Quantitative Spectroscopic Methods Research Articles

Quantification of energy-converting protein complexes in plant thylakoid membranes

Knowledge about the exact abundance and ratio of photosynthetic protein complexes in thylakoid membranes is central to understanding structure-function relationships in energy conversion. Recent modeling approaches for studying light harvesting and electron transport reactions rely on quantitative information on the constituent complexes in thylakoid membranes. Over the last decades several quantitative methods have been established and refined, enabling precise stoichiometric information on the five main energy-converting building blocks in the thylakoid membrane: Light-harvesting complex II (LHCII), Photosystem II (PSII), Photosystem I (PSI), cytochrome b6f complex (cyt b6f complex), and ATPase. This paper summarizes a few quantitative spectroscopic and biochemical methods that are currently available for quantification of plant thylakoid protein complexes. Two new methods are presented for quantification of LHCII and the cyt b6f complex, which agree well with established methods. In addition, recent improvements in mass spectrometry (MS) allow deeper compositional information on thylakoid membranes. The comparison between mass spectrometric and more classical protein quantification methods shows similar quantities of complexes, confirming the potential of thylakoid protein complex quantification by MS. The quantitative information on PSII, PSI, and LHCII reveal that about one third of LHCII must be associated with PSI for a balanced light energy absorption by the two photosystems.

Nanolayers of Poly(N,N'-Dimethylaminoethyl Methacrylate) with a Star Topology and Their Antibacterial Activity.

In this paper, we focus on the synthesis and characterization of novel stable nanolayers made of star methacrylate polymers. The effect of nanolayer modification on its antibacterial properties was also studied. A covalent immobilization of star poly(N,N′-dimethylaminoethyl methacrylate) (PDMAEMA) to benzophenone functionalized glass or silicon supports was carried out via a “grafting to” approach using UV irradiation. To date, star polymer UV immobilization has never been used for this purpose. The thickness of the resulting nanolayers increased from 30 to 120 nm with the molar mass of the immobilized stars. The successful bonding of star PDMAEMA to the supports was confirmed by surface sensitive quantitative spectroscopic methods. Next, amino groups in the polymer layer were quaternized with bromoethane, and the influence of this modification on the antibacterial properties of the obtained materials was analyzed using a selected reference strain of bacteria. The resulting star nanolayer surfaces exhibited higher antimicrobial activity against Bacillus subtilis ATCC 6633 compared to that of the linear PDMAEMA analogues grafted onto a support. These promising results and the knowledge about the influence of the topology and modification of PDMAEMA layers on their properties may help in searching for new materials for antimicrobial applications in medicine.

Spectral study of fluorone dyes adsorption on chitosan-based polyelectrolyte complexes

Polyelectrolyte complexes of the chitosan–chondroitin sulfate and chitosan–hyaluronate polycation–polyanion pairs were synthesized and characterized as potential dye adsorbents at different pH levels. Equilibrium isotherm analysis was applied to investigate the efficiency and the mechanism of the adsorption of fluorone dyes (fluorescein, eosinY, erythrosinB) on the synthesized complexes. The inefficiency of the fluorescein adsorption was proved by two different quantitative spectroscopic methods. The adsorption isotherm for both eosinY and erythrosinB was adequately described in terms of the Langmuir–Freundlich model. The observed room-temperature phosphorescence of the adsorbed erythrosinB was attributed to the surface inhomogeneity of the synthesized complexes. The revealed variation in the adsorption properties of fluorone dyes was related to the differences in their ionic forms as well as in their polarity and hydrophobicity.

Quantitative Ultrasound Evaluation of Tumor Cell Death Response in Locally Advanced Breast Cancer Patients Receiving Chemotherapy

Quantitative ultrasound techniques have been recently shown to be capable of detecting cell death through studies conducted on in vitro and in vivo models. This study investigates for the first time the potential of early detection of tumor cell death in response to clinical cancer therapy administration in patients using quantitative ultrasound spectroscopic methods. Patients (n = 24) with locally advanced breast cancer received neoadjuvant chemotherapy treatments. Ultrasound data were collected before treatment onset and at 4 times during treatment (weeks 1, 4, and 8, and preoperatively). Quantitative ultrasound parameters were evaluated for clinically responsive and nonresponding patients. Results indicated that quantitative ultrasound parameters showed significant changes for patients who responded to treatment, and no similar alteration was observed in treatment-refractory patients. Such differences between clinically and pathologically determined responding and nonresponding patients were statistically significant (P < 0.05) after 4 weeks of chemotherapy. Responding patients showed changes in parameters related to cell death with, on average, an increase in mid-band fit and 0-MHz intercept of 9.1 ± 1.2 dBr and 8.9 ± 1.9 dBr, respectively, whereas spectral slope was invariant. Linear discriminant analysis revealed a sensitivity of 100% and a specificity of 83.3% for distinguishing nonresponding patients by the fourth week into a course of chemotherapy lasting several months. This study reports for the first time that quantitative ultrasound spectroscopic methods can be applied clinically to evaluate cancer treatment responses noninvasively. The results form a basis for monitoring chemotherapy effects and facilitating the personalization of cancer treatment.

Oligomeric Carbocation-like Species from Protonation of Chloroalkanes

The protonation of chloroethane by the strongest known solid superacid, the carborane acid H(CHB(11)Cl(11)), has been studied by quantitative IR spectroscopic methods to track mass balance and uncover previously unobserved chemistry. In the first step, an intermediate EtCl·H(CHB(11)Cl(11)) species without full proton transfer to EtCl can be observed when d(5)-deuterated chloroethane is used. It rapidly eliminates HCl (but not DCl) to form ethyl carborane, Et(CHB(11)Cl(11)), which binds a second molecule of chloroethane to form the Et(2)Cl(+) chloronium ion. This undergoes a slower, previously unrecognized HCl elimination reaction to form a butyl carborane, Bu(CHB(11)Cl(11)), beginning an oligomerization process whereby unsymmetrical dialkylchloronium ions decompose to alkyl carboranes of formula Bu(C(2)H(4))(n)(CHB(11)Cl(11)) up to n = 4. Over time, a parallel competing process of de-oligomerization take place in the presence of free carborane acid that finishes with the formation of hexyl or butyl carboranes. Upon heating to 150 C, the final products are all converted to the remarkably stable tert-butyl cation carborane salt.

Inflammatory Response of Lung Derived Cells in Rat Pups Following Intratracheal Surfactant Instillation

Background: Neonatal respiratory-distress syndrome (nRDS) is a disease that affects the lung functions in premature infants who are born before their surfactant biosynthetic system has matured. Several surfactant-replacement therapies have been developed and are efficacious in treating the RDS. Objective: The study investigates the inflammatory response of the lung derived cells in rat pups with intratracheal instillation of surfactant (SF), isolated from porcine and the synthetic surfactant. Methods: Rat pups were divided into three groups: Controls were instilled with buffer, similarly P-SF and S-SF group of animals were instilled with porcine and synthetic surfactant, respectively. Bronchoalveolar fluid (BALF) was isolated and the different cytokines and reactive oxygen-nitrogen species (RONS) were measured using quantitative ELISA and fluorescence spectroscopic methods, respectively. Results: After 24 hrs, cytokines secretions were found to be elevated in the BALF with porcine and synthetic surfactant. Further, nitric oxide (NO) and citrulline levels as well as the reactive oxygen species (ROS) generation were also found to be increased with surfactant administration. Conclusion: In the treatment of respiratory distress syndrome, the anti-inflammatory response of surfactant may thus depend upon the specific preparation of the surfactant and its administration.

Spectroscopic Characterization of Linseed Oil Based Polymers

Linseed oil based polymers from cationic and thermal polymerizations have been investigated quantitatively through 1H NMR and FTIR spectroscopic analysis. The solubility of the samples ranges from 22 to 37% for cationic samples and from 4.23 to 53% for thermal samples. The content of the grafted linseed oil calculated from 1H NMR results ranges from 22.9 to 43.0% and from 0 to 10% for cationic and thermal samples. The grafted linseed oil contents from FTIR are 18.2−45.4% and 0−10.7% for cationic and thermal samples. The values obtained through quantitative 1H NMR and FTIR spectroscopic analysis methods are consistent and can be applied to other polymers also.

The Fastest Flights in Nature: High-Speed Spore Discharge Mechanisms among Fungi

BackgroundA variety of spore discharge processes have evolved among the fungi. Those with the longest ranges are powered by hydrostatic pressure and include “squirt guns” that are most common in the Ascomycota and Zygomycota. In these fungi, fluid-filled stalks that support single spores or spore-filled sporangia, or cells called asci that contain multiple spores, are pressurized by osmosis. Because spores are discharged at such high speeds, most of the information on launch processes from previous studies has been inferred from mathematical models and is subject to a number of errors.Methodology/Principal FindingsIn this study, we have used ultra-high-speed video cameras running at maximum frame rates of 250,000 fps to analyze the entire launch process in four species of fungi that grow on the dung of herbivores. For the first time we have direct measurements of launch speeds and empirical estimates of acceleration in these fungi. Launch speeds ranged from 2 to 25 m s−1 and corresponding accelerations of 20,000 to 180,000 g propelled spores over distances of up to 2.5 meters. In addition, quantitative spectroscopic methods were used to identify the organic and inorganic osmolytes responsible for generating the turgor pressures that drive spore discharge.Conclusions/SignificanceThe new video data allowed us to test different models for the effect of viscous drag and identify errors in the previous approaches to modeling spore motion. The spectroscopic data show that high speed spore discharge mechanisms in fungi are powered by the same levels of turgor pressure that are characteristic of fungal hyphae and do not require any special mechanisms of osmolyte accumulation.

Metabolite quantification and high-field MRS in breast cancer.

In vivo 1H MRS is rapidly developing as a clinical tool for diagnosing and characterizing breast cancers. Many in vivo and in vitro experiments have demonstrated that alterations in concentrations of choline-containing metabolites are associated with malignant transformation. In recent years, considerable efforts have been made to evaluate the role of 1H MRS measurements of total choline-containing compounds in the management of patients with breast cancer. Current technological developments, including the use of high-field MR scanners and quantitative spectroscopic analysis methods, promise to increase the sensitivity and accuracy of breast MRS. This article reviews the literature describing in vivo MRS in breast cancer, with an emphasis on the development of high-field MR scanning and quantitative methods. Potential applications of these technologies for diagnosing suspicious lesions and monitoring response to chemotherapy are discussed.

Quantitative analysis of biodegradable amphiphilic poly(L-lactide)-block-poly(ethyleneglycol)-blockpoly(L-lactide) by using TG, FTIR and NMR

The thermogravimetric analysis (TG) of two series of tri-block copolymers based on poly(L,L-lactide) (PLLA) and poly(ethyleneglycol) (PEG) segments, having molar mass of 4000 or 600 g mol–1, respectively, is reported. The prepared block copolymers presented wide range of molecular masses (800 to 47500 g mol–1) and compositions (16 to 80 mass% PEG). The thermal stability increased with the PLLA and/or PEG segment size and the tri-block copolymers prepared from PEG 4000 started to decompose at higher temperatures compared to those copolymers from PEG 600. The copolymers compositions were determined by thermogravimetric analysis and the results were compared to other traditional quantitative spectroscopic methods, hydrogen nuclear magnetic resonance spectrometry (1HNMR) and Fourier transform infrared spectrometry (FTIR). The PEG 4000 copolymer compositions calculated by TG and by 1HNMR, presented differences of 1%, demonstrating feasibility of using thermogravimetric analysis for quantitative purposes.

Measurements of Internal Distance Changes of the 30 S Ribosome Using FRET with Multiple Donor–Acceptor Pairs: Quantitative Spectroscopic Methods

We present analytical and experimental procedures for determining distance changes within the 30 S subunit of the Escherichia coli ribosome using Förster resonance energy transfer (FRET). We discuss ways to contend with complexities when using FRET to measure distance changes within large multi-subunit macromolecular complexes, such as the ribosome. Complications can arise due to non-stoichiometric labeling of donor and acceptor probes, as well as environmental effects that are specific to each conjugation site. We show how to account for changes in extinction coefficients, quenching, labeling stoichiometry and other variations in the spectroscopic properties of the dye to enable more accurate calculation of distances from FRET data. We also discuss approximations that concern the orientation of the transition moments of the two dye molecules, as well as the impact of other errors in the measurement of absolute distances. Thirteen dye-pair locations with different distances using 18 independent FRET pairs conjugated to specific 30 S protein residues have been used to determine distance changes within the 30 S subunit upon association with the 50 S subunit, forming the 70 S ribosome. Here, we explain the spectroscopic methods we have used, which should be of general interest in studies that aim at obtaining quantitative distance information from FRET.

Spectroscopic study on the interaction between methylene blue and chondroitin 4-sulfate and its analytical application.

The interaction of Methylene Blue (MB) with chondroitin-4-sulfate (CHS) has been investigated using spectroscopic techniques, including UV-Vis absorption, Rayleigh resonance scattering (RRS), and circular dichroism (CD). The addition of CHS caused a decrease in the absorbance of MB at 664 nm with a new absorption band appearing at 570 nm, enhanced RRS at 314 nm and 560 nm, and also resulted in an intense CD signal at 568 nm. The Scatchard model has been applied to calculating the binding constant and the number of binding sites. The calculated parameters are consistent with the experimental results. The factors affecting the interaction were investigated. Quantitative spectroscopic methods were developed for the first time. They are based on the fact that a decrease in the absorption at 664 nm and an enhancement of the RRS intensity at 314 nm are proportional to the concentration of CHS added in a certain range. Satisfactory results were obtained on the determination of synthetic samples.

Experimental and theoretical characterization of an inductively coupled plasma source

A typical planar inductively coupled plasma source was characterized by means of several diagnostic methods. Langmuir probe measurements as well as quantitative spectroscopic methods were used in order to determine electron density, mean electron energy, electron energy distribution functions, and the neutral particle temperature. A model based on hydrodynamic balance equations has been developed and used to predict parameters which cannot be determined by direct measurements. As the agreement of experimental and simulated data is excellent, the overall discharge efficiency, influences of substrate holder, fluxes as well as energies, and incident angle of ions on the substrate were determined.

Remote sensing of the earth from space

Abstract Remote sensing is the popular name for the measurement of physical quantities at a distance, usually by quantitative spectroscopic methods. Its application to the study of the Earth's atmosphere and surface from satellites is a rapidly growing field, with many applications, and new or improved techniques are constantly being developed. In this article we look at some of the basic principles and technical challenges involved, and at the benefits which are accruing in a number of research areas in earth science, with particular emphasis on the use of remote sensing to understand global change.

The secretion of protein material in the parasympathetic submaxillary saliva

This paper deals with a study of the secretion of protein material by the submaxillary gland of the cat in response to electrical stimulation of the chorda tympani nerve. In the attempt to make a more generous use of certain advantages offered by physical methods in research on the digestive glands, the submaxillary gland was chosen for the initial investigation because of the relative ease of physiological experimentation. The study is characterized by (1) the use of quantitative spectroscopic methods of analysis which permit an extension in the range of the investigation over that possible with a purely chemical procedure, and (2) the application of mathematical analysis in the interpretation of the results. The work of previous investigators (Heidenhain 1868, 1878, 1883; Werther 1886; Langley and Fletcher 1890; Gregerson and Ingalls 1931) has shown that with increasing intensity of the stimulus the para­-sympathetic submaxillary saliva becomes more concentrated in (1) inorganic salts which are presumably derived from the tissue fluids and ultimately from the blood, and (2) organic material, provided that the stores in the gland are not too much depleted by previous stimulation. The con­centration changes in the former group of substances are presumably connected with phenomena occurring at the glandular membranes; those in the latter group obviously cannot be accounted for by a simple washing out of stored material from the gland, but must result from the operation of a more complicated secretion mechanism. In the attempt to gain a better understanding of the fundamental nature of the secretion processes, we have determined spectroscopically the relative concentrations of protein material, and the Na and K concentrations in samples of saliva obtained in response to various intensities of stimulation of the chorda tympani nerve. Many of the samples were also analysed chemically for protein and non-protein nitrogen, chlorides, and acid-combining power. These sub­stances, along with water, are the main constituents of the submaxillary saliva as obtained in our experiments. A long series of relatively small samples of secretion was taken from each animal; this feature of the experimental technique is considered to be important, viz. under such conditions one may best determine the changes in composition resulting from the variation of one factor only—the intensity of the stimulus—since changes in other factors which are not directly controlled are small from sample to sample.

TRANSPORT OF MATERIAL IN SOURCES FOR SPECTROSCOPIC ANALYSIS

A study of the transport of material in the condensed d-c. spark discharge has been made, with the object of placing quantitative spectroscopic methods of analysis on a less empirical basis and thus permitting, (a) the recognition of certain conditions of source operation which lead to unreliable determinations, and (b) the determination of some of the criteria to be fulfilled for maximum precision in analytic measurements.Variations in the transport phenomena, as indicated by variations in the relative distributions of emitting atoms of various elements along the discharge axis on the addition of certain substances to a sample, appear to be closely connected with the ionization potentials and masses of the elements, and with the ionization potentials of other particles present in the discharge. The data indicate the necessity for the use of a spectroscopic buffer, and provide certain criteria for the choice of the buffer and of the internal standard element.The exact nature of the mechanism of transport is uncertain, but various considerations indicate that neither the motion of ions under the influence of the field nor diffusion in the ordinary sense is sufficient to account for observed features.