microM Range Research Articles

The two CO-dehydrogenases of Thermococcus sp. AM4

Ni-containing CO-dehydrogenases (CODHs) allow some microorganisms to couple ATP synthesis to CO oxidation, or to use either CO or CO2 as a source of carbon. The recent detailed characterizations of some of them have evidenced a great diversity in terms of catalytic properties and resistance to O2. In an effort to increase the number of available CODHs, we have heterologously produced in Desulfovibrio fructosovorans, purified and characterized the two CooS-type CODHs (CooS1 and CooS2) from the hyperthermophilic archaeon Thermococcus sp. AM4 (Tc). We have also crystallized CooS2, which is coupled in vivo to a hydrogenase. CooS1 and CooS2 are homodimers, and harbour five metalloclusters: two [Ni4Fe-4S] C clusters, two [4Fe-4S] B clusters and one interfacial [4Fe-4S] D cluster. We show that both are dependent on a maturase, CooC1 or CooC2, which is interchangeable. The homologous protein CooC3 does not allow Ni insertion in either CooS. The two CODHs from Tc have similar properties: they can both oxidize and produce CO. The Michaelis constants (Km) are in the microM range for CO and in the mM range (CODH 1) or above (CODH 2) for CO2. Product inhibition is observed only for CO2 reduction, consistent with CO2 binding being much weaker than CO binding. The two enzymes are rather O2 sensitive (similarly to CODH II from Carboxydothermus hydrogenoformans), and react more slowly with O2 than any other CODH for which these data are available.

Planar Microelectrode Chip for Synchronous Simulative Neurochemical and Neuroelectrial Monitoring

The microfabricated microelectrode arrays on glass substrate combined with homemade multichannel detection system as a powerful tool for synchronous neurochemical and neuroelectrial monitoring was described. The dual-mode (neurochemical and neuroelectrial: simultaneous recording of spikes, and dopamine overflow on the same spatiotemporal scale) recording was carried in PBS buffer. The neurochemical constant potential amperometry was carried at +0.4 V working potential for nM-microM dopamine detection and nM level dopamine concentration increase can be detected by the device. The microelectrode chip showed high sensitivity of 0.88 nA/nM mm2 during nM range and 0.43 nA/nM mm2 during microM range for the determination of DA. The amperometric method used to detect dopamine (DA) did not significantly influence electrophysiological activity at low concentration (< 200 nM) and have a quickly clear away strike at high concentration (> 2 microM). The system uniquely affords synchronous measurement of chemical and electrical neural activity at the adjacent recording sites on the same chip. The sensitivity and resolution of the device allows for simultaneous recoding of electrical and chemical signaling, which may be useful for examining specific spatiotemporal relationships between electrical and chemical signaling in vitro.

Synthetic Coprisin Analog Peptide, D-CopA3 has Antimicrobial Activity and Pro-Apoptotic Effects in Human Leukemia Cells

Recently, we reported that the synthetic Coprisin analog peptide 9-mer dimer CopA3 (consisted of all-L amino acid sequence) was designed based on a defensin-like peptide, Coprisin isolated from Copris tripartitus. The 9-mer dimer CopA3 (L-CopA3) had antibacterial activity and induced apoptosis in human leukemia cells via a caspase-independent pathway. In this study, all of amino acid sequences of L-CopA3 were modified to all D-form amino acids (D-CopA3) to develop a more effective antimicrobial peptide. We investigated whether D-CopA3 had antimicrobial activities against pathogenic microorganisms and proapoptotic effects in human leukemia cells (U937, Jurkat, and AML-2). The synthetic peptide D-CopA3 had antimicrobial activities against various pathogenic bacteria and yeast fungus with MIC values in the 4~64 microM range. Moreover, D-CopA3 caused cell growth inhibition, and increased the chromosomal DNA fragmentation and the expression of inflammatory cytokines, TNF-alpha and IL1-beta, transcripts in human leukemia cells. The all-D amino acid peptide D-CopA3 proved as effective as the L-CopA3 reported previously. These results provide the basis for developing D-CopA3 as a new antibiotic peptide.

Optimization of Chemical Vapor Deposition Diamond Films Growth on Steel: Correlation Between Mechanical Properties, Structure, and Composition

In the present work we perform optimization of mechanical and crystalline properties of CVD microcrystalline diamond films grown on steel substrates. A chromium-nitride (Cr-N) interlayer had been previously proposed to serve as a buffer for carbon and iron inter-diffusion and as a matching layer for the widely differing expansion coefficients of diamond and steel. However, adhesion and wear as well as crystalline perfection of diamond films are strongly affected by conditions of both Cr-N interlayer preparation and CVD diamond deposition. In this work we assess the effects of two parameters. The first one is the temperature of the Cr-N interlayer preparation: temperatures in the range of 500 degrees C-800 degrees C were used. The second one is diamond film thickness in the 0.5 microm-2 microm range monitored through variation of the deposition time from approximately 30 min to 2 hours. The mechanical properties of so deposited diamond films were investigated. For this purpose, scratch tests were performed at different indentation loads. The friction coefficient and wear loss were assessed. The mechanical and tribological properties were related to structure, composition, and crystalline perfection of diamond films which were extensively analyzed using different microscopic and spectroscopic techniques. It was found that relatively thick diamond film deposited on the Cr-N interlayer prepared at the temperature similar to that of the CVD process has the best mechanical and adhesion strength. This film was stable without visible cracks around the wear track during all scratch tests with different indentation loads. In other cases, cracking and delamination of the films took place at low to moderate indentation loads.

Vanadium Oxide Nanoparticles as Optical Sensors of Cysteine

We report on the synthesis of vanadium oxide nanoparticles using the laser ablation in solution technique. The particles were characterized by dynamic light scattering-DLS, transmission electron microscopy-TEM, X-ray diffraction-XRD, X-ray photoelectron spectroscopy-XPS and UV-Vis optical spectroscopy. The oxide nanoparticles are mainly composed of tetragonal V2O5, a semiconductor with a 2.2 eV band gap. The interaction of the nanoparticles with cysteine, a very important aminoacid present in proteins, was studied. Upon reaction with cysteine, the bandgap of the nanoparticles shifts to the ultraviolet region at 2.87 eV. This color change from yellow to transparent can be used for selective cysteine sensing. Additionally, the intervalence band of the optical absorption spectra shows capability for cysteine sensing in the microM range.

Investigation of BK Channel Gating using Mallotoxin

While large-conductance, BK K+ channels are activated by both voltage and by Ca2+ in the microM range, the channels can be activated by voltage even in the absence of Ca2+. BK channel β-subunits, several chemicals, and mutations can shift voltage activation toward more hyperpolarized potentials but in a rather complex manner. In contrast the accessory protein leucine-rich repeat containing protein 26 (LRRC26) and mallotoxin each appears to produce a simple shift. We have investigated the functional basis for the gating shift produced by mallotoxin in the absence and presence of the LRRC26 protein in the context of the Horrigan-Aldrich (HA) model. In the absence of intracellular Ca2+, we found that, in addition to a hyperpolarizing shift of BK activation, mallotoxin produced a large, hyperpolarizing shift of channel activation kinetics. This result suggests that a major action of mallotoxin is to sensitize the BK channel voltage sensors. We also found that the degree of gating shift of mallotoxin was significantly decreased when co-expressed with the LRRC26 protein or in native parotid acinar cells that endogenously express this accessory protein. These results suggest that there is limit to the degree of sensitization of the BK channel voltage sensors.

An Industry Perspective on the Monitoring of Subvisible Particles as a Quality Attribute for Protein Therapeutics

Concern around the lack of monitoring of proteinaceous subvisible particulates in the 0.1-10 microm range has been heightened (Carpenter et al., 2009, J Pharm Sci 98: 1202-1205), primarily due to uncertainty around the potential immunogenicity risk from these particles. This article, representing the opinions of a number of industry scientists, aims to further the discussion by developing a common understanding around the technical capabilities, limitations, as well as utility of monitoring this size range; reiterating that the link between aggregation and clinical immunogenicity has not been unequivocally established; and emphasizing that such particles are present in marketed products which remain safe and efficacious despite the lack of monitoring. Measurement of subvisible particulates in the <10 microm size range has value as an aid in product development and characterization. Limitations in measurement technologies, variability from container/closure, concentration, viscosity, history, and inherent batch heterogeneity, make these measurements unsuitable as specification for release and stability or for comparability, at the present time. Such particles constitute microgram levels of protein with currently monitored sizes >or=10 microm representing the largest fraction. These levels are well below what is detected or reported for other product quality attributes. Subvisible particles remain a product quality attribute that is also qualified in clinical trials.

Raman microscopy and surface‐enhanced Raman scattering (SERS) for in situ analysis of biofilms

Biofilms are communities of micro-organisms enclosed in a matrix of extracellular polymeric substances (EPS). They represent a ubiquitous form of microbial life on Earth. Detailed information on chemical composition and structure of the EPS matrix is relevant in medicine, industry and technological processes. Raman microscopy (RM) provides whole-organism fingerprints for biological samples with spatial resolution in the microm range and enables correlations between optical and chemical images to be made. Low water background makes RM beneficial for in situ studies of biofilms, since water is the major component of the biofilm matrix. In this paper we discuss the feasibility of RM for chemical characterization of different structures in a multispecies biofilm matrix, including microbial constituents and EPS. We show that by improving the sensitivity of RM with surface-enhanced Raman scattering (SERS) one can perform rapid biofilm analysis. In particular, by choosing appropriate SERS substrates and solving the problem of SERS measurement reproducibility one can carry out in situ study of different components in the complex biofilm matrix.

Design, synthesis, and biological evaluation of ketoprofen analogs as potent cyclooxygenase-2 inhibitors

A new series of ketoprofen analogs were synthesized to evaluate their biological activities as selective cyclooxygenase-2 (COX-2) inhibitors. In vitro COX-1 and COX-2 inhibition studies showed that all compounds were potent and selective inhibitors of the COX-2 isozyme with IC(50) values in the highly potent 0.057-0.085 microM range, and COX-2 selectivity indexes in the 115 to >1298.7 range. Compounds possessing azido pharmacophore group (8a and 8b) exhibited highly COX-2 inhibitory selectivity and potency even more than reference drug celecoxib. Molecular modeling studies indicated that the azido substituent can be inserted deeply into the secondary pocket of COX-2 active site for interactions with Arg(513).

Picosecond synchronously pumped ZnGeP_2 optical parametric oscillator

We report on the experimental operation of a picosecond, singly resonant, synchronously pumped optical parametric oscillator (SPOPO) based on a ZnGeP(2) (ZGP) crystal. The ZGP SPOPO is pumped with the idler radiation from a diffraction-grating-tuned periodically poled lithium niobate SPOPO emitting a continuous picosecond pulse train (Delta tau approximately 8 ps, f=76 MHz). The noncritically 90 degree phase-matched ZGP SPOPO is tuned in the 3.8-5.6 microm range by tuning the pump wavelength (2.25-2.5 microm). As high as approximately 800 mW of average power is emitted at 4 microm.

A Chemical Genetic Screen for Modulators of Asymmetrical 2,2′-Dimeric Naphthoquinones Cytotoxicity in Yeast

BackgroundDimeric naphthoquinones (BiQ) were originally synthesized as a new class of HIV integrase inhibitors but have shown integrase-independent cytotoxicity in acute lymphoblastic leukemia cell lines suggesting their use as potential anti-neoplastic agents. The mechanism of this cytotoxicity is unknown. In order to gain insight into the mode of action of binaphthoquinones we performed a systematic high-throughput screen in a yeast isogenic deletion mutant array for enhanced or suppressed growth in the presence of binaphthoquinones.Methodology/Principal findingsExposure of wild type yeast strains to various BiQs demonstrated inhibition of yeast growth with IC50s in the µM range. Drug sensitivity and resistance screens were performed by exposing arrays of a haploid yeast deletion mutant library to BiQs at concentrations near their IC50. Sensitivity screens identified yeast with deletions affecting mitochondrial function and cellular respiration as having increased sensitivity to BiQs. Corresponding to this, wild type yeast grown in the absence of a fermentable carbon source were particularly sensitive to BiQs, and treatment with BiQs was shown to disrupt the mitochondrial membrane potential and lead to the generation of reactive oxygen species (ROS). Furthermore, baseline ROS production in BiQ sensitive mutant strains was increased compared to wild type and could be further augmented by the presence of BiQ. Screens for resistance to BiQ action identified the mitochondrial external NAD(P)H dehydrogenase, NDE1, as critical to BiQ toxicity and over-expression of this gene resulted in increased ROS production and increased sensitivity of wild type yeast to BiQ.Conclusions/SignificanceIn yeast, binaphthoquinone cytotoxicity is likely mediated through NAD(P)H:quonine oxidoreductases leading to ROS production and dysfunctional mitochondria. Further studies are required to validate this mechanism in mammalian cells.

Complexation of metals by phytosiderophores revealed by CE‐ESI‐MS and CE‐ICP‐MS

CE-ESI-MS and CE-ICP-MS were implemented for studying three phytosiderophores (mugineic acid, epi-mugineic acid and deoxymugineic acid) and their metal complexes. Free ligands and ferric complexes were analyzed using the first methodology, while six free metals (Co(II), Cu(II), Fe(III), Mn(II), Ni(II) and Zn(II)) together with the corresponding complexes were investigated by the latter technique. CE separation was realized at a voltage of +25 kV employing a BGE containing 20 mM ammonium bicarbonate at pH 7.2. Both techniques revealed limits of detection in the high nM to low microM range. Standard additions to hydroponic samples of H. distichon, cv. Bodega (spring barley) showed regression coefficients for the metal-ligand complexes ranging from 0.984 to 0.999. Additionally, results of a competitivity study allowed the determination of relative metal-phytosiderophore complex stability constants of deoxymugineic/mugineic acid.

Nitric Oxide in Cancer Therapeutics: Interaction with Cytotoxic Chemotherapy

Nitric oxide is a key second messenger in most tissues, where it is generated at low concentrations, predominantly by the catalytic action of two constitutively expressed isoforms of nitric oxide synthase (NOS). Both of these are found in tumours, but malignancy is also associated with the expression of high levels of the inducible isoform of NOS, which is responsible for generation of high NO(*) concentrations, not associated with normal physiology. This has profound consequences for the aetiology and malignant progression of primary cancer and metastatic dissemination. It also ensures that tumour vasculature remains highly dilated, so maintaining the abnormally high growth rates, characteristic of malignant disease. This dependency on NO(*) can be targeted therapeutically by administering NOS inhibitors to block NO(*) production, so reducing the availability of metabolic substrates and slowing tumour growth. However, there is now clear evidence that the effects of NO(*) in tumours are bimodal such that intermediate levels optimise tumour growth, and interventions to raise or lower NO(*) concentrations can inhibit it. Concentrations in the high microM range generated by NOS gene therapy or NO(*) donor drugs induce apoptosis in solid tumours in vivo and slow their growth dramatically. These interventions are also potent enhancers of the anticancer effects of cytotoxic chemotherapy, particularly with the anthracyclines and platinum compounds. There is also clear evidence for specificity against malignant compared with normal cells, associated with the specific generation of peroxynitrite. Recent clinical trials have demonstrated both the safety and efficacy of nitric oxide therapy against lung and prostate cancer.

A compact multipurpose nanomanipulator for use inside a scanning electron microscope

A compact, two-stage nanomanipulator was designed and built for use inside a scanning electron microscope. It consists of a fine stage employing piezostacks that provide a 15 microm range in three dimensions and a coarse stage based on commercially available stick-slip motors. Besides the fabrication of enhanced probes for scanning probe microscopy and the enhancement of electron field emitters, other novel manipulation processes were developed, such as locating, picking up, and positioning small nanostructures with an accuracy of approximately 10 nm. In combination with in situ I-V experiments, welding, and etching, this results in a multipurpose nanofactory, enabling a new range of experiments.

Design and Synthesis of Novel Quinone Inhibitors Targeted to the Redox Function of Apurinic/Apyrimidinic Endonuclease 1/Redox Enhancing Factor-1 (Ape1/Ref-1)

The multifunctional enzyme apurinic endonuclease 1/redox enhancing factor 1 (Ape1/ref-1) maintains genetic fidelity through the repair of apurinic sites and regulates transcription through redox-dependent activation of transcription factors. Ape1 can therefore serve as a therapeutic target in either a DNA repair or transcriptional context. Inhibitors of the redox function can be used as either therapeutics or novel tools for separating the two functions for in vitro study. Presently there exist only a few compounds that have been reported to inhibit Ape1 redox activity; here we describe a series of quinones that exhibit micromolar inhibition of the redox function of Ape1. Benzoquinone and naphthoquinone analogues of the Ape1-inhibitor E3330 were designed and synthesized to explore structural effects on redox function and inhibition of cell growth. Most of the naphthoquinones were low micromolar inhibitors of Ape1 redox activity, and the most potent analogues inhibited tumor cell growth with IC(50) values in the 10-20 microM range.

Aegyptin displays high‐affinity for the von Willebrand factor binding site (RGQOGVMGF) in collagen and inhibits carotid thrombus formation in vivo

Aegyptin is a 30 kDa mosquito salivary gland protein that binds to collagen and inhibits platelet aggregation. We have studied the biophysical properties of aegyptin and its mechanism of action. Light-scattering plot showed that aegyptin has an elongated monomeric form, which explains the apparent molecular mass of 110 kDa estimated by gel-filtration chromatography. Surface plasmon resonance identified the sequence RGQOGVMGF (where O is hydroxyproline) that mediates collagen interaction with von Willebrand factor (vWF) as a high-affinity binding site for aegyptin, with a K(D) of approximately 5 nM. Additionally, aegyptin interacts with the linear peptide RGQPGVMGF and heat-denatured collagen, indicating that the triple helix and hydroxyproline are not a prerequisite for binding. However, aegyptin does not interact with scrambled RGQPGVMGF peptide. Aegyptin also recognizes the peptides (GPO)(10) and GFOGER with low affinity (microM range), which respectively represent glycoprotein VI and integrin alpha2beta1 binding sites in collagen. Truncated forms of aegyptin were engineered, and the C-terminus fragment was shown to interact with collagen and to attenuate platelet aggregation. In addition, aegyptin prevents laser-induced carotid thrombus formation in the presence of Rose Bengal in vivo, without significant bleeding in rats. In conclusion, aegyptin interacts with distinct binding sites in collagen, and is useful tool to inhibit platelet-collagen interaction in vitro and in vivo.

Risedronate reduces intracortical porosity in women with osteoporosis

Nonvertebral fractures account for 80% of all fractures and their accompanying morbidity and mortality. Despite this, the effect of drug therapy on cortical morphology has received limited attention, partly because cortical bone is believed to remodel less and decrease less with age than trabecular bone. However, the haversian canals traversing the cortex provide a surface for remodeling that produces bone loss, porosity, and cortical fragility. We developed a new method of 3D micro-computed tomography (microCT) to quantify intracortical porosity and the effects of treatment. Women with osteoporosis randomized to risedronate (5 mg/day, n = 28) or placebo (n = 21) had paired transiliac biopsies at baseline and 5 years imaged using 3D microCT. Pores determined from 8 to 12 slices were stratified by their minor axis length into those 25 to 100 microm (closing cone of haversian canals), 100 to 300 microm (cutting cone of haversian canals), and >300 microm (coalescent cavities). Porosity was analyzed as pore area (percent bone area) and pore density (pore number/mm(2)). Medians are reported. Risedronate reduced pore area in the 25 to 100, 100 to 300, and 300 to 500 microm ranges over 5 years (p = .0008, .04, NS, respectively) corresponding to an 18% to 25% reduction. In the placebo group, pore area was unchanged. At 5 years, pore area and pore number/mm(2) in the 25 to 100 microm range were each 17% lower in the risedronate group than in the placebo group (p = .02 and .04, respectively). Risedronate is likely to maintain bone strength and reduce nonvertebral fracture risk in part by reducing remodeling and therefore the number and size of intracortical cavities.

9-Arylpurines as a Novel Class of Enterovirus Inhibitors

Here we report on a novel class of enterovirus inhibitors that can be structurally described as 9-arylpurines. These compounds elicit activity against a variety of enteroviruses in the low microM range including Coxsackie virus A16, A21, A24, Coxsackie virus B3, and echovirus 9. Structure-activity relationship (SAR) studies indicate that a chlorine or bromine atom is required at position 6 of the purine ring for antiviral activity. The most selective compounds in this series inhibited Coxsackie virus B3 replication in a dose-dependent manner with EC(50) values around 5-8 microM. No toxicity on different cell lines was observed at concentrations up to 250 microM. Moreover, no cross-resistance to TBZE-029 and TTP-8307 CVB3 resistant strains was detected.

Abstract 5786: Prostaglandin E 2 and EP3 Activation Potentiate Human Platelet Responsiveness

Activated human platelets synthesize prostaglandin (PG) E 2 , although at much lower rates than thromboxane A 2 . PGE 2 binds different receptors (EP1–4). EP3-deleted mice displayed lower platelet response to sub-threshold concentrations of agonists and less susceptibility to experimental thrombosis than wild type mice. However, the role of PGE 2 in human platelet function remains poorly characterized. We investigated the effect of PGE 2 and EP-agonists on human platelet responsiveness to other agonists. Platelets were incubated with increasing concentrations of PGE 2 or different EP agonists, and stimulated by aggregating doses of adenosine diphosphate (ADP), collagen or arachidonic acid (AA). Aggregation was measured by the optical method. EP expression was studied by immunohistochemistry in platelets and bone marrow megakaryocytes. PGE 2 in the microM range (1–200 microM) dose-dependently inhibited 10microM ADP-induced aggregation. However nanoM PGE 2 concentrations (2–200 nanoM), dose-dependently increased the slope (velocity) of the secondary wave of 8 microM ADP-induced platelet aggregation, with an EC 50 of 25±6 nanoM (n=15), without affecting maximal aggregation. At lower (4 microM) ADP concentration, PGE 2 reverted the reversible aggregation, with an EC 50 of 29±10 nanoM (n=6). In in vitro aspirinated platelets, 200 nanoM PGE 2 in part counteracted the effect of aspirin in abolishing the secondary wave of 8 microM ADP-induced aggregation. In addition, PGE 2 dose-dependently abolished shape change caused by 10 microg/ml collagen and 50 microg/ml AA, with an IC 50 of 157±4 nanoM. The EP3 agonist, 11-deoxy-16,16-dimetyl PGE 2 , mimicked the PGE 2 effect on the secondary wave of ADP-induced aggregation, with an EC 50 58±1 nanoM (n=10), while the EP2 agonist butaprost was ineffective. The EP3 agonist at microM concentrations, dose-dependently induced spontaneous platelet aggregation with an EC 50 of 5.5±1 microM. Immunostains revealed EP3 expression in human platelets and megakaryocytes. We conclude that PGE 2 -EP3 interaction potentiates human platelet response to physiological concentrations of common agonists. EP3 might represent a novel pharmacological target for inhibiting platelet responsiveness in atherothrombotic diseases.

Cucurbit[7]uril Complexation Drives Thermal trans–cis‐Azobenzene Isomerization and Enables Colorimetric Amine Detection

Complexation of yellow diaminoazobenzenes 1 and 3 inside cucurbit[7]uril (CB[7]) results in the formation of purple-colored CB[7].cis-1.2H+ and CB[7].cis-3.2H+ complexes, respectively. The high binding affinity and selectivity displayed by CB[7] toward 1 and 3 pays the >10 kcal mol(-1) thermodynamic cost for this isomerization. We investigated the behavior of these complexes as a function of pH and observed large pK(a) shifts and high pH responsiveness, which are characteristic of cucurbit[n]uril molecular containers. The remarkable yellow to purple color change was utilized in the construction of an indicator displacement assay for biologically active amines 4-10. This indicator displacement assay is capable of quantifying the pseudoephedrine (5) content in Sudafed tablets over the 5-350 microM range.