UV Absorption Measurements Research Articles

Spectroscopic Study on the Effect of Imidazophenazine Tethered to 5′-End of Pentadecathymidilate on Stability of Poly(dA)·(dT)15 Duplex

The effect of imidazo[4,5-d]phenazine (Pzn) attached to the 5(')-end of (dT)(15) oligonucleotide via a flexible linker on the thermal stability of poly(dA)·(dT)(15) duplex was studied in aqueous buffered solution containing 0.1 М NaCl at the equimolar ratio of adenine and thymine bases (100 μM each) using spectroscopic techniques. Duplex formation was investigated by measuring UV absorption and fluorescence melting curves for the Pzn-modified system. Tethered phenazine derivative enhances the thermostability of poly(dA)·(dT)(15) duplex increasing the helix-to-coil transition temperature by 4.5 °С due to an intercalation of the dye chromophore between AT-base pairs. The thermodynamic parameters of the transition for non-modified and modified systems were estimated using "all-or-none" model. The modification of the (dT)(15) results in a decrease in the transition enthalpy, however, the observed gain in the Gibbs free energy of complex formation, ΔG, is provided with the corresponding decrease in entropy change. The increase of ΔG value at 37 °C in consequence of (dT)(15) modification was found to be equal to 1.3 kcal/mol per oligonucleotide strand.

Role of curcumin on the determination of the critical micellar concentration by absorbance, fluorescence and fluorescence anisotropy techniques

Curcumin (C21H20O6) is a natural antioxidant which has a wide range of physiological and pharmacological actions. Here, first time this is employed for the determination of the critical micellar concentration (cmc) of both ionic and nonionic surfactants using the measurements of UV absorption, fluorescence spectroscopy and fluorescence polarization anisotropy. Results on a number of surfactants have agreed with those evaluated from conductometric, tensiometric and calorimetric methods. The absorbance and fluorescence intensities of curcumin are enhanced by different micellar solutions. The profiles of the absorbance and fluorescence intensities as a function of concentration of surfactant can be described by sigmoidal function to evaluate the cmc of the studied amphiphiles. However, we have found that some of the anisotropy profiles are sigmoidal in nature.

Electrochemical and spectroscopic studies of the interaction between the neuroleptic drug, gabapentin, and DNA

The interaction between gabapentin (GP), a neuroleptic drug, and calf-thymus DNA was investigated. Cyclic voltammetry and different spectroscopic methods (UV/vis spectrophotometry, spectroflourimetry and circular dichroism) were used to study the process. The oxidation current of GP was considerably decreased in the presence of calf-thymus DNA. The negative shift of the peak potential in cyclic voltammetry suggested an electrostatic interaction, while spectroscopic methods show minor groove binding as the predominant mode. The values of binding constants obtained from UV absorption, spectrofluorimetry and voltammetric measurements were in close agreement. Standard Gibbs free energy change, ΔG(0), of the interaction was calculated to be -21.99 kJ mol(-1) which indicated the spontaneity of the binding interaction. A hyperchromic effect in the maximum absorption band of DNA as well as quenching in fluorescence experiments were observed in the presence of GP.

Simplified analysis of lipoprotein lipase activity: Evaluation of lipasemic activity of low molecular weight heparin in rats

A simple procedure for measuring lipoprotein lipase activity was developed by using newly formulated substrate with turbidimetry method. The activity of lipoprotein lipase was expressed as Y (value) (%) that was calculated by measuring UV absorbance (600 nm) at two time points (30 sec and 15 min). Lipid emulsions as the substrate and other factors affecting the lipolytic activity of lipase were studied. The optimal conditions for an in vitro experiment were found to be with LIPOMCT as lipid substrate at 37°C in tris-HCl buffer (pH 7.4) in the presence of BSA. To evaluate an in vivo applicability, low molecular weight heparin (LMWH)-containing drug, Sulodexide, was administered to the rats. The serum from LMWH-administered rats was incubated in an optimized analytical condition without BSA. As expected, increasing the amount of LMWH administered led to higher lipase activity. The newly developed method was successfully applied to an in vivo model suggesting the potential to be applicable for the pharmacodynamic studies of commercially available products of LPL analogues in human subjects, and for the diagnosis of acute pancreatitis in the clinical laboratory.

Modification of the Zonal Elution Method for Detection of Transient Protein–Protein Interactions Involving Ligand Exchange

A new affinity chromatography method was developed by modifying a zonal elution method. The new method targets transient protein-protein interactions, such as those encountered during direct ligand transfer between the ligand transporter and its cognate receptor. A ligand-loaded transport protein is immobilized on the solid support, and a plug containing a putative receptor is flowed through the column. Elution profiles of proteins not interacting with the immobilized transporter can be approximated with a simple Gaussian curve, while the elution profiles of cognate receptors show significant delay and exhibit complex shape. Ligand transfer from the immobilized transporter molecules to the receptors is verified by both UV absorbance measurements and mass spectrometry. The sensitivity of the method is demonstrated using retinoic acid (RA) transfer from various isoforms of cellular RA binding proteins (CRABPs) and RA receptor γ (RARγ). Although these interactions have been hypothesized long ago to proceed via direct mechanism (i.e., via transient docking of the receptor and the transporter), the existing biophysical techniques failed to detect the presence of the transporter-receptor complexes. However, the modified zonal elution method provides unequivocal evidence of direct interaction between RARγ and one of the CRABP isoforms (CRABP II) during the ligand transfer to the receptor.

Carbamazepine removal from water by dielectric barrier discharge: Comparison of ex situ and in situ discharge on water

Dielectric barrier discharges (DBD) were used for the degradation of carbamazepine (CBZ) in aqueous solution. The electric discharge was generated either ex situ or in situ directly on the water surface. To maintain the same ozone concentration of 40ppm in both instances, the power injected was 0.7W in the ex situ discharge and 12W in the in situ discharge. The results showed 100% CBZ removal after 3min of treatment with the ex situ discharge, while the in situ discharge only removed 81% of the CBZ after 60min. According to measurements of UV absorbance at 285nm and 254nm, and of total organic carbon, the ex situ discharge system also proved to be more effective than the in situ system. The measurement of nitrogen oxides in both gaseous and liquid phases indicated that high energy in situ discharges produced a large amount of NOx. These species contributed to decreased pH and significantly slowed the CBZ oxidation rate, due to their competition with ozone. Production of NOx should be avoided when using the DBD technique for wastewater treatment.

Preparation of dissolvable albumin stents for vascular anastomosis with a 1.9 µm laser and in vitro mechanical strength assessments

We present a clinically relevant method for producing and sterilizing dissolvable albumin stents to provide intraluminal support during vascular anastomosis, and a method for photothermally soldering vessels using a 1.9 µm diode laser with a 200-µm spot size, albumin solder, and water as the chromophore. Our aim in this study was to assess the mechanical integrity of soldered vessels, and to determine if gamma-irradiation affected the solubility of the stents. The axial tensile strength and burst pressure of 3.75 ± 0.3 mm inner-diameter vessels soldered with varied swath width (1-7 mm), laser power (430-610 mW), solder concentration (22-46%w/w), and solder layering (1-3 layers) was tested in vitro. Stent dissolution was monitored by weight in blood, and with UV absorbance measurements in phosphate buffered saline (PBS). Solubility was measured for stents sterilized by 25 kGy gamma-irradiation, and stents with varied diameter and wall thickness. Optimized soldering parameters yielded tensile strengths of 4.4 ± 1.2 N and burst pressures of 400 ± 90 mm Hg with stay sutures. Differences in stent solubility in blood and PBS were not statistically significant (p = 0.99). Sterilization by 25 kGy gamma-irradiation did not cause significant changes (p > 0.6) in stent solubility, which was primarily volume-dependent. Under simulated intravascular flow conditions, 3 mm stents dissolved completely with 2.7 ± 0.7 ml/mg. The results show that fast-dissolving stents can be produced reliably using the extrusion technique, and sterilized by gamma-irradiation. Without stay sutures, soldered vessels exhibited low tensile strength, but burst pressures comparable to sutured vessels. It was concluded that stay sutures would be necessary in vivo due to degradation of the tensile strength of soldered vessels with exposure to moisture.

Mixed-surface, lipid-tethered quantum dots for targeting cells and tissues

Quantum dots (QDs), with their variable luminescent properties, are rapidly transcending traditional labeling techniques in biological imaging and hold vast potential for biosensing applications. An obstacle in any biosensor development is targeted specificity. Here we report a facile procedure for creating QDs targeted to the cell membrane with the goal of cell-surface protease biosensing. This procedure generates water-soluble QDs with variable coverage of lipid functional groups. The resulting hydrophobicity is quantitatively controlled by the molar ratio of lipids per QD. Appropriate tuning of the hydrophobicity ensures solubility in common aqueous cell culture media, while providing affinity to the lipid bilayer of cell membranes. The reaction and exchange process was directly evaluated by measuring UV absorption spectra associated with dithiocarbamate formation. Cell membrane binding was assessed using flow cytometry and total internal reflection fluorescence imaging with live cells, and tissue affinity was measured using histochemical staining and fluorescence imaging of frozen tissue sections. Increases in cell and tissue binding were found to be regulated by both QD hydrophobicity and surface charge, underlying the importance of QD surface properties in the optimization of both luminescence and targeting capability.

Microstructure and surface property of macroscopic gradient polymer initiated by polysiloxane benzophenone photoinitiators with different silicone chain lengths

This paper reported three kinds of polysiloxane benzophenone photoinitiators (HBP-Si-A/B/C) with different silicon contents, which were synthesized based on traditional photoinitiator 4-hydroxy benzophenone (HBP) and epoxy polysiloxane. The effect of silicon content in the photoinitiator molecule on its photopolymerization property, the ability to float up and surface morphology and the property of the gradient polymer film initiated by it was investigated. It is proved by UV absorption and gel permeation chromatography (GPC) measurements that HBP-Si-A/B/C with different silicon content had different abilities to float up. As a result, the gradient polymer with more significant molecular weight gradients could be obtained by using the polysiloxane benzophenone photoinitiator with higher silicon content, which had a better ability to float up caused by lower surface tension and energy, and the surface property was connected with the surface microstructure. The increase of silicon content in the polysiloxane-based photoinitiator could decrease the dispersion surface energy of gradient polymer film initiated by it and generate a more hydrophobic surface by a change of microstructure. In addition, the polysiloxane-based photoinitiator could also restrain the forming of wrinkling on the surface of the UV-curable film due to mitigation of oxygen inhibition in radical photopolymerization, and it may have potential in controlling patterned microstructures and surface property.

Stereochemical characterization of fluorinated 2-(phenanthren-1-yl)propionic acids by enantioselective high performance liquid chromatography analysis and electronic circular dichroism detection

Enantioselective high performance liquid chromatography (HPLC) coupled with a detection system based on the simultaneous measurement of UV absorption and electronic circular dichroism (ECD) allows a complete stereochemical characterization of chiral compounds, once the relationship between sign of the chiroptical properties and absolute configuration is determined. In the present communication, the development of enantioselective HPLC methods for the resolution of a series of fluorinated 2-phenanthrenylpropionic acids (1–6) is reported. Different chiral stationary phases (CSPs) were tested: Chiralcel® OJ, Chiralcel® OD, Chiralpak® AD, (S,S)-Whelk-O® 1, Chirobiotic™ T and α1-acid glycoprotein (AGP). The results allow the application of the methods to a reliable determination of the enantiomeric excess for all the examined compounds; the highest enantioselectivity values were obtained with the Hibar® [(S,S)-Whelk-O® 1] column for some of the examined compounds. In the case of rac-2-(6-fluorophenanthren-1-yl)propionic acid (1), the relationship between circular dichroism and absolute configuration of the enantiomeric fractions was determined by ECD analysis and time-dependent density functional theory (TD-DFT) calculations. The experimental ECD spectrum of the second-eluted fraction of 1 on the Hibar® [(S,S)-Whelk-O® 1] column was found to be in excellent agreement with the theoretical ECD spectrum of (S)-1; therefore, the absolute configuration of the first- and second-eluted enantiomers on the (S,S)-Whelk-O® 1 CSP was assessed as (R) and (S), respectively, and the elution orders of the enantiomeric forms of 1 were determined on all the different CSPs.

Automated microfluidic sample-preparation platform for high-throughput structural investigation of proteins by small-angle X-ray scattering

A new microfluidic sample-preparation system is presented for the structural investigation of proteins using small-angle X-ray scattering (SAXS) at synchrotrons. The system includes hardware and software features for precise fluidic control, sample mixing by diffusion, automated X-ray exposure control, UV absorbance measurements and automated data analysis. As little as 15 µl of sample is required to perform a complete analysis cycle, including sample mixing, SAXS measurement, continuous UV absorbance measurements, and cleaning of the channels and X-ray cell with buffer. The complete analysis cycle can be performed in less than 3 min. Bovine serum albumin was used as a model protein to characterize the mixing efficiency and sample consumption of the system. The N2 fragment of an adaptor protein (p120-RasGAP) was used to demonstrate how the device can be used to survey the structural space of a protein by screening a wide set of conditions using high-throughput techniques.

Testing by photometric measurement and camera study of theoretical prediction of microvolume universal sessile dropshape

The approach to the theory of sessile dropshapes held on a cylindrical drophead is discussed. It reveals an 'undifferentiable' universal micro-dropshape for volumes below 3μL. Camera studies demonstrate the veracity of this prediction exploited in the design of a new microvolume spectrometer. The mean pathlength of light injected through a microvolume sessile drop has been determined both from the model and from experiment. Drop volumes determine accurately the mean pathlength and with this Beer's law relationship is experimentally confirmed. The Transmitted Light Drop Analyser uses this universal 'natural cuvette' to deliver both high-performance UV spectra and absorbance measurements at discrete wavelengths.

Simple HPLC method for detection of trace ephedrine and pseudoephedrine in high‐purity methamphetamine

A simple and sensitive HPLC technique was developed for the qualitative determination of ephedrine and pseudoephedrine (ephedrines), used as precursors of clandestine d-methamphetamine hydrochloride of high purity. Good separation of ephedrines from bulk d-methamphetamine was achieved, without any extraction or derivatization procedure on a CAPCELLPACK C18 MGII (250 × 4.6 mm) column. The mobile phase consisted of 50 mM KH2 PO4-acetonitrile (94:6 v/v %) using an isocratic pump system within 20 min for detecting two analytes. One run took about 50 min as it was necessary to wash out overloaded methamphetamine for column conditioning. The analytes were detected by UV absorbance measurement at 210 nm. A sample (20 mg) was simply dissolved in 1 mL of water, and a 50 μL aliquot of the solution was injected into the HPLC. The detection limits for ephedrine and pseudoephedrine in bulk d-methamphetamine were as low as 3 ppm each. This analytical separation technique made it possible to detect ephedrine and/or pseudoephedrine in seven samples of high-purity d-methamphetamine hydrochloride seized in Japan. The presence of trace ephedrines in illicit methamphetamine may strongly indicate a synthetic route via ephedrine in methamphetamine profiling. This method is simple and sensitive, requiring only commonly available equipment, and should be useful for high-purity methamphetamine profiling.

Synthesis of Modified Guanidine-Based Polymers and their Antimicrobial Activities Revealed by AFM and CLSM

Modified guanidine-based polymers with chain extension were synthesized by condensation and cross-linking polymerizations in an attempt to increase molecular weight and charge density of the antimicrobial polymers. The antimicrobial activity and the corresponding mechanisms were investigated by several approaches. The results indicated that the antimicrobial activities of the modified guanidine-based polymer, based on the minimum inhibition concentration (MIC) against E.coli, varied with alkyl monomer ratios. UV absorption at 260 nm further quantified the amount of intracellular components leaked into bacteria suspension. The UV absorption measurements were also used to monitor inhibition processes dynamically. It was found that the modified guanidine-based polymer inhibited the growth of bacteria by causing membrane compromised and intracellular leaked. Dual fluorescent dyes were used to stain all bacteria including the dead ones, which enabled us to utilize CLSM to visualize the viability of bacteria in the presence of various modified guanidine-based polymers without causing any damage. The morphologies of bacteria untreated and treated with modified guanidine-based polymer were observed using an atomic force microscope (AFM), which further demonstrated the damage of E.coli membrane and the leakage of intracellular component induced by the modified guanidine-based polymers.

Tracing Organic Footprints from Industrial Effluent Discharge in Recalcitrant Riverine Chromophoric Dissolved Organic Matter

Excitation–emission matrix fluorescence spectroscopy, combined with parallel factor analysis and measurements of UV absorption and dissolved organic carbon (DOC) concentrations, was used to trace the footprints of industrial effluents discharged into the lower Kishon River (Israel). The lower Kishon River typifies streams that are affected by seawater tidal intrusion and represents an extreme case of severe long-term pollution caused mainly by a variety of industrial effluents. The industrial effluents may contribute about 90%, in terms of biochemical oxygen demand, of the total organic carbon discharged into the lower Kishon River. Water samples were collected along the river, including the points of effluent discharge from industrial plants, between November 2005 and September 2006. Two types of fluorescent components characterized the fluorescence of the lower Kishon River water: component I corresponded to humic-like matter and component II spectrally resembled material known to be associated with biological productivity, but different from typical tryptophan-like fluorophore. These fluorescent components and other substances that absorbed light at 254 nm contributed to the DOC pool that resisted riverine microbial degradation under laboratory conditions, and that constitutes up to 70% of the overall riverine DOC. The variations in DOC concentration, absorbance at 254 nm, and concentration of humic-like matter (characterized by component I) correlated with the distance from the sea and the water electrical conductivity, and were linked to seawater tidal intrusion. The increased concentration of component II, as well as its enlarged fraction in the overall riverine DOC pool, was found to be associated with the location of major inputs of the industrial effluents. These findings support the use of this fluorescent component as an indicator of industrial pollution in such severely contaminated riverine systems.

Solution Structures of Ca2+-CIB1 and Mg2+-CIB1 and Their Interactions with the Platelet Integrin αIIb Cytoplasmic Domain

The calcium- and integrin-binding protein 1 (CIB1) is a ubiquitous Ca(2+)-binding protein and a specific binding partner for the platelet integrin αIIb cytoplasmic domain, which confers the key role of CIB1 in hemostasis. CIB1 is also known to be involved in apoptosis, embryogenesis, and the DNA damage response. In this study, the solution structures of both Ca(2+)-CIB1 and Mg(2+)-CIB1 were determined using solution-state NMR spectroscopy. The methyl groups of Ile, Leu, and Val were selectively protonated to compensate for the loss of protons due to deuteration. The solution structure of Ca(2+)-CIB1 possesses smaller opened EF-hands in its C-domain compared with available crystal structures. Ca(2+)-CIB1 and Mg(2+)-CIB1 have similar structures, but the N-lobe of Mg(2+)-CIB1 is slightly more opened than that of Ca(2+)-CIB1. Additional NMR experiments, such as chemical shift perturbation and methyl group solvent accessibility as measured by a nitroxide surface probe, were carried out to further characterize the structures of Ca(2+)-CIB1 and Mg(2+)-CIB1 as well as their interactions with the integrin αIIb cytoplasmic domain. NMR measurements of backbone amide proton slow motion (microsecond to millisecond) dynamics confirmed that the C-terminal helix of Ca(2+)-CIB1 is displaced upon αIIb binding. The EF-hand III of both Ca(2+)-CIB1 and Mg(2+)-CIB1 was identified to be directly involved in the interaction of CIB1 with αIIb. Together, these data illustrate that CIB1 behaves quite differently from related EF-hand regulatory calcium-binding proteins, such as calmodulin or neuronal calcium sensor proteins.

Analysis and Modeling of Gas‐Phase Processes in a CHF3/Ar Discharge

AbstractThe present work deals with the diagnostics of fluorocarbon plasmas by different experimental methods and complementary numerical analysis of the plasma. The plasma diagnostics were performed with non‐invasive methods. In this paper, we present results obtained by laser‐induced fluorescence and UV absorption measurements. The complementary numerical simulations accounted for the electron–neutral interactions, discharge dynamics, and chemical reactions. Several insights were obtained from the combined experimental and numerical approaches, especially concerning the conclusiveness of the results and previous observations from the literature. As important initial neutral species, CF3 and H originate from electron collisions with CHF3. The atomic hydrogen is responsible for fluorine abstraction reactions which result in the formation of hydrofluoric acid as well as in unsaturated carbon bonds. It was found from the simulations that in the examined discharges, this reaction is the most important channel for the high‐rate production of the CF molecule in the gas phase via ${\rm CF}_{2} + {\rm H}\to {\rm CF} + {\rm HF}$. For this molecule, no anisotropy in the density was found and the production solely occurs in the gas phase. Contrary to that, the production of CF2 via the abstraction of fluorine from CF3 in the gas phase was found to be of less importance for the overall concentration of this molecule. Instead, chemical reactions at the chamber walls which release CF2 were identified experimentally as major sources. According to the simulations, the unsaturated fluorocarbon molecules react to oligomers, such that the net reaction can be roughly described by $n{\rm CHF}_{3} \to {\rm (CF}_{{\rm 2}} {\rm )}_{n} + n{\rm HF}$. A good agreement between the simulated and the experimentally observed densities and rates was obtained, which confirms the interpretation of the ongoing plasma‐chemical processes as well as the underlying physical discharge parameters consistently. magnified image

Thermophoretic melting curves quantify the conformation and stability of RNA and DNA

Measuring parameters such as stability and conformation of biomolecules, especially of nucleic acids, is important in the field of biology, medical diagnostics and biotechnology. We present a thermophoretic method to analyse the conformation and thermal stability of nucleic acids. It relies on the directed movement of molecules in a temperature gradient that depends on surface characteristics of the molecule, such as size, charge and hydrophobicity. By measuring thermophoresis of nucleic acids over temperature, we find clear melting transitions and resolve intermediate conformational states. These intermediate states are indicated by an additional peak in the thermophoretic signal preceding most melting transitions. We analysed single nucleotide polymorphisms, DNA modifications, conformational states of DNA hairpins and microRNA duplexes. The method is validated successfully against calculated melting temperatures and UV absorbance measurements. Interestingly, the methylation of DNA is detected by the thermophoretic amplitude even if it does not affect the melting temperature. In the described setup, thermophoresis is measured all-optical in a simple setup using a reproducible capillary format with only 250 nl probe consumption. The thermophoretic analysis of nucleic acids shows the technique’s versatility for the investigation of nucleic acids relevant in cellular processes like RNA interference or gene silencing.

Nuclear Tau, a Key Player in Neuronal DNA Protection

Tau, a neuronal protein involved in neurodegenerative disorders such as Alzheimer disease, which is primarily described as a microtubule-associated protein, has also been observed in the nuclei of neuronal and non-neuronal cells. However, the function of the nuclear form of Tau in neurons has not yet been elucidated. In this work, we demonstrate that acute oxidative stress and mild heat stress (HS) induce the accumulation of dephosphorylated Tau in neuronal nuclei. Using chromatin immunoprecipitation assays, we demonstrate that the capacity of endogenous Tau to interact with neuronal DNA increased following HS. Comet assays performed on both wild-type and Tau-deficient neuronal cultures showed that Tau fully protected neuronal genomic DNA against HS-induced damage. Interestingly, HS-induced DNA damage observed in Tau-deficient cells was completely rescued after the overexpression of human Tau targeted to the nucleus. These results highlight a novel role for nuclear Tau as a key player in early stress response.

Control of concentration gradient and initiating gradient photopolymerization of polysiloxane benzophenone photoinitiator

This paper reported a polysiloxane benzophenone photoinitiator (HBP-Si-A), which was synthesized based on traditional photoinitiator 4-Hydroxybenzophenone (HBP) and epoxy polysiloxane. Its structure was confirmed by proton nuclear magnetic resonance (1H-NMR), fourier transform infrared spectroscopy (FT-IR) and gel permeation chromatography (GPC). The kinetics of photopolymerization initiated by HBP-Si-A was studied by real-time infrared spectroscopy (RTIR). More importantly, it is proved by X-ray photoelectron spectroscopy (XPS), UV absorption and GPC measurements that the polysiloxane benzophenone photoinitiator (HBP-Si-A) had good ability to float up. As a result, the HBP-Si-A could easily form concentration gradient in photopolymerization system and initiate gradient photopolymerization. A convenient and simple method for preparing gradient polymer initiated by HBP-Si-A was successfully developed. The surface morphology and elemental composition of each segment of poly(tripropylene glycol diacrylate) (PTPGDA) were studied by scanning electron microscopy (SEM) and energy dispersive spectrometer (EDS) respectively. In addition, HBP-Si-A could also more effectively mitigate inhibition of oxygen in radical photopolymerization.