EFFECT OF NITROGEN STARVATION ON OPTICAL PROPERTIES,PIGMENTS, AND ARACHIDONIC ACID CONTENT OF THE UNICELLULAR GREEN ALGA PARIETOCHLORIS INCISA (TREBOUXIOPHYCEAE,CHLOROPHYTA)1

Spectral properties of cell suspensions, individual cells, and extracts of the unicellular green alga Parietochloris incisa (Reisigl) Shin Watan. grown under low light were studied. Long‐term nitrogen (N) deprivation resulted in a decrease of chloroplast volume, appearance of numerous large cytoplasmic oil bodies, and the deposition of triacylglycerols with a high proportion of arachidonic acid. Chlorophylls a and b underwent a synchronous decline, whereas carotenoids (Car) showed a relative increase. Simultaneously, significant qualitative changes in the spectral properties of P. incisa individual cells, cell extracts, and cell suspensions were observed. To a large extent, the spectral changes observed in cell suspension could be attributed to a decrease in overall pigment content, leading to a gradual weakening of the so‐called package effect and accumulation of additional amounts of Car over chl, most probably, in oil bodies. Several optical characteristics of cell suspensions could serve as sensitive indicators of N‐deficiency in P. incisa. Furthermore, the absorption ratios, A₄₇₆/A₆₇₆ and A₆₅₀/A_676, showed close correlations with the Car‐to‐chl ratio and relative arachidonic acid (AA) content, respectively. The latter makes it possible to suggest that the increase in AA percentage in P. incisa proceeds in parallel with a decrease in cell chl content, accounting for the weakening of the package effect. N‐replenishment resulted in complete recovery of cell optical properties. The possible significance of the changes in cell ultrastructure, pigments, lipids, and optical properties is discussed with special reference to the ability of algae to adapt to and survive under conditions of long‐term nutrient deficiency.     

Rapid techniques for assessing fibre quality of flax breeding lines and cultivars using visible and near infrared spectroscopy, and thermal analysis

Fifteen different flax cultivars and breeding lines (E‐68, Baltu?iai, Belinka, Vega 2, Ilona, Elise, Kasty?iai, Evelin, 1963‐3, Ariane, Hermes, 01057‐12, 1698‐13a, 2017‐3, 1864‐24) were cultivated, harvested, water‐retted, scutched and resulting fibres passed through pin frames to produce representative samples for each variety. The aim of this investigation was to develop rapid techniques for assessing quality of fibre obtained from a comparative agronomical trial. The fibres produced were then assessed using visible and near infrared spectroscopy (Vis–NIRS), thermal analysis, scanning electron microscopy and airflow method for measuring fibre fineness. The relationships between agronomical characteristics, fibre fineness, thermal and spectral results were assessed using principal component analysis and partial least squares regression methods. The micrographs of the samples revealed the presence of residual pectic and cuticular tissues on all fibres, and significant differences between the 15 fibre samples were not observed. A significant relationship of the differences in fibre fineness of the test samples as measured by airflow method and Vis–NIR spectra was observed with an R² of 0.97 and standard error of calibration (SEC) of 1.69 dtex, and the former parameter also correlated with the measured thermal combustion parameters showing an R² of 0.91 and SEC of 2.86 dtex, indicating that the two rapid techniques could be used for the assessment of fibre quality of selected plants from the breeding programme. The advantages of using the two instrumental techniques compared with the existing airflow method are briefly discussed.     

Vibrational CD (VCD) and atomic force microscopy (AFM) study of DNA interaction with Cr3+ ions: VCD and AFM evidence of DNA condensation

The interaction of natural calf thymus DNA with Cr³⁺ ions was studied at room temperature by means of vibrational CD (VCD) and infrared absorption (ir) spectroscopy, and atomic force microscopy (AFM). Cr³⁺ ion binding mainly to N₇ (G) and to phosphate groups was demonstrated. ψ‐Type VCD spectra resembling electronic CD (ECD) spectra, which appear during ψ‐type DNA condensation, were observed. These spectra are characterized mainly by an anomalous, severalfold increase of VCD intensity. Such anomalous VCD spectra were assigned to DNA condensation with formation of large and dense particles of a size comparable to the wavelength of the probing ir beam and possessing large‐scale helicity. Atomic force microscopy confirmed DNA condensation by Cr³⁺ ions and the formation of tight DNA particles responsible for the ψ‐type VCD spectra. Upon increasing the Cr³⁺ ion concentration the shape of the condensates changed from loose flower‐like structures to highly packed dense spheres. No DNA denaturation was seen even at the highest concentration of Cr³⁺ ions studied. The secondary structure of DNA remained in a B‐form before and after the condensation. VCD and ir as well as AFM proved to be an effective combination for investigating DNA condensation. In addition to the ability of VCD to determine DNA condensation, VCD and ir can in the same experiment provide unambiguous information about the secondary structure of DNA contained in the condensed particles. © 2002 Wiley Periodicals, Inc. Biopolymers 61: 243–260, 2002     

Insights into the bile acid transportation system: the human ileal lipid-binding protein-cholyltaurine complex and its comparison with homologous structures

Bile acids are generated in vivo from cholesterol in the liver, and they undergo an enterohepatic circulation involving the small intestine, liver, and kidney. To understand the molecular mechanism of this transportation, it is essential to gain insight into the three-dimensional (3D) structures of proteins involved in the bile acid recycling in free and complexed form and to compare them with homologous members of this protein family. Here we report the solution structure of the human ileal lipid-binding protein (ILBP) in free form and in complex with cholyltaurine. Both structures are compared with a previously published structure of the porcine ILBP-cholylglycine complex and with related lipid-binding proteins. Protein structures were determined in solution by using two-dimensional (2D)- and 3D-homo and heteronuclear NMR techniques, leading to an almost complete resonance assignment and a significant number of distance constraints for distance geometry and restrained molecular dynamics simulations. The identification of several intermolecular distance constraints unambiguously determines the cholyltaurine-binding site. The bile acid is deeply buried within ILBP with its flexible side-chain situated close to the fatty acid portal as entry region into the inner ILBP core. This binding mode differs significantly from the orientation of cholylglycine in porcine ILBP. A detailed analysis using the GRID/CPCA strategy reveals differences in favorable interactions between protein-binding sites and potential ligands. This characterization will allow for the rational design of potential inhibitors for this relevant system.     

Simultaneous and rapid monitoring of biomass and biopolymer production by Sphingomonas paucimobilis using Fourier transform-near infrared spectroscopy

The application of Fourier Transform near infrared spectroscopy (FT-NIRS) to near real-time monitoring of polysaccharide and biomass concentration was investigated using a gellan-producing strain of Sphingomonas paucimobilis grown in a stirred tank reactor. Successful models for both biomass and gellan were constructed despite the physichochemical complexity of the viscous process fluid. Modelling of biomass proved more challenging than for gellan, partly because of the low range of biomass concentration but a model with a good correlation coefficient (0.94) was formulated based on second derivative spectra. The gellan model was highly satisfactory, with an excellent correlation coefficient (0.98), again based on second derivative spectra. No sample pre-treatment was required and all spectral scanning was carried out on whole broth. Additionally, both models should be robust in practice since both were formulated using low numbers of factors. Thus, the near real time simultaneous monitoring of gellan and biomass in this highly complex matrix using FT-NIRS potentially opens the way to greatly improved process control strategies.     

Structural changes of low density lipoproteins with Cu2+ and glucose induced oxidation

The compositional and structural changes of lipids and apolipoproteins during in vitro oxidation of low density lipoprotein (LDL) are investigated in this study by IR spectroscopy. For comparison, LDL samples containing either copper or glucose at physiological or pathological concentrations are considered in order to know the separate affects of these chemical factors on LDL oxidation. The results show that the initial steps of lipid oxidation proceed through hydrogen atom loss from methylene groups, as well as loss of cholesteryl ester molecules, and later a recovering of carbonyl compounds resulting from aldehyde formation that generally occurs in autooxidation processes. Lipid oxidation is induced by copper ions, and glucose enhances metal ion induced LDL oxidation as determined by conjugated diene formation. As to the protein conformational changes, IR spectroscopy reveals for the first time that LDL oxidation involves formation of beta-sheet structures, these being more abundant in LDL samples with pathological concentrations of glucose or copper. Consequently, the LDL structural changes may contribute to the recognition of oxidized LDL particles by scavenger receptors.     

Predicting the conformational states of cyclic tetrapeptides

Biologically active cyclic tetrapeptides, usually found among fungi metabolites, exhibit phytotoxic or cytostatic activities that are likely to be governed by specific conformations adopted in solution. For conformational studies and drug design, there is a strong interest in using fast and reliable methods to determine correctly the conformational population of cyclotetrapeptides. We show here that standard molecular mechanics computational approach gives satisfactory results. The method was validated step by step by experimental data either obtained after synthesis and NMR analysis, or found in the literature. The cyclo(Gly)(4), cyclo(Ala)(4), cyclo(Sar)(4), and cyclo(SarGly)(2) peptides were used to evaluate the prediction of the peptide backbone conformation, and the detailed conformational analysis of tentoxin, a natural phytotoxic cyclotetrapeptide in which N-alkylated peptide bonds alternate with regular secondary ones, was used to validate the computation of conformers proportions. From the knowledge of an initial cyclic primary structure and of the D or L configuration of the amino acids, we show that it is possible to determine the exact orientation of carbonyl groups and to predict the nature of conformers present in solution. The proportion of each conformer can be inferred from a statistical thermodynamics approach by using the potential energy values of each conformer, computed by molecular mechanics methods with the TRIPOS force field, which allowed us to account for the solvent. The solvent contribution was processed by two different methods according to the nature of the interactions: whether through the dielectric constant introduced in the electrostatic potential, when interaction with solute molecules are weak or negligible, or through the computation of free energy of solvation using the algorithm SILVERWARE for solvents explicitly interacting with the solute. When applied to tentoxin, this conformational analysis yielded results in very good agreement with the experimental data reported by Pinet et al. (Biopolymers, 1995, Vol. 36, pp. 135-152), on both the nature of existing conformers and their relative proportions, whatever the nature of the considered solvent.     

Structural characterization of the second TSP1-module of human thrombospondin

The TSP1-module has been first identified as the type 1 repeat of thrombospondin-1. Members of this extracellular module-family have since been shown to be present in several hundred metazoan proteins as well as in proteins of some protists. Despite the widespread occurrence and biological importance of this module-type, relatively little is known about their three-dimensional structure. To define the structural features of this important module-family, we have expressed the second TSP1-domain of human thrombospondin 1 in Escherichia coli. Amino acid sequencing of proteolytic fragments of the recombinant protein have shown that its disulfide bonds connect the six conserved cysteines in a 1-5, 2-6, 3-4 pattern. Circular dichroism studies on the recombinant protein indicate that the disulfide-bonded TSP1-module consists primarily of distorted beta-strands.     

Selectivity in heavy metal- binding to peptides and proteins

The metal-binding affinities and three-dimensional structures of three synthetic 18-residue peptides with sequences derived from that of the highly conserved metal-binding motif MXCXXC found in many heavy metal-binding proteins were determined. A change in register of the cysteines and alanines of the sequence from the periplasmic mercury-binding protein, MerP, i.e., CAAC, CACA, and CCAA, affects the specificity of metal binding, in particular, the peptide with vicinal cysteines binds only mercury. The three-dimensional structures of the mercury-bound forms of the three peptides determined in solution by NMR spectroscopy peptides differ considerably, even though they are all linear bicoordinate complexes. The three-dimensional structure of the peptide with CAAC bound to Cd(II) demonstrates that the metal-binding loop is malleable enough to accommodate modes of coordination other than linear bicoordinate.