nm are observed with the blue light excitation, but not with orange light excitation where the signal rose only slightly to a maximum. The induction of F650 was not caused by an admixture of the F685 fluorescence and thus our data show light-inducible and dark-reversible changes of phycobilin fluorescence in vivo. We discuss possible interpretations of this new observation.     

Induction of metamorphosis in the Asian shore crab Hemigrapsus sanguineus: Characterization of the cue associated with biofilm from adult habitat

Recruitment of crabs to nursery habitat requires settlement of the megalopal stage on suitable substratum followed by metamorphosis into the first juvenile stage. Reducing the time to metamorphosis may result in higher recruitment and survival. Previous work has shown that metamorphosis of the Asian shore crab is accelerated by cues from three different sources: (a) water-soluble exudate produced by conspecific adult crabs; (b) biofilm covering rocks in natural habitat for this species; and (c) abiotic rock from natural habitat. The objective of the present investigation was to characterize the metamorphic cue associated with biofilm from rocky intertidal habitat and to compare the three metamorphic cues (exudate from conspecific adults, biofilm from rocky intertidal, and texture of substratum) that have been identified for H. sanguineus. Results of our study show that megalopae of the Asian shore crab respond strongly to biofilm associated with rocky intertidal habitat that has developed for at least 8 days. We also found that megalopae respond to textured rock surfaces from natural habitat, even when those surfaces had been rendered abiotic. The cue remains active after the biofilm has been exposed to − 20 ºC for 12 h, but is de-activated by a few minutes exposure to 100 °C. Moreover, the biofilm cue appears to work in synergy with cues from other sources, but requires actual contact with the biofilm. Our findings show that addition of biofilm to an abiotic textured rock surface significantly decreases mean time to metamorphosis, and simultaneous exposure of megalopae to biofilm-covered rock and to exudate from adult H. sanguineus decreases mean time to metamorphosis even further. The response of this species to multiple cues—and particularly to biofilm in the absence of adult conspecifics—provides a clear advantage in the colonization of virgin habitat and helps explain the very rapid spread of this invasive species along the majority of the east coast of the United States in only two decades.     

Thermal treatment enhances the stability and biological activity of a truncated tilapia somatotropin contained in Pichia pastoris culture supernatant

The importance of somatotropin as a growth promoting agent and immune-stimulator has long been recognized and its potential application in the fish farming industry has been an active research area. In the work reported here, we sought to improve the stability of a previously obtained truncated somatotropin by applying a 60 °C heat shock to the culture supernatant containing this molecule, and then compared its effects with and without heat shock on larval growth and immune functions. We observed that the treatment with heat shock at 60 °C enhanced protein stability, growth and innate immune functions in tilapia larvae.     

Intrinsic Disorder Mediates Cooperative Signal Transduction in STIM1

Intrinsically disordered domains have been reported to play important roles in signal transduction networks by introducing cooperativity into protein–protein interactions. Unlike intrinsically disordered domains that become ordered upon binding, the EF-SAM domain in the stromal interaction molecule (STIM) 1 is distinct in that it is ordered in the monomeric state and partially unfolded in its oligomeric state, with the population of the two states depending on the local Ca^2 + concentration. The oligomerization of STIM1, which triggers extracellular Ca^2 + influx, exhibits cooperativity with respect to the local endoplasmic reticulum Ca^2 + concentration. Although the physiological importance of the oligomerization reaction is well established, the mechanism of the observed cooperativity is not known. Here, we examine the response of the STIM1 EF-SAM domain to changes in Ca^2 + concentration using mathematical modeling based on in vitro experiments. We find that the EF-SAM domain partially unfolds and dimerizes cooperatively with respect to Ca^2 + concentration, with Hill coefficients and half-maximal activation concentrations very close to the values observed in vivo for STIM1 redistribution and extracellular Ca^2 + influx. Our mathematical model of the dimerization reaction agrees quantitatively with our analytical ultracentrifugation-based measurements and previously published free energies of unfolding. A simple interpretation of these results is that Ca^2 + loss effectively acts as a denaturant, enabling cooperative dimerization and robust signal transduction. We present a structural model of the Ca^2 +-unbound EF-SAM domain that is consistent with a wide range of evidence, including resistance to proteolytic cleavage of the putative dimerization portion.     

Effect of human beta-defensin-3 on the proliferation of fibroblasts on periodontally involved root surfaces

Human beta-defensin-3 (HBD-3) has versatile antibacterial activity against oral bacteria and can promote the proliferation of fibroblasts. The goal of the present study was to investigate the effect of HBD-3 on attachment and proliferation of periodontal ligament cells (PDL) onto the periodontitis affected root surfaces. PDL cells were seeded onto healthy and diseased root specimens with scaling and root planing (SRP), SRP & HBD-3 (100 ng/ml), or SRP & HBD-3 (200 ng/ml) treatment for 1, 3, and 7 days incubation. The results showed that HBD-3, especially in the 200 ng/ml group, significantly promoted fibroblast attachment and proliferation onto the diseased root surfaces. The cell number in the HBD-3 group was much greater than in the group treated with SRP alone. On day 7, the cells in the HBD-3 were well-spread and formed a network similar to those on the surfaces of the healthy root specimens. These results suggest that HBD-3 could play an important role in antibacterial activity and fibroblast proliferation, thus promoting periodontal regeneration. Meanwhile, HBD-3 might act as a potent regeneration-promoter in infectious diseases.     

Engineered Si(1 0 0) surfaces for the gas-phase anchoring of metal β-diketonate complexes

A synthetic strategy for the covalent anchoring of nickel β-diketonate complexes on Si(1 0 0) has been examined. Engineered Si(1 0 0) surfaces were prepared by the Si-grafting of 10-undecylenic acid methyl ester followed by hydrolysis of the ester to free the carboxylic functions suited for the anchoring of the Ni complex. Bis(pentane-2,4-dionate)Ni(II) was bonded to the functionalized surface from the gas phase by the exchange of the acetylacetonate ligand with the grafted acid. The surface density of the anchored Ni complex was controlled by tuning the surface concentration of carboxylic groups adopting a mixed monolayer of undecylenic acid and 1-decene used as a spectator spacer. The nickel decorated silicon surfaces were characterized by attenuate total reflectance infrared absorption spectroscopy (ATR-IRAS) and angle resolved X-ray photoelectron spectroscopy (AR-XPS).     

Lactococcus lactis as a cell factory: a twofold increase in phosphofructokinase activity results in a proportional increase in specific rates of glucose uptake and lactate formation

Despite the fact that the area of glycolysis in Lactococcus lactis has been intensively studied, only a limited number of studies have been focused on the regulation of uptake of glucose itself. Using the tool of the glucostat fed-batch mode of culture, it was demonstrated in our earlier work that the concentration of glucose regulates its uptake rate and that the control of the glycolytic flux resides to a large extent in processes outside the pathway itself, like glucose transport and the ATP consuming reactions, while allosteric properties of key enzymes like phosphofructokinase (PFK) have a significant influence on the control. Extending our work, we report here the results of fermentations with engineered L. lactis strains with altered PFK activity in which the pfkA gene from Aspergillus niger, and its truncated version pfk13 that encodes a shorter PFK1 fragment were cloned. The results in this study suggest that, under the optimum for the microorganism applied microaerobic conditions, the glycolytic capacity of L. lactis was significantly increased in engineered strains with increased PFK activity. The transformant strain in which the truncated pfk13 gene of A. niger was expressed performed more efficiently as it was able to grow successfully in glucostat cultures with 277 mM glucose - while the optimum glucose concentration for the parental strain was 55 mM. The present work demonstrates the direct effect of PFK activity on the glycolytic flux in L. lactis since a twofold increase in specific PFK activity (from 7.1 to 14.5 U/OD₆₀₀) resulted in a proportional increase of the maximum specific rates of glucose uptake (from 0.8 to 1.7 μM s⁻¹ g CDW⁻¹) and lactate formation (from 15 to 22.8 g lactate (g CDW)⁻¹ h⁻¹).     

Syn-anti conformational switching: Synthesis and X-ray structures of tweezer and anti form in a zinc porphyrin dimer induced by axial ligands

Stepwise addition of 1,2-diaminobenzene to a Zn-1,2-bis(meso-octaethylporphyrinyl)ethane produces both tweezer and anti-form of the complex depending on the concentration of the axial ligand which exhibit two major equilibrium steps (with two step-wise binding constants): first, guest ligation leading to the formation of 1:1 host-guest tweezer structure (K₁) and, second, guest molecule ligation (K₂) forming 1:2 host-guest anti species and the corresponding binding constants are 1.82 × 10³ M⁻¹ and 1.34 × 10² M⁻¹, respectively. However, when guest like 1,4-diaminobenzene and 4-CN-pyridine are used, the ligand geometry prevents its entry into interporphyrin cavity to form a tweezer structure, thus producing only the 1:2 anti complex. Single crystal X-ray structures of both tweezer and anti form produced in a single Zn-bisporphyrin are reported here for the first time. The nonbonding Zn···Zn distance within a molecule is 5.55 and 10.01 Å in tweezer and anti form, respectively. Although the average Zn-N (por) distances are comparable for both the forms, the Zn-N (1,2-diaminobenzene/4-CN-pyridine) distances and the displacement of Zn from the mean porphyrin planes are larger in tweezer compared to anti conformation.     

Glyoxal causes inflammatory injury in human vascular endothelial cells

To explore mechanisms of diabetes-associated vascular endothelial cells (ECs) injury, human umbilical vein ECs were treated for 24 h with high glucose (HG; 26 mM), advanced glycation end-products (AGEs; 100 μg/ml) or their intermediate, glyoxal (GO: 50-5000 μM). HG and AGEs had no effects on ECs morphology and inflammatory states as measured by vascular cell adhesion molecule (VCAM)-1 and cyclooxygenase (COX)-2 expressions. GO (500 μM, 24 h) induced cytotoxic morphological changes and protein expression of COX-2 but not VCAM-1. GO (500 μM, 24 h) activated ERK but not JNK, p38 or NF-κB. However, ERK inhibitor PD98059 was ineffective to GO-induced COX-2. While EUK134, synthetic combined superoxide dismutase/catalase mimetic, had no effect on GO-mediated inflammation, sodium nitroprusside inhibited it. The present results indicate that glyoxal, a metabolite of glucose might be a more powerful inducer for vascular ECs inflammatory injury. Nitric oxide but not anti-oxidant is preventive against GO-mediated inflammatory injury.