Current limits of structural biology: The transient interaction between cytochrome c 6 and photosystem I.

Trimeric photosystem I from the cyanobacterium Thermosynechococcus elongatus (TePSI) is an intrinsic membrane protein, which converts solar energy into electrical energy by oxidizing the soluble redox mediator cytochrome c 6 (Cyt c 6 ) and reducing ferredoxin. Here, we use cryo-electron microscopy and small angle neutron scattering (SANS) to characterize the transient binding of Cyt c 6 to TePSI. The structure of TePSI cross-linked to Cyt c 6 was solved at a resolution of 2.9 Å and shows additional cofactors as well as side chain density for 84% of the peptide chain of subunit PsaK, revealing a hydrophobic, membrane intrinsic loop that enables binding of associated proteins. Due to the poor binding specificity, Cyt c 6 could not be localized with certainty in our cryo-EM analysis. SANS measurements confirm that Cyt c 6 does not bind to TePSI at protein concentrations comparable to those for cross-linking. However, SANS data indicate a complex formation between TePSI and the non-native mitochondrial cytochrome from horse heart (Cyt c HH ). Our study pinpoints the difficulty of identifying very small binding partners (less than 5% of the overall size) in EM structures when binding affinities are poor. We relate our results to well resolved co-structures with known binding affinities and recommend confirmatory methods for complexes with K M values higher than 20 µM.

[Skin-to-skin care in the delivery room: impact of SpO2 monitoring]. / Peau-à-peau en salle de naissance : impact du monitorage de la saturation pulsée en oxygène.

BACKGROUND: Skin-to-skin care in the delivery room increases mother-newborn bonding, reduces the newborn's stress level, and facilitates breastfeeding. However, a few reports of life-threatening events in newborn infants during skin-to-skin care have prompted suggestions that SpO2 monitoring may be of value in the delivery room. The present study compared SpO2 monitoring with standard clinical practices during skin-to-skin care in the delivery room. The midwife's opinion and the mother's anxiety level were assessed for both procedures. MATERIALS AND METHODS: The midwife's opinion was measured on a Likert scale and the mother's anxiety level was measured on the State-Trait Anxiety Inventory Y-A and Y-B scales. Two procedures (standard clinical practice vs. SpO2 monitoring) were compared prospectively in two consecutive 3-month periods. RESULTS: Seventy case report forms were completed for the "standard clinical practice" group and 62 were completed for the "SpO2 monitoring" group. The care procedure was considered to be satisfactory or quite satisfactory in 60 cases (96.8%) in the "SpO2 monitoring" group and in 57 cases (81.4%; P<0.05) in the "standard clinical practice" group. There was no significant difference between the groups in terms of the mean maternal anxiety level. CONCLUSION: SpO2 monitoring during skin-to-skin care in the delivery room was well accepted by the midwife. Relative to standard clinical practice alone, SpO2 monitoring was not associated with elevated maternal anxiety levels.

Patterns of phenylpropanoids in non-inoculated and potato virus Y-inoculated leaves of transgenic tobacco plants expressing yeast-derived invertase.

The patterns of secondary metabolites in leaves of yeast invertase-transgenic tobacco plants (Nicotiana tabacum L. cv. Samsun NN) were analyzed. Plants expressing cytosolic yeast-derived invertase (cytInv) or apoplastic (cell wall associated) yeast invertase (cwInv) showed a characteristic phytochemical phenotype compared to untransformed controls (wild-type plants). The level of phenylpropanoids decreased in the cytInv plants but increased in the cwInv plants, which showed an induced de novo synthesis of a caffeic acid amide, i.e. N-caffeoylputrescine. In addition, the level of the coumarin glucoside scopolin was markedly enhanced. Increased accumulation of scopolin in the cwInv plants is possibly correlated with the induction of defense reactions and the appearance of necrotic lesions similar to the hypersensitive response caused by avirulent pathogens. This is consistent with results from potato virus Y-infected plants. Whereas there was no additional increase in the coumarins in leaves following infection in cwInv plants, wild-type plants showed a slight increase and cytInc a marked increase.

Cloning and heterologous expression of a rape cDNA encoding UDP-glucose:sinapate glucosyltransferase.

A cDNA encoding a UDP-glucose:sinapate glucosyltransferase (SGT) that catalyzes the formation of 1-O-sinapoylglucose, was isolated from cDNA libraries constructed from immature seeds and young seedlings of rape (Brassica napus L.). The open reading frame encoded a protein of 497 amino acids with a calculated molecular mass of 55,970 Da and an isoelectric point of 6.36. The enzyme, functionally expressed in Escherichia coli, exhibited broad substrate specificity, glucosylating sinapate, cinnamate, ferulate, 4-coumarate and caffeate. Indole-3-acetate, 4-hydroxybenzoate and salicylate were not conjugated. The amino acid sequence of the SGT exhibited a distinct sequence identity to putative indole-3-acetate glucosyltransferases from Arabidopsis thaliana and a limonoid glucosyltransferase from Citrus unshiu, indicating that SGT belongs to a distinct subgroup of glucosyltransferases that catalyze the formation of 1-O-acylglucosides (beta-acetal esters).

[Incidence of Legionella in warm water systems in Saxony-Anhalt]. / Vorkommen von Legionellen in Warmwassersystemen in Sachsen-Anhalt.

The incidence of Legionella in warm water systems of Sachsen-(Saxony-)Anhalt was investigated. The Legionella were isolated from water samples using plate cultures followed by serotyping methods. In high-risk areas of Legionella infections such as hospitals and homes for the aged, 48% respectively 43% of the samples were positive. Warm water systems of 61% of the hospitals and 43% of homes for aged people were found to be contaminated with Legionella. The number of Legionella were most frequently (50.7%) between 10 and 100 colony-forming units/ml (cfu/ml). High-level Legionella contamination (> 1000 cfu/ml) were detected only in 0.6% of the samples. Legionella pneumophila serogroup 1 (L.p. SG 1) was identified rarely. The reasons for positive Legionella findings are old drinking water heating systems and conduits. To decrease Legionella growth, reconstruction of the old systems according to the recommendations [1, 2] is imperative.

Molecular cloning and heterologous expression of acridone synthase from elicited Ruta graveolens L. cell suspension cultures.

Cell suspension cultures of Ruta graveolens L. produce a variety of acridone alkaloids, and the accumulation can be stimulated by the addition of fungal elicitors. Acridone synthase, the enzyme catalyzing the synthesis of 1,3-dihydroxy-N-methylacridone from N-methylanthraniloyl-CoA and malonyl-CoA, had been isolated from these cells, and the partial enzyme polypeptide sequence, elucidated from six tryptic fragments, revealed homology to heterologous chalcone synthases. Poly(A)+ RNA was isolated from Ruta cells that had been treated for 6 h with a crude cell wall elicitor from Phytophthora megasperma f. sp. glycinea, and a cDNA library was constructed in lambda 2AP. Clones harboring acridone synthase cDNA were isolated from the library by screening with a synthetic oligonucleotide probe complementary to a short stretch of sequence of the enzyme peptide with negligible homology to chalcone synthases. The identity of the clones was substantiated by DNA sequencing and by recognition of five additional peptides, determined previously from tryptic acridone synthase digests, in the translated sequence. An insert of roughly 1.4 kb encoded the complete acridone synthase, and alignments at both DNA and protein levels corroborated the high degree of homology to chalcone synthases. Expression of the enzyme in vector pET-11c in the Escherichia coli pLysS host strain proved the identity of the cloned cDNA. The heterologous enzyme in the crude E. coli extract exhibit high acridone but no chalcone synthase activity. The results were fully supported by northern blot hybridizations which revealed that the specific transcript abundance did not increase but rather decreased upon white light irradiation of cultured Ruta graveolens L. cells, a condition that commonly induces the abundance of chalcone synthase transcripts.

Acridone Alkaloids from Cell Suspension Cultures of Thamnosma montana.

Cell Suspension cultures of THAMNOSMA MONTANA were grown in Gamborg B5 medium which was best suited for acridone alkaloid formation. From the lyophilized cell material nine acridones and three acridone-monoglucosides have been isolated which were found for the first time in the genus THAMNOSMA. Moreover, N-methylacridone which is known from the intact plant was found. Gravacridonol monoglucoside was identified as a new alkaloid. Interestingly, most isolated alkaloids belong to the dihydrofuroacridones.

Purification and properties of acridone synthase from cell suspension cultures of Ruta graveolens L.

Acridone synthase has been purified from cell suspension cultures of Ruta graveolens using a combination of gel filtration and ion exchange chromatography. The purified enzyme has an apparent molecular weight of 69 kDa on gel filtration and a subunit structure on SDS-PAGE of 40 kDa. The apparent Km-values are 10.64 microM and 32.8 microM for N-methylanthraniloyl-CoA and malonyl-CoA, respectively. Tryptic digestion of the homogeneous acridone synthase was performed. Seven of the peptides were chosen for microsequencing. The homology of the amino acid sequences from this particular polypeptide and corresponding peptides from chalcone synthase 3 from garden pea amounted to 76%.

[The kinetics of vaccine antibodies against tetanus toxoid, diphtheria toxoid, measles virus, poliomyelitis virus and pneumococcus after allogeneic and autologous bone marrow transplantation and revaccination. 3: The kinetics of vaccine antibodies against tetanus toxoid and diphtheria toxoid after allogeneic and autologous bone marrow transplantation and combined revaccination against diphtheria and tetanus]. / Untersuchungen zur Kinetik der Impfantikörper gegen Tetanustoxoid, Diphtherietoxoid, Masern-Virus, Poliomyelitis-Virus und Pneumokokken nach allogener und autologer Knochenmarktransplantation und Wiederholungsimpfung. Teil 3: Kinetik der Impfantikörper gegen Tetanustoxoid und Diphtherietoxoid nach allogener und autologer Knochenmarktransplantation und kombinierter Wiederholungsimpfung gegen Diphtherie und Tetanus.

The 3rd part of the paper deals with the results of a combined revaccination against diphtheria and tetanus in a group of 25 children after allogeneic bone marrow transplantation (BMT) with and without graft versus host disease (GvHD) and after autologous transplantation. It can be shown that in the allogeneic transplanted groups with and without GvHD it is possible to build up a tetanus and diphtheria antitoxin titre in a safe protective cause by a 2nd basic immunisation consisting of 3 single vaccinations starting about 9 to 12 months later. For autologous transplanted children only 1 to 2 vaccinations at a later term than for the allogeneic transplanted children may possibly be sufficient.

[Kinetics of vaccine antibodies to tetanus toxoid, diphtheria toxoid, measles virus, poliomyelitis virus and pneumococci after allogenic and autologous bone marrow transplantation and booster immunization. 1: The kinetics of vaccine antibodies to tetanus toxoid after allogenic and autologous bone marrow transplantation]. / Untersuchungen zur Kinetik der Impfantikörper gegen Tetanustoxoid, Diphtherietoxoid, Masern-Virus, Poliomyelitis-virus und Pneumokokken nach allogener und autologer Knochenmarktransplantation und Wiederholungsimpfung. Teil 1: Kinetik der Impfantikörper gegen Tetanustoxoid nach allogener und autologer Knochenmarktransplantation.

Today BMT belongs to the established methods of treatment in haematology and oncology. Because of the constant increase of healthy long-term survivors after BMT the problem of immunological reconstitution and eventual possible late effects gets more and more importance. One problem, which til now has been few attention paid to, is that of the protection by vaccination after BMT. We report on the kinetics of the tetanus-antitoxin in 20 patients after allogeneic or autologous BMT and demonstrate the influence of a graft-versus-host disease and its therapy on the antibody kinetics. In the group of allogeneic transplanted children without a GvHD the tetanus-antitoxin titers felt below their detection range after a time of about 8 months whereas in the group with GvHD this effect already occurred after nearly 4 months. The autologous transplanted patients have a positive antibody level til the time of 20 months after BMT. As a consequence of the lost protection by vaccination after BMT follows the necessity of revaccinations respectively of boostering after immunological reconstitution.

Biotransformation of a [14CH3]-2-Methylaminobenzophenone by Plant Cell Cultures.

2-[ (14)C]-Methylamino-2',4'-dimethoxy-6'-hydroxybenzophenone ( 4) was synthesized and administered to RUTA GRAVEOLENS cell suspension cultures. Compound 4 was not incorporated into acridone alkaloids but glucosylated giving 7. This reaction takes place also in cell suspension cultures of ADHATODA VASICA and PEGANUM HARMALA.

N-Methylation of Anthranilic Acid to N-Methylanthranilic Acid by Cell-free Extracts of Ruta graveolens Tissue Cultures.

Cell-free extracts from acridone synthesizing cell suspension cultures of RUTA GRAVEOLENS L. catalyze the N-methylation of anthranilic acid using S-adenosyl-L-methionine as methyl donor. The stability of enzyme preparations was remarkably high during storage at -20 degrees C. Optimum activity was exhibited at pH 8.2, Mg (2+) was not required for maximum activity and EDTA did not affect the reaction rate. The rate of N-methylanthranilic acid formation was shown to be linear for about 45 min and was proportional to the protein concentration up to at least 0.350 mg of the enzyme preparation. In a number of suspension cultures of plant species not belonging to the Rutaceae this particular N-methyltransferase was not found. Apparantly N-methylation of anthranilic acid is the first pathway-specific reaction in acridone alkaloid biosynthesis.

Biosynthesis of rutacridone: the N-methylation step.

The N-methylation of rutacridone was investigated. This dihydrofuroacridone derivative ist the main alkaloid in cell suspension cultures of RUTA GRAVEOLENS, strain R-20. The N-methyl group is provided by L-methionine. N-methylanthranilic acid serves as excellent precursor of rutacridone. Furthermore this particular precursor could be trapped after feeding anthranilic acid in short term experiments.

Biosynthesis of rutacridone in tissue cultures of Ruta graveolens L.

The dihydrofuroacridone, rutacridone, is the main alkaloid in tissue cultures of Ruta graveolens L. strain R-19. The biosynthesis of this particular acridone alkaloid was investigated by using calluses and suspension cultures of strain R-19. Anthranilic acid is specifically incorporated into ring A of rutacridone. Some further evidence was provided that acetate via a polyketide is involved in acridone biosynthesis. Mevalonic acid gave a poor incorporation into rutacridone. Thus the origin of the isopropyldihydrofuran moiety of the investigated alkaloid is still obscure.