Ultrasound delivery of Surface Enhanced InfraRed Absorption active gold-nanoprobes into fibroblast cells: a biological study via Synchrotron-based InfraRed microanalysis at single cell level.

Ultrasound (US) induced transient membrane permeabilisation has emerged as a hugely promising tool for the delivery of exogenous vectors through the cytoplasmic membrane, paving the way to the design of novel anticancer strategies by targeting functional nanomaterials to specific biological sites. An essential step towards this end is the detailed recognition of suitably marked nanoparticles in sonoporated cells and the investigation of the potential related biological effects. By taking advantage of Synchrotron Radiation Fourier Transform Infrared micro-spectroscopy (SR-microFTIR) in providing highly sensitive analysis at the single cell level, we studied the internalisation of a nanoprobe within fibroblasts (NIH-3T3) promoted by low-intensity US. To this aim we employed 20 nm gold nanoparticles conjugated with the IR marker 4-aminothiophenol. The significant Surface Enhanced Infrared Absorption provided by the nanoprobes, with an absorbance increase up to two orders of magnitude, allowed us to efficiently recognise their inclusion within cells. Notably, the selective and stable SR-microFTIR detection from single cells that have internalised the nanoprobe exhibited clear changes in both shape and intensity of the spectral profile, highlighting the occurrence of biological effects. Flow cytometry, immunofluorescence and murine cytokinesis-block micronucleus assays confirmed the presence of slight but significant cytotoxic and genotoxic events associated with the US-nanoprobe combined treatments. Our results can provide novel hints towards US and nanomedicine combined strategies for cell spectral imaging as well as drug delivery-based therapies.

Vibrational spectroscopic monitoring and biochemical analysis of pericellular matrix formation and maturation in a 3-dimensional chondrocyte culture model.

Isolated and monolayer expanded chondrocytes are not the ideal cell form to produce a cartilage matrix. In articular cartilage, each chondrocyte is surrounded by a 2-4 µm thick collagen VI-rich pericellular matrix (PCM) forming a chondron. Freshly extracted chondrons form a more cartilage-like extracellular matrix (ECM) than chondrocytes and their surrounding PCM is thought to maintain the chondrocyte phenotype. To regenerate articular cartilage, preserving and/or regenerating a functional PCM is essential. In this study, a highly biomimicking hyaluronic acid (HA) hydrogel was used as a 3-dimensional system to culture freshly isolated bovine chondrons (with an intact PCM) and chondrocytes (without a PCM) for up to 21 days. We assessed the HA hydrogel's capacity to maintain and potentially re-generate PCM formation by both biochemical and immunological analyses of the key components of the PCM. For the first time, synchrotron based Fourier transform infrared (SR-FTIR) microspectroscopy was utilised to reveal the dynamic process of PCM re-generation. At day 1, highly specific collagen VI staining was visible within chondron containing HA hydrogels. In contrast, collagen VI was absent at day 1 but punctate, focal staining increased during the culture period of chondrocyte containing HA hydrogels. Chondron containing HA hydrogels produced more collagen II and GAGs than the chondrocyte containing HA hydrogels. Principal component analysis (PCA) of spectra in fingerprint regions of the chondrocyte-containing constructs at day 7, 14 and 21 culturing showed clear spectral differences. The clusters of day 14 and day 21 samples were closer to the chondron samples, while the day 7 samples were closer to chondrocytes. PCA scores in the lipid region revealed no major differences between chondrocyte and chondron samples, but showed that the cultured chondrocyte samples at day 7, day 14 and day 21 clustered together. These data would indicate that SR-FTIR microspectroscopy can help to better understand the PCM formation and maturation in tissue engineered models, which involves subtle changes in collagen and aggrecan.

Generating single metalloprotein crystals in well-defined redox states: electrochemical control combined with infrared imaging of a NiFe hydrogenase crystal.

We describe an approach to generating and verifying well-defined redox states in metalloprotein single crystals by combining electrochemical control with synchrotron infrared microspectroscopic imaging. For NiFe hydrogenase 1 from Escherichia coli we demonstrate fully reversible and uniform electrochemical reduction from the oxidised inactive to the fully reduced state, and temporally resolve steps during this reduction.

Effects of nilotinib on leukaemia cells using vibrational microspectroscopy and cell cloning.

Over the last few years, both synchrotron-based FTIR (S-FTIR) and Raman microspectroscopies have helped to better understand the effects of drugs on cancer cells. However, cancer is a mixture of cells with different sensitivity/resistance to drugs. Furthermore, the effects of drugs on cells produce both chemical and morphological changes, the latter could affect the spectra of cells incubated with drugs. Here, we successfully cloned sensitive and resistant leukaemia cells to nilotinib, a drug used in the management of leukaemia. This allowed both the study of a more uniform population and the study of sensitive and resistant cells prior to the addition of the drug with both S-FTIR and Raman microspectroscopies. The incubation with nilotinib produced changes in the S-FTIR and Raman spectra of both sensitive and resistant clones to nilotinib. Principal component analysis was able to distinguish between cells incubated in the absence or presence of the drug, even in the case of resistant clones. The latter would confirm that the spectral differences between the so-called resistant clonal cells prior to and after adding a drug might reside on those more or less sensitive cells that have been able to remain alive when they were collected to be studied with S-FTIR or Raman microspectroscopies. The data presented here indicate that the methodology of cell cloning can be applied to different types of malignant cells. This should facilitate the identification of spectral biomarkers of sensitivity/resistance to drugs. The next step would be a better assessment of sensitivity/resistance of leukaemia cells from patients which could guide clinicians to better tailor treatments to each individual patient.

Chemotherapeutic response to cisplatin-like drugs in human breast cancer cells probed by vibrational microspectroscopy.

Studies of drug-cell interactions in cancer model systems are essential in the preclinical stage of rational drug design, which relies on a thorough understanding of the mechanisms underlying cytotoxic activity and biological effects, at a molecular level. This study aimed at applying complementary vibrational spectroscopy methods to evaluate the cellular impact of two Pt(ii) and Pd(ii) dinuclear chelates with spermine (Pt2Spm and Pd2Spm), using cisplatin (cis-Pt(NH3)2Cl2) as a reference compound. Their effects on cellular metabolism were monitored in a human triple-negative metastatic breast cancer cell line (MDA-MB-231) by Raman and synchrotron-radiation infrared microspectroscopies, for different drug concentrations (2-8 µM) at 48 h exposure. Multivariate data analysis was applied (unsupervised PCA), unveiling drug- and concentration-dependent effects: apart from discrimination between control and drug-treated cells, a clear separation was obtained for the different agents studied - mononuclear vs. polynuclear, and Pt(ii) vs. Pd(ii). Spectral biomarkers of drug action were identified, as well as the cellular response to the chemotherapeutic insult. The main effect of the tested compounds was found to be on DNA, lipids and proteins, the Pd(ii) agent having a more significant impact on proteins while its Pt(ii) homologue affected the cellular lipid content at lower concentrations, which suggests the occurrence of distinct and unconventional pathways of cytotoxicity for these dinuclear polyamine complexes. Raman and FTIR microspectroscopies were confirmed as powerful non-invasive techniques to obtain unique spectral signatures of the biochemical impact and physiological reaction of cells to anticancer agents.

Towards microfluidic reactors for in situ synchrotron infrared studies.

Anodically bonded etched silicon microfluidic devices that allow infrared spectroscopic measurement of solutions are reported. These extend spatially well-resolved in situ infrared measurement to higher temperatures and pressures than previously reported, making them useful for effectively time-resolved measurement of realistic catalytic processes. A data processing technique necessary for the mitigation of interference fringes caused by multiple reflections of the probe beam is also described.

Unsupervised explorative data analysis of normal human leukocytes and BCR/ABL positive leukemic cells mid-infrared spectra.

We proved the ability of Fourier Transform Infrared microspectroscopy (microFTIR) complemented by Principal Component Analysis (PCA) to detect protein phosphorylation/de-phosphorylation in mammalian cells. We analyzed by microFTIR human polymorphonuclear neutrophil (PMNs) leukocytes, mouse-derived parental Ba/F3 cells (Ba/F3#PAR), Ba/F3 cells transfected with p210(BCR/ABL) (Ba/F3#WT) and expressing high levels of protein tyrosine kinase (PTK), and human-derived BCR/ABL positive K562 leukemic cell sub-clones engineered to differently express receptor-type tyrosine-protein phosphatase gamma (PTPRG). Synchrotron radiation (SR) and conventional (globar) IR sources were used to perform microFTIR respectively, on single cells and over several cells within the same sample. Ex vivo time-course experiments were run, inducing maximal protein phosphorylation in PMNs by 100 nM N-formylated tripeptide fMLP. Within the specific IR fingerprint 1800-850 cm(-1) frequency domain, PCA identified two regions with maximal signal variance. These were used to model and test the robustness of PCA in representing the dynamics of protein phosphorylation/de-phosphorylation processes. An IR signal ratio marker reflecting the homeostatic control by protein kinases and phosphatases was identified in normal leukocytes. The models identified by microFTIR and PCA in normal leukocytes also distinguished BCR/ABL positive Ba/F3#WT from BCR/ABL negative Ba/F3#PAR cells as well as K562 cells exposed to functionally active protein tyrosine phosphatase recombinant protein ICD-Tat transduced in cells by HIV-1 Tat technology or cells treated with the PTK inhibitor imatinib mesylate (IMA) from cells exposed to phosphatase inactive (D1028A)ICD-Tat recombinant protein and untreated control cells, respectively. The IR signal marker correctly reflected the degrees of protein phosphorylation associated with abnormal PTK activity in BCR/ABL positive leukemic cells and in general was inversely related to the expression/activity of PTPRG in leukemic sub-clones. In conclusion, we have described a new, reliable and simple spectroscopic method to study the ex vivo protein phosphorylation/de-phosphorylation balance in cell models: it is suitable for biomedical and pharmacological research labs but it also needs further optimization and its evaluation on large cohorts of patients to be proposed in the clinical setting of leukemia.

Mapping the intake of different elements in vegetal tissues by dual-energy X-ray imaging at DaPhine synchrotron light source.

This article reports on the first utilization of the soft X-ray beamline at the DaPhine synchrotron light source for mapping the intake of different elements in plant tissues. As a test, the method of dual-energy X-ray microradiography was applied to the investigation of the natural sulfur content in dried leaf and root samples. Our ultimate goal was to monitor the pollutant lead and its intake, which was added in controlled doses to the hydroponic medium of laboratory-controlled samples of vegetal species. The results obtained by the nondestructive X-ray radiographic analysis are compared to the values of concentrations determined by a standard chemical analysis utilizing atomic absorption spectroscopy. From this comparison the validity of the X-ray detection of heavy metals in biological samples has been confirmed. The superposition of the dual energy results on the simple planar radiography shows the representation of the pollutant intake directly on the sample structures. It should be pointed out that this method, developed here for plant root and leaves could be applied to any biological sample of interest, but the preparation and observation conditions necessitate different strategies according to the type of sample under analysis.

Monitoring of the heavy-metal hyperaccumulation in vegetal tissues by X-ray radiography and by femto-second laser induced breakdown spectroscopy.

This article reports on the utilization of X-ray microradiography and laser induced breakdown spectroscopy (LIBS) techniques for investigation of the metal accumulation in different part of leaf samples. The potential of the LIBS-analysis for finding the proper plant species for phytoremediation is compared with the results of microradiography measurements at the HERCULES source at ENEA, Rome (Italy) and X-ray microradiography experiments at the ELETTRA Synchrotron, Trieste (Italy).

Functional architecture of the major light-harvesting complex from higher plants.

Light-harvesting complexes (Lhc) catalyse sunlight harvesting for photosynthesis as well as other essential functions, including photoprotection by quenching of harmful chlorophyll triplet states and prevention of photoinhibition by dissipation of excitation energy in excess. In addition, folding of Lhc proteins depends on the availability of both xanthophylls and carotenoids, thus preventing the potential formation of harmful chlorophyll-protein complexes lacking photoprotectors. We have used the mutation analysis in order to study the association of the different functions to three protein domains, each composed of a xanthophyll molecule and of neighbour chlorophylls a and b, within the major antenna complex of photosystem II, i.e. LHCII. We have found that the xanthophyll to chlorophyll energy transfer is a shared property of the whole pigment-protein complex, and occurs with similar efficiency in each of the three structural domains. Photoprotection by quenching of chlorophyll triplets is catalysed mainly by lutein bound to site L1, and occurs via energy transfer from chlorophylls A1 and B1. This domain is essential for pigment-induced protein folding. The domains L2 and N1 weakly influence either the protein stability or the photoprotection; however, replacement of xanthophyll species bound to these structural domains modulates the fluorescence quantum yield of LHCII, and suggests that non-radiative dissipation of excess energy can be regulated through allosteric modification of the protein structure by exchanging xanthophylls in these sites.

IP videoconferencing using a quality of service public network.

Videoconferencing in the medical world has been successfully used for quite a few years. Nevertheless it has not spread significantly in daily use. Some of the problems rely on the infrastructure needed to set up a video session: one or more ISDN lines or a fast Internet connection. The first is not easily available everywhere in a building; the latter is rarely so fast to allow for a smooth operation with no quality drops. The use of Videonet, the first European commercial public Internet link with guaranteed Quality of Service (QoS), has the potential to be a breakthrough in videoconferencing. We describe this new system and its applications, with the first tests in a hospital environment. Our results show that there are still problems to be solved in order to achieve a quality comparable to ISDN.

Time-resolved fluorescence analysis of the photosystem II antenna proteins in detergent micelles and liposomes.

We have studied the time-resolved fluorescence properties of the light-harvesting complexes (Lhc) of photosystem II (Lhcb) in order to obtain information on the mechanism of energy dissipation (non-photochemical quenching) which is correlated to the conversion of violaxanthin to zeaxanthin in excess light conditions. The chlorophyll fluorescence decay of Lhcb proteins LHCII, CP29, CP26, and CP24 in detergent solution is mostly determined by two lifetime components of 1.2-1.5 and 3.6-4 ns while the contribution of the faster component is higher in CP29, CP26, and CP24 with respect to LHCII. The xanthophyll composition of Lhc proteins affects the ratio of the lifetime components: when zeaxanthin is bound into the site L2 of LHCII, the relative amplitude of the faster component is increased and, consequently, the chlorophyll fluorescence quenching is enhanced. Analysis of quenching in mutants of Arabidopsis thaliana, which incorporate either violaxanthin or zeaxanthin in their Lhc proteins, shows that the extent of quenching is enhanced in the presence of zeaxanthin. The origin of the two fluorescence lifetimes was analyzed by their temperature dependence: since lifetime heterogeneity was not affected by cooling to 77 K, it is concluded that each lifetime component corresponds to a distinct conformation of the Lhc proteins. Upon incorporation of Lhc proteins into liposomes, a quenching of chlorophyll fluorescence was observed due to shortening of all their lifetime components: this indicates that the equilibrium between the two conformations of Lhcb proteins is displaced toward the quenched conformation in lipid membranes or thylakoids with respect to detergent solution. By increasing the protein density in the liposomes, and therefore the probability of protein-protein interactions, a further decrease of fluorescence lifetimes takes place down to values typical of quenched leaves. We conclude that at least two major factors determine the quenching of chlorophyll fluorescence in Lhcb proteins, i.e., intrasubunit conformational change and intersubunit interactions within the lipid membranes, and that these processes are both important in the photoprotection mechanism of nonphotochemical quenching in vivo.

The chloroplast gene ycf9 encodes a photosystem II (PSII) core subunit, PsbZ, that participates in PSII supramolecular architecture.

We have characterized the biochemical nature and the function of PsbZ, the protein product of a ubiquitous open reading frame, which is known as ycf9 in Chlamydomonas and ORF 62 in tobacco, that is present in chloroplast and cyanobacterial genomes. After raising specific antibodies to PsbZ from Chlamydomonas and tobacco, we demonstrated that it is a bona fide photosystem II (PSII) subunit. PsbZ copurifies with PSII cores in Chlamydomonas as well as in tobacco. Accordingly, PSII mutants from Chlamydomonas and tobacco are deficient in PsbZ. Using psbZ-targeted gene inactivation in tobacco and Chlamydomonas, we show that this protein controls the interaction of PSII cores with the light-harvesting antenna; in particular, PSII-LHCII supercomplexes no longer could be isolated from PsbZ-deficient tobacco plants. The content of the minor chlorophyll binding protein CP26, and to a lesser extent that of CP29, also was altered substantially under most growth conditions in the tobacco mutant and in Chlamydomonas mutant cells grown under photoautotrophic conditions. These PsbZ-dependent changes in the supramolecular organization of the PSII cores with their peripheral antennas cause two distinct phenotypes in tobacco and are accompanied by considerable modifications in (1) the pattern of protein phosphorylation within PSII units, (2) the deepoxidation of xanthophylls, and (3) the kinetics and amplitude of nonphotochemical quenching. The role of PsbZ in excitation energy dissipation within PSII is discussed in light of its proximity to CP43, in agreement with the most recent structural data on PSII.

Energy transfer among CP29 chlorophylls: calculated Förster rates and experimental transient absorption at room temperature.

The energy transfer rates between chlorophylls in the light harvesting complex CP29 of higher plants at room temperature were calculated ab initio according to the Förster mechanism (Förster T. 1948, Ann. Physik. 2:55-67). Recently, the transition moment orientation of CP29 chlorophylls was determined by differential linear dichroism and absorption spectroscopy of wild-type versus mutant proteins in which single chromophores were missing (Simonetto R., Crimi M., Sandonà D., Croce R., Cinque G., Breton J., and Bassi R. 1999. Biochemistry. 38:12974-12983). In this way the Q(y) transition energy and chlorophyll a/b affinity of each binding site was obtained and their characteristics supported by reconstruction of steady-state linear dichroism and absorption spectra at room temperature. In this study, the spectral form of individual chlorophyll a and b ligands within the protein environment was experimentally determined, and their extinction coefficients were also used to evaluate the absolute overlap integral between donors and acceptors employing the Stepanov relation for both the emission spectrum and the Stokes shift. This information was used to calculate the time-dependent excitation redistribution among CP29 chlorophylls on solving numerically the Pauli master equation of the complex: transient absorption measurements in the (sub)picosecond time scale were simulated and compared to pump-and-probe experimental data in the Q(y) region on the native CP29 at room temperature upon selective excitation of chlorophylls b at 640 or 650 nm. The kinetic model indicates a bidirectional excitation transfer over all CP29 chlorophylls a species, which is particularly rapid between the pure sites A1-A2 and A4-A5. Chlorophylls b in mixed sites act mostly as energy donors for chlorophylls a, whereas site B5 shows high and bidirectional coupling independent of the pigment hosted.

The Soret absorption properties of carotenoids and chlorophylls in antenna complexes of higher plants.

The absorption spectra of two light harvesting complexes from higher plants, CP29 and LHC II, have been analysed in the Soret region in order to obtain a description in terms of the absorption spectra of the individual pigments. This information is of great practical use when applying spectroscopic techniques to the study of energy transfer in photosynthesis such as time-resolved spectroscopy thus allowing determination of the relative absorption cross-section for the different chromophores in the system as a function of wavelength. In this study, recombinant Lhc proteins carrying point mutations in pigment-binding residues have been used in order to obtain the spectral shape of individual chromophores by differential spectroscopy with respect to the WT protein. Combinations of spectra thus obtained were then used to fit the absorption spectra of WT and mutant pigment-proteins according to the constraints posed by stoichiometry of pigments as derived by biochemical analysis. This procedure allowed identification of each pigment in term of its wavelength position, spectral shape and extinction coefficient. The data obtained by this procedure have been successfully applied to the description of other higher plant Lhc proteins thus supporting the view that the Lhc superfamily members share specific pigment-protein interactions as suggested by sequence homology.

Absorption spectra of chlorophyll a and b in Lhcb protein environment.

The spectral forms of the two chlorophyll species in higher plant Photosystem II antenna proteins have been experimentally determined within their protein environment. Recombinant CP29 and LHC II antenna proteins missing individual chromophores were obtained by over-expression in bacteria without any changing of the primary protein sequence and in vitro reconstitution. Difference absorption spectroscopy with respect to the corresponding proteins binding the complete pigment complement yielded the spectral shape and extinction of single chlorophyll a and b. A functional relation of their absorption was given by Gaussian subband decomposition covering the entire Q(x) and Q(y) optical region together with the absolute value of the molar extinction coefficient. With respect to analogous determinations reported in the literature for organic solvents, this information is valuable for further understanding the in-protein chlorophyll excited states and excited state dynamics: in particular, for the calculation of Förster transfer rates by means of chlorophyll-chlorophyll overlap integral employing the Stepanov relation for emission and single chromophore transition energies according to the results of mutational analysis of chlorophyll binding sites [Bassi et al. (1999) Proc Natl Acad Sci USA 96: 10056-10061; Remelli et al. (1999) J Biol Chem 274: 33510-33521].

Orientation of chlorophyll transition moments in the higher-plant light-harvesting complex CP29.

The Q(y) transition dipole moment vectors of all eight chlorophylls in the higher-plant antenna protein CP29 were calculated by an original method on the basis of linear dichroism and absorption spectroscopy. The contribution of individual chromophores was determined from difference spectra between wild type and mutant proteins in which a single chlorophyll has been removed by mutating pigment-binding residues. Recombinant proteins were constructed by overexpressing the apoprotein in bacteria and refolding of the pigment-protein complex in vitro [Bassi, R., Croce, R., Cugini, D., and Sandonà, D. (1999) Proc. Natl. Acad. Sci. U.S.A. (in press)]. The spectroscopic data are interpreted on the basis of a protein structural model obtained via the homology with the major antenna complex LHCII [Kuhlbrandt, W., Wang, D. N., and Fujiyoshi, Y. (1994) Nature 367, 614-621]. The results allow us to determine the orientation of six chromophores within the protein structure. The orientations of the two remaining chromophores are inferred by considering the symmetry properties of CP29 and fitting steady state absorption and linear dichroism spectra by independent chlorophyll spectral forms. As a consequence, four "mixed" sites with different chlorophyll a and b binding affinities are identified in CP29. Geometrical data and the Förster mechanism for energy transfer suggest that excitation energy equilibrates rapidly among chlorophyll "pure" sites while energy preferentially flows outward from chlorophyll "mixed" sites. The orientation of the dipole moments of two chlorophyll molecules symmetrically located at the center of the protein and parallel to the carotenoid transition vectors suggests a role in energy transfer from xanthophyll to chlorophyll.

The TSH-dependent variation of the essential elements iodine, selenium and zinc within human thyroid tissues.

Instrumental Neutron Activation Analysis was used in order to measure iodine, selenium and zinc concentration in thyroid samples. A pair of samples of normal and nodular tissue were collected from the thyroid gland from 72 patients selected on the basis of pathological criteria (44 cases of multinodular goiter, 12 of chronic lymphocytic thyroiditis (CLT), 6 of thyroid adenoma (TA) and 12 of thyroid cancer (TC)). The check for tissue homogeneity and sampling error was performed by means of the coefficient of variation (CV%) of the elements in replicate samples of normal and altered tissues. High CV% values (> 15%) for iodine reflected a functional variability in thyroid follicles, while low CV% values (< 10%) for selenium and zinc indicated that the composition of selected tissues was rather homogeneous. The variation of the element's concentration was compared in normal and altered tissues. The mean element concentrations had values close to those already reported in the literature; furthermore, our patients had marginal iodine and selenium deficiency. Both normal and nodular tissues in CLT showed statistically significant lower zinc values as compared with the other thyroid diseases. To evaluate the thyroid function, thyroid stimulating hormone (TSH) and thyroxine (T4) levels were measured in the serum of patients. Two arbitrary serum-TSH threshold levels (TSH < 1.0 and > 4.0 mU/L) were introduced in order to classify, respectively, hyperthyroidism and hypothyroidism, as well as euthyroid conditions (1.0 < TSH < 4.0 mU/L), and each patient was assigned to one of these groups. The influence of TSH in the variation of the concentration of iodine, selenium and zinc in normal and altered human thyroid tissues was significant.

In vitro reconstitution of the recombinant photosystem II light-harvesting complex CP24 and its spectroscopic characterization.

The light-harvesting chlorophyll a/b protein CP24, a minor subunit of the photosystem II antenna system, is a major violaxanthin-binding protein involved in the regulation of excited state concentration of chlorophyll a. This subunit is poorly characterized due to the difficulty in isolation and instability during purification procedures. We have used an alternative approach in order to gain information on the properties of this protein; the Lhcb6 cDNA has been overexpressed in bacteria in order to obtain the CP24 apoprotein, which was then reconstituted in vitro with xanthophylls, chlorophyll a, and chlorophyll b, yielding a pigment-protein complex with properties essentially identical to the native protein extracted from maize thylakoids. Although all carotenoids were supplied during refolding, the recombinant holoprotein exhibited high selectivity in xanthophyll binding by coordinating violaxanthin and lutein but not neoxanthin or beta-carotene. Each monomer bound a total of 10 chlorophyll a plus chlorophyll b and two xanthophyll molecules. Moreover, the protein could be refolded in the presence of different chlorophyll a to chlorophyll b ratios for yielding a family of recombinant proteins with different chlorophyll a/b ratios but still binding the same total number of porphyrins. A peculiar feature of CP24 was its refolding capability in the absence of lutein, contrary to the case of other homologous proteins, thus showing higher plasticity in xanthophyll binding. These characteristics of CP24 are discussed with respect to its role in binding zeaxanthin in high light stress conditions. The spectroscopic analysis of a recombinant CP24 complex binding eight chlorophyll b molecules and a single chlorophyll a molecule by Gaussian deconvolution allowed the identification of four subbands peaking at wavelengths of 638, 645, 653, and 659 nm, which have an increased amplitude with respect to the native complex and therefore identify the chlorophyll b absorption in the antenna protein environment. Gaussian subbands at wavelengths 666, 673, 679, and 686 nm are depleted in the high chlorophyll b complex, thus suggesting they derive from chlorophyll a.

Structure-activity relationship of new growth inhibitors of Trypanosoma cruzi.

Several drugs bearing the 4-phenoxyphenoxy skeleton and other closely related structures were designed, synthesized, and evaluated as antiproliferative agents against Trypanosoma cruzi, the etiologic agent of Chagas' disease. The new class of drugs was envisioned by modifying the nonpolar 4-phenoxyphenoxy moiety replacing selected aromatic protons by different groups via electrophilic aromatic substitution reactions as well as introducing a sulfur atom at the polar extreme. Of the designed compounds, sulfur-containing derivatives were shown to be potent antireplicative agents against T. cruzi. Among these drugs, 4-phenoxyphenoxyethyl thiocyanate (compound 56) proved to be an extremely active growth inhibitor of the epimastigote forms of T. cruzi and displayed an IC50 of 2.2 microM. Under the same assay conditions, this drug was much more active than Nifurtimox, one of the drugs currently in clinical use to control this disease. This thiocyanate derivative was also a very active inhibitor against the intracellular form of the parasite at the nanomolar level. Other sulfur derivatives prepared also exhibited very potent antiproliferative action against T. cruzi. The presence of a sulfur atom at the polar extreme for this family of compounds seems to be very important for biological action because this atom was always associated with high inhibition values. 4-Phenoxyphenoxyethyl thiocyanate presents very good prospective not only as a lead drug but also as a potential chemotherapeutic agent.