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We analysed deposits collected on the lower divertor of WEST after the first high fluence campaign performed in 2023. Deposits were collected on the high field side (thick deposition area) of 2 ITER-grade plasma facing units (PFUs), located on different toroidal positions. Using focused ion beam cross sectioning, the deposits were found to be very thick (12–55 µm). A significant difference was observed in the toroidal direction with thicker deposits for the PFU located at the maximal heat load in the inner side, showing a deposition pattern due to the toroidal magnetic field modulation. Deposits present a complex layer-by-layer structure with dense layers, some melted parts and porosities within the layers. The deposition is mainly composed of tungsten with oxygen, boron, carbon and traces of nitrogen whose composition vary along the radial direction. We identified W-rich deposits in the high plasma flux area near the inner strike point and deposits rich in O, B, C and N in the low plasma flux area further away from this strike point. W dense layers of about 5 up to 40 µm thick in the thick deposit area near the strike point were attributed to the high fluence campaign. Pure boron layers resulting from wall conditioning and processed by plasma wall interactions were also observed.

The evaluation of the impact of plasma-facing units (PFUs) damage on subsequent plasma operation is an important issue for ITER. This is one of the topic addressed by the operation of dedicated PFUs in WEST. From the start of WEST in 2017, few ITER grade divertor PFUs were tested in the lower divertor for short plasma exposure (experimental campaigns C1–C5). A PFU is an assembly of tungsten blocks on CuCrZr tube via pure copper interlayer. Since 2022, WEST operates with a lower divertor fully equipped with ITER grade PFUs (experimental campaign C6–C7) with long duration plasma exposure capabilities. To test the ageing of the ITER-grade PFUs on an accelerated basis, well controlled damage was implemented on purpose with electron beam loading on the plasma facing surface of two different PFUs (pre-damage#1 and #2 featuring different level of damage). Damage tackled in this study is induced by the loading with numerous transient heat loading events to mimic the ELM impact on the ITER grade PFU. This paper presents the damage before and after WEST plasma exposure and shows the tungsten sources and temperature of the block measured during the operation. On damaged area, surface roughening is heterogeneous. With WEST campaigns (C3, C4 and C7) respectively ended in (2018, 2019 and 2023) a global smoothing of the pre-damaged surfaces, in particular those located in the high loaded erosion area, is observed. No evolution of the crack is observed in the electron beam loaded area, while new cracking are observed on non pre-damaged area on the tungsten top surfaces in highly loaded areas (outer strike point) after significant plasma duration and cumulated energy (first observation in C4 and tends to be confirmed with preliminary observations after the C7 campaign). Smoothing is less pronounced after the 2018 campaign, which may be due to the related short plasma duration. The surface smoothing of blocks also depends on their poloidal position. For PFU which has no shaping, toroidal positioning within a block has no influence on the surface smoothing. After 2019 and 2023 campaigns, equivalent relative smoothing is obtained (between 19 and 32%). For the explored plasma conditions, damage has no influence on the maximum brightness of W.

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This study demonstrates that Raman microscopy is a suitable technique for future post mortem analyses of JET and ITER plasma facing components. We focus here on laboratory deposited and bombarded samples of beryllium and beryllium carbides and start to build a reference spectral databases for fusion relevant beryllium-based materials. We identified the beryllium phonon density of states, its second harmonic and E2G and B2G second harmonic and combination modes for defective beryllium in the spectral range 300–700 and 700–1300 cm−1, lying close to Be–D modes of beryllium hydrides. We also identified beryllium carbide signature, Be2C, combining Raman microscopy and DFT calculation. We have shown that, depending on the optical constants of the material probed, in depth sensitivity at the nanometer scale can be performed using different wavelengths. This way, we demonstrate that multi-wavelength Raman microscopy is sensitive to in-depth stress caused by ion implantation (down to  ≈30 nm under the surface for Be) and Be/C concentration (down to 400 nm or more under the surface for Be+C), which is a main contribution of this work. The depth resolution reached can then be adapted for studying the supersaturated surface layer found on tokamak deposits. [ABSTRACT FROM AUTHOR]

Recently, the surface of carbon fibre composite tiles of the toroidal pump limiter of Tore Supra has been analysed by scanning electron and atomic force microscopies. In regions where fibres are perpendicular to the surface, a specific erosion pattern has been observed. It is constituted of a striation oriented with an angle oblique with respect to the magnetic field. The characteristic wavelength of this structure is micrometric, and similar to the fibre size. Modelling has been undertaken to reproduce this micrometric pattern. It is shown to originate from the carbon composite structure, for which it has been found by measurement using laboratory plasma that the erosion rate of the fibres is different from that of the surrounding matrix. Modelling emphasizes the effect of the impinging flux angle distributions of deuterium ions and carbon impurities that are preliminarily determined from computation of the magnetic sheath. In the case of deuterium the sheath is shown to have little effect on the particle trajectories for the simulation parameters considered here, although when impurities are included the sheath deflection is significant. Furthermore this study shows how the fibre organization in the composite influences the striation direction and points out the importance of the angular dependence of the sputtering yield. [ABSTRACT FROM AUTHOR]

Purpose: Group B Streptococcus (GBS) is the leading cause of invasive infections in newborns. The prevention of GBS neonatal disease relies on the administration of an intrapartum antibiotic prophylaxis to GBS-colonized women. In recent years, rapid intrapartum detection of GBS vaginal colonization using real-time nucleic acid amplification tests (NAATs) emerged as an alternative to antenatal culture screening methods., Methods: We compared the performances of two loop-mediated isothermal amplification (LAMP) tests, the Ampliflash® GBS and the PlusLife® GBS tests, to standard culture for GBS detection in vaginal specimens from pregnant women. The study was conducted from April to July 2023 in a French hospital of the Paris area., Results: A total of 303 samples were analyzed, including 85 culture-positive samples (28.1%). The Ampliflash® GBS test and the PlusLife® GBS tests gave a result for 100% and 96.3% tests, respectively. The performances of the tests were as follows: sensitivity 87.1% (95% confidence interval (CI) 78.3-92.6) and 98.7% (95% CI 93.0-99.8), specificity 99.1% (95% CI 96.7-99.8), and 91.9% (95% CI 87.3-95.0), respectively. False negative results of the Ampliflash® GBS test correlated with low-density GBS cultures. Time-to-results correlated with GBS culture density only for the PlusLife® GBS test (p < 0.001)., Conclusion: Both techniques provide excellent analytical performances with high sensitivity and specificity together with a short turnaround time and results available in 10 to 35 min. Their potential to further reduce the burden of GBS neonatal disease compared with antenatal culture screening needs to be assessed in future clinical studies., (© 2024. The Author(s).)

Docosahexaenoic acid (DHA) is a semiessential polyunsaturated fatty acid (PUFA) for eukaryotic cells that is found in natural sources such as fish and algal oils and widely used as an ingredient for omega-3 containing foods or supplements. DHA effects are connected to its natural structure with six cis double bonds, but geometrical monotrans isomers can be formed during distillation or deodorization processes, as an unwanted event that alters molecular characteristics and annihilates health benefits. The characterization of the six monotrans DHA regioisomers is an open issue to address for analytical, biological, and nutraceutical applications. Here we report the preparation, separation, and first identification of each isomer by a dual approach consisting of the following: (i) the direct thiyl radical-catalyzed isomerization of cis-DHA methyl ester and (ii) the two-step synthesis from cis-DHA methyl ester via monoepoxides as intermediates, which are separated and identified by nuclear magnetic resonance spectroscopy, followed by elimination for the unequivocal assignment of the double bond position. This monotrans DHA isomer library with NMR and GC analytical characterization was also used to examine the products of thiyl-radical-catalyzed isomerization of a fish oil sample and to evaluate the trans isomer content in omega-3 containing supplements commercially available in Italy and Spain.

The use of liposomes has been crucial for investigations in biomimetic chemical biology as a membrane model and in medicinal chemistry for drug delivery. Liposomes are made of phospholipids whose biophysical characteristics strongly depend on the type of fatty acid moiety, where natural unsaturated lipids always have the double bond geometry in the cis configuration. The influence of lipid double bond configuration had not been considered so far with respect to the competence of liposomes in delivery. We were interested in evaluating possible changes in the molecular properties induced by the conversion of the double bond from cis to trans geometry. Here we report on the effects of the addition of trans -phospholipids supplied in different amounts to other liposome constituents (cholesterol, neutral phospholipids and cationic surfactants), on the size, ζ-potential and stability of liposomal formulations and on their ability to encapsulate two dyes such as rhodamine B and fluorescein. From a biotechnological point of view, trans -containing liposomes proved to have different characteristics from those containing the cis analogues, and to influence the incorporation and release of the dyes. These results open new perspectives in the use of the unnatural lipid geometry, for the purpose of changing liposome behavior and/or of obtaining molecular interferences, also in view of synergic effects of cell toxicity, especially in antitumoral strategies., Competing Interests: The authors declared no conflicts of interest.

Apolipoprotein E (apoE) is a major lipid carrier of the lipoprotein transport system that plays critical roles in various pathologies. Human apoE has three common isoforms, the apoE4 being associated with Alzheimer's disease. This is the first study in the literature investigating the effects of apoE (apoE3 and apoE4 isoforms) on membrane fatty acid profile in neuroblastoma SK-N-SH cells. Fatty acid analyses were carried out by gas chromatography of the corresponding methyl esters (FAME). We observed the occurrence of membrane fatty acid remodeling in the presence of each of the two apoE isoforms. ApoE3 increased the membrane level of stearic acid and dihomo-gamma-linolenic acid (DGLA), whereas apoE4 had opposite effects. Both apoE3 and apoE4 increased saturated and monounsaturated fatty acids (SFA and MUFA), omega-6/omega-3 ratio and decreased total polyunsaturated fatty acid (PUFA) amount, but with various intensities. Moreover, both apoE isoforms decreased membrane homeostasis indexes such as PUFA balance, unsaturation index and peroxidation index. Our results highlight membrane property changes connected to the apoE isoforms suggesting membrane lipidomics to be inserted in further model studies of apolipoproteins in health and disease., (Copyright © 2017. Published by Elsevier B.V.)

Membranes attract attention in medicine, concerning lipidome composition and fatty acid correlation with neurological diseases. Hyperspectral dark field microscopy (HDFM), a biophotonic imaging using reflectance spectra, provides accurate characterization of healthy adult RBC identifying a library of 8 spectral end-members. Here we report hyperspectral RBC imaging in children affected by Autism Spectrum Disorder (ASD) (n = 21) compared to healthy age-matched subjects (n = 20), investigating if statistically significant differences in their HDFM spectra exist, that can comprehensively map a membrane impairment involved in disease. A significant difference concerning one end-member (spectrum 4) was found (P value = 0.0021). A thorough statistical treatment evidenced: i) diagnostic performance by the receiving operators curve (ROC) analysis, with cut-offs and very high predictive values (P value = 0.0008) of spectrum 4 for identifying disease; ii) significant correlations of spectrum 4 with clinical parameters and with the RBC membrane deficit of the omega-3 docosahexaenoic acid (DHA) in ASD patients; iii) by principal component analysis, very high affinity values of spectrum 4 to the factor that combines behavioural parameters and the variable "cc" discriminating cases and controls. These results foresee the use of biophotonic methodologies in ASD diagnostic panels combining with molecular elements for a correct neuronal growth.

Fatty acids, as structural components of membranes and inflammation/anti-inflammatory mediators, have well-known protective and regulatory effects. They are studied as biomarkers of pathological conditions, as well as saturated and unsaturated hydrophobic moieties in membrane phospholipids that contribute to homeostasis and physiological functions. Lifestyle, nutrition, metabolism and stress-with an excess of radical and oxidative processes-cause fatty acid changes that are examined in the human body using blood lipids. Fatty acid-based membrane lipidomics represents a powerful diagnostic tool for assessing the quantity and quality of fatty acid constituents and also for the follow-up of the membrane fatty acid remodeling that is associated with different physiological and pathological conditions. This review focuses on fatty acid biomarkers with two examples of recent lipidomic research and health applications: (i) monounsaturated fatty acids and the analytical challenge offered by hexadecenoic fatty acids (C16:1); and (ii) the cohort of 10 fatty acids in phospholipids of red blood cell membranes and its connections to metabolic and nutritional status in healthy and diseased subjects.

Graft-versus-host disease (GVHD) is among the most challenging complications in unrelated donor hematopoietic cell transplantation (HCT). The highly polymorphic MHC class I chain-related gene A, MICA, encodes a stress-induced glycoprotein expressed primarily on epithelia. MICA interacts with the invariant activating receptor NKG2D, expressed by cytotoxic lymphocytes, and is located in the MHC, next to HLA-B Hence, MICA has the requisite attributes of a bona fide transplantation antigen. Using high-resolution sequence-based genotyping of MICA, we retrospectively analyzed the clinical effect of MICA mismatches in a multicenter cohort of 922 unrelated donor HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 10/10 allele-matched HCT pairs. Among the 922 pairs, 113 (12.3%) were mismatched in MICA MICA mismatches were significantly associated with an increased incidence of grade III-IV acute GVHD (hazard ratio [HR], 1.83; 95% confidence interval [CI], 1.50-2.23; P < .001), chronic GVHD (HR, 1.50; 95% CI, 1.45-1.55; P < .001), and nonelapse mortality (HR, 1.35; 95% CI, 1.24-1.46; P < .001). The increased risk for GVHD was mirrored by a lower risk for relapse (HR, 0.50; 95% CI, 0.43-0.59; P < .001), indicating a possible graft-versus-leukemia effect. In conclusion, when possible, selecting a MICA-matched donor significantly influences key clinical outcomes of HCT in which a marked reduction of GVHD is paramount. The tight linkage disequilibrium between MICA and HLA-B renders identifying a MICA-matched donor readily feasible in clinical practice., (© 2016 by The American Society of Hematology.)

We herein report on the formation of BeD2 nanocrystalline domes on the surface of a beryllium sample exposed to energetic deuterium ions. A polycrystalline beryllium sample was exposed to D ions at 2 keV/atom leading to laterally averaged deuterium areal densities up to 3.5 10(17) D cm(-2), and studied using nuclear reaction analysis, Raman microscopy, atomic force microscopy, optical microscopy and quantum calculations. Incorporating D in beryllium generates a tensile stress that reaches a plateau at  ≈1.5 10(17) D cm(-2). For values higher than 2.0 10(17) cm(-2), we observed the growth of  ≈90 nm high dendrites, covering up to 10% of the surface in some zones of the sample when the deuterium concentration was 3  ×  10(17) D cm(-2). These dendrites are composed of crystalline BeD2, as evidenced by Raman microscopy and quantum calculations. They are candidates to explain low temperature thermal desorption spectroscopy peaks observed when bombarding Be samples with D ions with fluencies higher than 1.2 10(17) D cm(-2).

Lipids are important antigens that induce T cell-mediated specific immune responses. They are presented to T lymphocytes by a specific class of MHC-I like proteins, termed CD1. The majority of the described CD1-presented mycobacterial antigens are presented by the CD1b isoform. We previously demonstrated that the stimulation of CD1b-restricted T cells by the hexamannosylated phosphatidyl-myo-inositol (PIM(6)), a family of mycobacterial antigens, requires a prior partial digestion of the antigen oligomannoside moiety by α-mannosidase and that CD1e is an accessory protein absolutely required for the generation of the lipid immunogenic form. Here, we show that CD1e behaves as a lipid transfer protein influencing lipid immunoediting and membrane transfer of PIM lipids. CD1e selectively assists the α-mannosidase-dependent digestion of PIM(6) species according to their degree of acylation. Moreover, CD1e transfers only diacylated PIM from donor to acceptor liposomes and also from membranes to CD1b. This study provides new insight into the molecular mechanisms by which CD1e contributes to lipid immunoediting and CD1-restricted presentation to T cells.

CD1e is the only human CD1 protein existing in soluble form in the late endosomes of dendritic cells, where it facilitates the processing of glycolipid antigens that are ultimately recognized by CD1b-restricted T cells. The precise function of CD1e remains undefined, thus impeding efforts to predict the participation of this protein in the presentation of other antigens. To gain insight into its function, we determined the crystal structure of recombinant CD1e expressed in human cells at 2.90-Å resolution. The structure revealed a groove less intricate than in other CD1 proteins, with a significantly wider portal characterized by a 2 Å-larger spacing between the α1 and α2 helices. No electron density corresponding to endogenous ligands was detected within the groove, despite the presence of ligands unequivocally established by native mass spectrometry in recombinant CD1e. Our structural data indicate that the water-exposed CD1e groove could ensure the establishment of loose contacts with lipids. In agreement with this possibility, lipid association and dissociation processes were found to be considerably faster with CD1e than with CD1b. Moreover, CD1e was found to mediate in vitro the transfer of lipids to CD1b and the displacement of lipids from stable CD1b-antigen complexes. Altogether, these data support that CD1e could have evolved to mediate lipid-exchange/editing processes with CD1b and point to a pathway whereby the repertoire of lipid antigens presented by human dendritic cells might be expanded.

The plasma membrane proteins CD1a, CD1b and CD1c are expressed by human dendritic cells, the professional antigen-presenting cells of the immune system, and present lipid antigens to T lymphocytes. CD1e belongs to the same family of molecules, but accumulates as a membrane-associated form in the Golgi compartments of immature dendritic cells and as a soluble cleaved form in the lysosomes of mature dendritic cells. In lysosomes, the N-terminal propeptide of CD1e is also cleaved, but the functional consequences of this step are unknown. Here, we investigated how the pH changes encountered during transport to lysosomes affect the structure of CD1e and its ligand-binding properties. Circular dichroism studies demonstrated that the secondary and tertiary structures of recombinant CD1e were barely altered by pH changes. Nevertheless, at acidic pH, guanidium chloride-induced unfolding of CD1e molecules required lower concentrations of denaturing agent. The nonfunctional L194P allelic variant was found to be structurally less stable at acidic pH than the functional forms, providing an explanation for the lack of its detection in lysosomes. The number of water-exposed hydrophobic patches that bind 8-anilinonaphthalene-1-sulfonate was higher in acidic conditions, especially for the L194P variant. CD1e molecules interacted with lipid surfaces enriched in anionic lipids, such as bis(monoacylglycero)phosphate, a late endosomal/lysosomal lipid, especially at acidic pH, or when the propeptide was present. Altogether, these data indicate that, in the late endosomes/lysosomes of DCs, the acid pH promotes the binding of lipid antigens to CD1e through increased hydrophobic and ionic interactions., (© 2011 The Authors Journal compilation © 2011 FEBS.)

This paper tells the history of two brothers, almost a generation apart in age, who met again, after having followed different academic paths, to introduce biophysical research in photosynthesis at the University of Padova. The development of two research groups, one in the Chemistry Department, the other in the Biology Department led to a comprehensive interdisciplinary group across academic barriers. The group of Giovanni Giacometti developed in Physical Chemistry, during the years before his retirement, with some roots which can be traced to the famous Linus Pauling school of the mid 1950s, and made possible, by the work of many students (especially Donatella Carbonera and Marilena Di Valentin) and of an older associate (Giancarlo Agostini). The group participated quite actively with a number of European and American laboratories in the application of physical techniques, especially Electron Spin Resonance (EPR) associated with Optical Spectroscopy (Optically Detected Magnetic Resonance; ODMR), and contributed to the development of the understanding of the structure-function relationships in photosynthetic membrane complexes, stimulated by the determination of the X-ray structure of the purple photosynthetic reaction center in the mid 1980s ( J. Deisenhofer, H. Michel, R. Huber and others). The younger brother of Giovanni, Giorgio Mario Giacometti, came to Padova after obtaining biochemical knowledge from the Rossi-Fanelli school in Rome, where Jeffries Wyman, Eraldo Antonini and Maurizio Brunori were the world masters of hemoglobin research. In Padova, together with a group of young scientists (at first Roberto Bassi and Roberto Barbato, now leaders of their own groups in Verona and in Alessandria respectively, followed soon by brilliant coworkers such as Fernanda Rigoni, Elisabetta Bergantino and more recently Ildikò Szabò and Paola Costantini), Giorgio approached more biochemical themes of oxygenic photosynthesis, such as purification and characterization of antenna chlorophyll-protein complexes, Photosystem II (PS II) particles and subunits, having always in mind structural and molecular problems at the level of the largest integrated particles, which are more difficult to investigate in detail by the spectroscopic techniques.

Rapid-scan Fourier transform infrared (FTIR) difference spectroscopy was used to investigate the electron transfer reaction Q(A-)Q(B)-->Q(A)Q(B-) (k(AB)(1)) in mutant reaction centers of Rhodobacter sphaeroides, where Asp-L210 and/or Asp-M17 have been replaced with Asn. Mutation of both residues decreases drastically k(AB)(1)), attributed to slow proton transfer to Glu-L212, which becomes rate limiting for electron transfer to Q(B) [M.L. Paddock et al., Biochemistry 40 (2001) 6893]. In the double mutant, the FTIR difference spectrum recorded during the time window 4-29 ms following a flash showed peaks at 1670 (-), 1601 (-) and 1467 (+) cm(-1), characteristic of Q(A) reduction. The time evolution of the spectra shows reoxidation of Q(A-) and concomitant reduction of Q(B) with a kinetics of about 40 ms. In native reaction centers and in both single mutants, formation of Q(B-) occurs much faster than in the double mutant. Within the time resolution of the technique, protonation of Glu-L212, as characterized by an absorption increase at 1728 cm(-1) [E. Nabedryk et al., Biochemistry 34 (1995) 14722], was found to proceed with the same kinetics as reduction of Q(B) in all samples. These rapid-scan FTIR results support the model of proton uptake being rate limiting for the first electron transfer from Q(A-) to Q(B) and the identification of Glu-L212 as the main proton acceptor in the state Q(A)Q(B-).

Optically detected magnetic resonance of chlorosome-containing membranes from the green filamentous bacterium Chloroflexus aurantiacus has been performed both by fluorescence and absorption detection. Triplet states localized in the chlorosomes and in the B808-866 complex have been characterized. After chemical reduction with ascorbate followed by illumination at 200 K, recombination triplet state localized in the primary donor becomes largely populated under illumination at low temperature while all the antenna triplet states, which are localized in carotenoids and BChl a molecules, are strongly quenched. We were able to obtain the T-S spectrum of the primary donor P870 surrounded by all the antenna complexes connected to the RC via energy transfer and then in its intact environment. We found clear spectroscopic evidence for exciton interaction between the RC and the B808-866 antenna complex. This evidence was provided by the comparison of the T-S spectrum of P870 in the membranes with that of isolated RC. The analogy of some features of the difference spectra with those previously found in the same kind of experiments for Rb. sphaeroides, allows to predict a similar coupling among the primary donor and the nearby antenna BChl a molecules, assembled as circular aggregate.

Chemical oxidation of the chlorosomes from Chloroflexus aurantiacus and Chlorobium tepidum green bacteria produces bacteriochlorophyll radicals, which are characterized by an anomalously narrow EPR signal compared to in vitro monomeric BChl c (.+) [Van Noort PI, Zhu Y, LoBrutto R and Blankenship RE (1997) Biophys J 72: 316-325]. We have performed oxidant concentration and temperature-dependent X-band EPR measurements in order to elucidate the line narrowing mechanism. The linewidth decreases as the oxidant concentration is increased only for Chloroflexus indicating that for this system Heisenberg spin exchange is at least partially responsible for the EPR spectra narrowing. For both species the linewidth is decreasing on increasing the temperature. This indicates that temperature-activated electron transfer is the main narrowing mechanism for BChl radicals in chlorosomes. The extent of the electron transfer process among different BChl molecules has been evaluated and a comparison between the two species representative of the two green bacteria families has been made. In parallel, high frequency EPR experiments have been performed on the oxidized chlorosomes of Chloroflexus and Chlorobium at 110 and 330 GHz in the full temperature range investigated at X-band. The g-tensor components obtained from the simulation of the 330 GHz EPR spectrum from Chlorobium show the same anisotropy as those of monomeric Chl a (.+) [Bratt PJ, Poluektov OG, Thurnauer MC, Krzystek J, Brunel LC, Schrier J, Hsiao YW, Zerner M and Angerhofer A (2000) J Phys Chem B 104: 6973-6977]. The spectrum of Chloroflexus has a nearly axial g-tensor with reduced anisotropy compared to Chlorobium and monomeric Chl a in vitro. g-tensor values and temperature dependence of the linewidth have been discussed in terms of the differences in the local structure of the chlorosomes of the two families.

Two histidines provide the axial ligands of the two chlorophyll a (Chl a) molecules which form the primary electron donor (P700) of photosystem I (PSI). Histidine 676 in the protein subunit PsaA, His(A676), and histidine 656 in subunit PsaB, His(B656), were replaced in the green algae Chlamydomnas reinhardtii by site-directed mutagenesis with nonpolar, uncharged polar, acidic, and basic amino acid residues. Only the substitutions with uncharged polar residues led to a significant accumulation of PSI in the thylakoid membranes. These PSI complexes were isolated and the physical properties of the primary donor characterized. The midpoint potential of P700(+)(*)/P700 was increased in all mutants (up to 140 mV) and showed a dependence on size and polarizability of the residues when His(B656) was substituted. In the light-minus-dark absorbance spectra, all mutations in PsaB exhibited an additional bleaching band at 665 nm at room temperature comparable with the published spectrum for the replacement of His(B656) with asparagine [Webber, A. N., Su Hui, Bingham, S. E., Käss, H., Krabben, L., Kuhn, M., Jordan, R., Schlodder, E., and Lubitz, W. (1996) Biochemistry 35, 12857-12863]. Substitutions of His(A676) showed an additional shoulder around 680 nm. In the low-temperature absorbance difference spectra of P700(+)(*)/P700, a blue shift of the main bleaching band by 2 nm and some changes in the spectral features around 660 nm were observed for mutations of His(B656) in PsaB. The analogous substitution in PsaA showed only a shift of the main bleaching band. Similar effects of the mutations were found in the (3)P700/P700 absorbance difference spectra at low temperatures (T = 2 K). The zero-field splitting parameters of (3)P700 were not significantly changed in the mutated PSI complexes. The electron spin density distribution of P700(+)(*), determined by ENDOR spectroscopy, was only changed when His(B656) was replaced. In all measurements, two general observations were made. (i) The replacement of His(B656) had a much stronger impact on the physical properties of P700 than the mutation of His(A676). (ii) The exchange of His(B656) with glutamine induces the smallest changes in the spectra or the midpoint potential, whereas the other replacements exhibited a stronger but very similar influence on the spectroscopic features of P700. The data provide convincing evidence that the unpaired electron in the cation radical and the triplet state of P700 are mainly localized on the Chl a of the dimer which is axially coordinated by His(B656).

An ADMR T-S spectrum of the primary donor (P680) of photosystem II (PSII) was obtained from anaerobically photoreduced particles. The spectrum is the best resolved obtained so far having a main bleaching band at 684 nm with a linewidth of only 100 cm-1. The view that this spectrum is produced by native homogeneous P680 unlike those obtained before is defended. A small bleaching observed at 678 nm is discussed in terms of the reaction center structure. One possible interpretation of the observations is that P680 is a very loose dimer with an exciton splitting of only 144 cm-1 corresponding to a dimer center-to-center distance of roughly 11.5 A.

In this follow-up investigation, the authors studied all Swiss women in the canton of Zurich who developed anorexia nervosa between ages 12 and 25 years and who were hospitalized for the first time with this diagnosis between 1983 and 1985. Data were compared with those from an earlier study, which focused on the periods 1956-1958, 1963-1965, and 1973-1975. The incidence of anorexia nervosa did not increase significantly during 1983-1985 compared to 1973-1975, in contrast to the constant increase found between 1956 and 1975. However, the more frequent use of vomiting and abuse of laxatives in 1983-1985 may indicate an increase in cases with mixed features of anorexia nervosa and bulimia.

Resonance Raman and optical absorption spectra of ligand-free (deoxy) myoglobin and CO-bound myoglobin (MbCO) at pH 2.6 have been measured by using continuous-flow/rapid-mixing techniques. The spectra of deoxy myoglobin at low pH within 6 ms of the pH drop demonstrate that the iron-histidine bond has been ruptured but that the heme is still five-coordinate. Comparison with data from model complexes indicates that a weak-field ligand, such as a water molecule, is coordinated at the fifth position. The Raman spectrum of MbCO at low pH has an Fe-CO stretching mode that is characteristic of a six-coordinate heme with an unhindered Fe-CO moiety. Immediately following the pH drop in this case, there is no indication that the iron-proximal histidine bond is broken. Three different structural changes are detected at low pH: (i) the iron-proximal histidine (F8) bond in ligand-free myoglobin is broken and replaced by a weak-field ligand, (ii) the distal pocket in MbCO is opened, and (iii) protein constraints on the heme group in MbCO are relaxed. Previous conclusions that the kinetics of CO-binding in hemoproteins at low pH is modified by rupturing the iron-proximal histidine bond are supported by these new results which, however, demand a more complete reevaluation of the phenomenon.

A/Port Chalmers 1/73 and A/Victoria 3/75 live vaccines were well tolerated after oral administration and showed good immunogenic properties in subjects devoid of immune-defence or having low antibody titer. A/Victoria 3/75 vaccine was prepared before the epidemiological appearance of the corresponding virus which demonstrates that the preparation of a vaccine with new strains can be made in a comparatively short time. Our experiments and controls allow us to establish that the oral administration of influenza virus, grown on chicken kidney embryo, elicits the appearance of HI antibodies in a high number of susceptible subjects. A 60% seroconversion rate obtained after administration of two doses represents a favourable result, the more so as the vaccine can be considered absolutely safe in very young subjects.

1. The decay of oxyferroperoxidase to ferriperoxidase was studied by rapidly mixing solutions of ferroperoxidase with various amounts of oxygen and following the time-course of appearance of oxyferroperoxidase and its subsequent decay to ferriperoxidase by reaction with ferroperoxidase. 2. The scheme can be accommodated by and [Formula: see text] and occurs without detectable intermediates being observed in the millisecond time-range. 3. Analogue-computer simulation of the reaction is in agreement with the initial rate-limiting reaction being an intermolecular, not intramolecular, electron-donating process, and analysis of the data leads to quantitative values for the rate constants of the overall process. 4. The reaction of oxyferroperoxidase with ferroperoxidase is a model of a terminal oxidase, and the results are discussed in terms of the possible importance of this reaction in peroxidase function, and also in the light it throws on autoxidation of haem compounds.

The reaction of Octopus vulgaris hemocyanin with nitrite was studied under a variety of conditions in which the green half-met derivative is formed. Analytical evidence shows that the amount of chemically detectable nitrite in various samples of the derivative is not proportional to the cupric copper detected by EPR. The kinetics of oxidation of hemocyanin as a function of protein concentration and pH, in the presence of nitrite and ascorbate, is consistent with a scheme in which NO2 is the reactive oxidant. We suggest that the green half-methemocyanin contains a metal center with one cuprous and one cupric copper without an exogenous nitrogen oxide ligand.

151 subjects, vaccinated by oral route with live influenza viruses attenuated in the USSR (bivalent A/England/42/72-B/Hong Kong/5/72 vaccine) and 66 non-vaccinated cohabitants have been studied. The vaccination did not cause any important side effect. The vaccine showed good immunizing activity: HI antibodies appeared in 91% of the subjects initially devoid of antibodies against A type and in 70% against B type. No viral diffusion occurred from the vaccinated to the cohabitants. During an A/Port Chalmers/1/73 epidemic the morbidity (3.3%) among the orally vaccinated patients turned out significantly lower than that among the non-vaccinated cohabitants (12.1%). The protection index was 72.6%.

A mathod is described for the purification, on an industrial scale, of influenza viruses grown in allantoic cavity of embryonated eggs. The mehtod consists of combining continuous-flow centrifugation with zonal centrifugation in a sucrose (36.6 per cent-52.5 per cent w/v) density gradient. The sample flow rate is approximately 3.7 litres/h and the volumes treated vary between 3 and 33 litres of allantoic fluid. Both the recovery of the virus and the degree of concentration and purification result satisfactory.

Ligand-binding equilibria, kinetics and (13)C n.m.r. spectra of bound (13)CO, of the haemoglobins from two fishes that are very distant on the evolutionary scale, i.e. the fourth haemoglobin component from Salmo irideus and the single component from Osteoglossum bicirrhosum, were studied. The C-terminal sequence was also determined for the haemoglobin from Osteoglossum. The results show that (i) the C-terminal residues of both chains are not directly responsible for the characteristic heterotropic effect known as Root effect, since for both fish haemoglobins these residues are identical with those of human haemoglobins. (ii) In all haemoglobins characterized by the Root effect a dependence of the (13)CO n.m.r. resonances on pH is observed. However, the extent of the shift(s) depends on the particular protein, and is probably the result of a combination of both tertiary and quaternary conformational changes. (iii) Both haemoglobins from trout and Osteoglossum manifest a functional heterogeneity between the two types of chains in the tetramer, which increases with proton activity. For CO, the effect is very small for trout haemoglobin IV, and very marked for Osteoglossum haemoglobin; for O(2) strongly heterogeneous binding curves were obtained at approx. pH6.2 with both haemoglobins. (iv) Estimations of the relative values of the affinity constants for the alpha and beta chains in the tetramer were obtained for both haemoglobins from (13)CO n.m.r. spectra at low fractional saturation. On the basis of these findings the molecular mechanism underlying the Root effect is discussed.

The immunizing capacity and the tolerability of two purified vaccines containing the inactivated A/New Jersey/76 ( x 53 recombinant) swine-like strain were studied. A trivalent vaccine (A/New Jersey/76-300 I.U.; A/Victoria 3/75-300 I.U.; B/Hong Kong 5/75-300 I.U.) was administered to 31 subjects over 50 years of age. No side-effects were observed and it caused a good HI antibody response to A/New Jersey/76, fairly good for B strain and modest for A/Victoria 3/75. A monovalent vaccine (A/New Jersey/76-400 I.U.) was administered to 32 subjects in the age range from 16 to 43 years. HI antibodies occurred in 84% of the subjects, devoid of antibodies before vaccination. Younger subjects (between 16-20 years) generally developed fairly low antibody titres; one among them (16-year-old) had a temperature rise within 48 h after vaccination. The Authors expect that the X 53 recombinant with the surface antigens of the A/New Jersey/76 strain may be employed for the preparation of the inactivated vaccine to be used in an emergency situation and discuss the composition of the vaccine more adapt for the present epidemiological situation.

The luminescence lifetime of Cu-metallothionein from the fungus Neurospora crassa has been studied by the frequency-domain emission technique. Lifetimes of 10.3 and 3.4 microseconds have been found for the protein in the absence and in the presence of oxygen respectively. Binding of Hg(II) results in a quenching of the luminescence correlated to the shortening of lifetime to 0.3-0.4 microsecond. No quenching by oxygen is found for the Hg(II)-Cu-metallothionein adduct. By analogy to model compounds, luminescence emission is attributed to a triplet excited state of a Cu(I)-to-sulphur charge-transfer complex. The comparison of bimolecular quenching constants for O2 and acrylamide indicates a highly compact structure of the protein.