Total Synthesis and Structure Correction of the Cyclic Lipodepsipeptide Orfamide A.

A total synthesis of the cyclic lipodepsipeptide natural product orfamide A was achieved. By developing a synthesis format using an aminoacid ester building block and SPPS protocol adaptation, a focused library of target compounds was obtained, in high yield and purity. Spectral and LC-HRMS data of all library members with the isolated natural product identified the 5 Leu residue to be d- and the 3'-OH group to be R-configured. The structural correction of orfamide A by chemical synthesis and analysis was confirmed by biological activity comparison in Chlamydomonas reinhardtii, which indicated compound configuration to be important for bioactivity. Acute toxicity was also found against Trypanosoma brucei, the parasite causing African sleeping sickness.

Enzyme-Primed Native Chemical Ligation Produces Autoinducing Cyclopeptides in Clostridia.

Clostridia coordinate many important processes such as toxin production, infection, and survival by density-dependent communication (quorum sensing) using autoinducing peptides (AIPs). Although clostridial AIPs have been proposed to be (thio)lactone-containing peptides, their true structures remain elusive. Here, we report the genome-guided discovery of an AIP that controls endospore formation in Ruminiclostridium cellulolyticum. Through a combination of chemical synthesis and chemical complementation assays with a mutant strain, we reveal that the genuine chemical mediator is a homodetic cyclopeptide (cAIP). Kinetic analyses indicate that the mature cAIP is produced via a cryptic thiolactone intermediate that undergoes a rapid S→N acyl shift, in a manner similar to intramolecular native chemical ligation (NCL). Finally, by implementing a chemical probe in a targeted screen, we show that this novel enzyme-primed, intramolecular NCL is a widespread feature of clostridial AIP biosynthesis.

Synthesis and catalytic activity of tridentate N-(2-pyridylethyl)-substituted bulky amidinates of calcium and strontium.

Metalation of the formamidine Dipp-N[double bond, length as m-dash]C(H)-N(H)-C2H4-Py (1a) and benzamidine Dipp-N[double bond, length as m-dash]C(Ph)-N(H)-C2H4-Py (1b) with [(thf)2M{N(SiMe3)2}2] (M = Ca, Sr) yields the corresponding homoleptic complexes [M{Dipp-N[double bond, length as m-dash]C(R)-N-C2H4-Py}2] (M/R = Ca/H (2a), Ca/Ph (2b), and Sr/Ph (3b)) regardless of the applied stoichiometry. Only during calciation of Dipp-N[double bond, length as m-dash]C(H)-N(H)-C2H4-Py (1a), the heteroleptic intermediate [{(Me3Si)2N}Ca{Dipp-N[double bond, length as m-dash]C(R)-N-C2H4-Py}]2 (2a') has been observed. The formamidinate complex of strontium crystallizes as a tmeda adduct of the type [(tmeda)Sr{Dipp-N[double bond, length as m-dash]C(H)-N-C2H4-Py}2] (3a). Metalation of the pivalamidine Dipp-N[double bond, length as m-dash]C(tBu)-N(H)-C2H4-Py (1c) leads to the formation of heteroleptic mononuclear [{(Me3Si)2N}M{Dipp-N[double bond, length as m-dash]C(tBu)-N(H)-C2H4-Py}] (M = Ca (2c) and Sr (3c)) with a side-on bonding of the Dipp group to the alkaline earth metals. Calciation of chiral Dipp-N[double bond, length as m-dash]C(tBu)-N(H)-CH2CH(Me)-Py (R)-1d and (S)-1d with [(thf)2Ca{N(SiMe3)2}2] yields the homoleptic complexes [Ca{Dipp-N[double bond, length as m-dash]C(tBu)-N-CH2CH(Me)-Py}2] with the enantiomeric forms (R,R)-2d and (S,S)-2d regardless of the applied stoichiometry. The complexes 2c and 2d catalyze the intramolecular hydroamination of the aminoalkene 2,2-diphenylpent-4-enylamine yielding 2-methyl-4,4-diphenylpyrrolidine but the stereochemistry cannot be influenced by the chiral compounds (R,R)-2d and (S,S)-2d.

Structure Elucidation and Activity of Kolossin†A, the D-/L-Pentadecapeptide Product of a Giant Nonribosomal Peptide Synthetase.

The largest continuous bacterial nonribosomal peptide synthetase discovered so far is described. It consists of 15 consecutive modules arising from an uninterrupted, fully functional gene in the entomopathogenic bacterium Photorhabdus luminescens. The identification of its cryptic biosynthesis product was achieved by using a combination of genome analysis, promoter exchange, isotopic labeling experiments, and total synthesis of a focused collection of peptide candidates. Although it belongs to the growing class of D-/ L-peptide natural products, the encoded metabolite kolossin A was found to be largely devoid of antibiotic activity and is likely involved in interspecies communication. A stereoisomer of this peculiar natural product displayed high activity against Trypanosoma brucei rhodesiense, a recalcitrant parasite that causes the deadly disease African sleeping sickness.

Phthalate exposure in pregnant women and newborns - the urinary metabolite excretion pattern differs distinctly.

Some phthalates are endocrine disruptors and reproductive and developmental toxicants. Data on newborn phthalate exposure and elimination characteristics are scarce. We determined 21 urinary phthalate metabolites (indicating exposure to 11 parent phthalates) in two study approaches: in the first approach we collected the urine of 20 healthy newborns at days 2-5 post partum together with 47 urine samples of 7 women during pregnancy. In the second fine tuned approach we collected first urine samples of 9 healthy newborns together with their mother's urine shortly before birth. To ensure full and contamination free collection of the newborns first urines we used special adhesive urine bags for children. All urine samples revealed ubiquitous exposures to phthalates comparable to other populations. Metabolite levels in the newborns first day urine samples were generally lower than in all other samples. However, the newborns urines (both first and day 2-5 urines) showed a metabolite pattern distinctly different from the maternal and general population samples: in the newborns urines the carboxy-metabolites of the long chain phthalates (DEHP, DiNP, DiDP) were the by far dominant metabolites with a relative share in the metabolite spectrum up to 6 times higher than in maternal urine. Oppositely, for the short chain phthalates (DBP, DiBP) oxidized metabolites seemed to be less favored than the simple monoesters in the newborns urines. The skewed metabolite distribution in the newborns urine warrants further investigation in terms of early phthalate metabolism, the quantity of internal phthalate exposure of the fetus/newborn and its possible health effects.

Fatty acid distribution of cord and maternal blood in human pregnancy: special focus on individual trans fatty acids and conjugated linoleic acids.

BACKGROUND: Maternal nutrition in pregnancy has a crucial impact on the development of the fetus. Dietary trans fatty acids (tFA) are known to have adverse health effects, especially during pregnancy. However, the distribution of tFA produced via partial hydrogenation of vegetable oils (mainly elaidic acid; t9) differs compared to ruminant-derived tFA (mainly vaccenic acid; t11). Recent findings indicate that they may have different impact on human health.Therefore, in this study, plasma and erythrocytes of mother-child pairs (n = 55) were sampled to investigate the distribution of tFA, including individual trans C18:1 fatty acids and conjugated linoleic acids (CLA) in fetal related to maternal lipids; with additional consideration of maternal dairy fat intake. RESULTS: Portion of t9 and t11, but also of c9,t11 CLA was higher in maternal than in fetal blood lipids. The portion of t9 in maternal and fetal lipids differed only slightly. In contrast, the portion of fetal t11 was only half of that in maternal blood. This led to a fetal t9/t11-index in plasma and erythrocytes being twice as high compared to the maternal values. A high dairy fat intake resulted in elevated portions of t11 and its Δ9-desaturation product c9,t11 CLA in maternal blood. In contrast, in the respective fetal blood lipids only c9,t11 CLA, but not t11 was increased. Nevertheless, a positive association between maternal and fetal plasma exists for both t11 and c9,t11 CLA. Furthermore, in contrast to t9, t11 was not negatively associated with n-3 LC-PUFA in fetal blood lipids. CONCLUSIONS: Fetal blood fatty acid composition essentially depends on and is altered by the maternal fatty acid supply. However, in addition to dietary factors, other aspects also contribute to the individual fatty acid distribution (oxidation, conversion, incorporation). The lower portion of fetal t11 compared to maternal t11, possibly results from Δ9-desaturation to c9,t11 CLA and/or oxidation. Based on the fatty acid distribution, it can be concluded that t11 differs from t9 regarding its metabolism and their impact on fetal LC-PUFA.

Cell-specific RNA interference by peptide-inhibited-peptidase-activated siRNAs.

The use of chemically-synthesized short interfering RNAs (siRNAs) is the key method of choice to manipulate gene expression in mammalian cell cultures and in vivo. Several previous studies have aimed at inducing cell-specific RNA interference (RNAi) in order to use siRNA molecules as therapeutic reagents. Here, we used peptide-inhibited siRNAs that were activated after cleavage by cell-specific peptidases. We show that siRNAs with bound peptide at the antisense strand could be activated in target cells and were able to induce RNAi in a cell-specific manner. Green Fluorescent Protein (GFP) and Signal Transducer and Activator of Transcription (STAT)-3 gene expression were selectively reduced in a JEG-3 human choriocarcinoma cell line expressing the activating enzyme caspase-4, whereas the effect was absent in HEK cells which lacked the enzyme. In JEG-3 cells, reduction of STAT3 gene expression by conventional and peptide-inhibited siRNA led to a decrease in cell proliferation. This suggests that peptide-inhibited siRNAs provide improved cell specificity and offers new opportunities for their therapeutic use.

Development of a human model to study homing behavior of immune cells into decidua and placental villi under ex vivo conditions.

PROBLEM: Homing of lymphocytes and NK cells into the decidua and its regulation has been very controversially discussed. Therefore, we aimed to establish an in vivo simulation method for analysis of homing behavior, which might be also useful for other cells such as stem or tumor cells. METHOD OF STUDY: A human term placenta has been perfused with medium to elute blood and then with maternal autologous carboxyfluorescein succinimidyl ester (CFSE)-labeled peripheral blood lymphocytes for 3 hr and rinsed for another 2 hr. Tissue was analysed histologically for detection of labeled cells. Labeled lymphocytes and beads in perfusate have been identified and counted by flow cytometry. RESULTS: At the moment of tissue fixation for histology, the perfusate was free of labeled cells. Labeled perfused lymphocytes have been found adhered and integrated in vessel wall structures, in decidual stroma and as colonies in individual villi. CONCLUSION: Placenta perfusion with a lymphocyte suspension is feasible without plugging the tube system. Time is sufficient for cells to adhere and to migrate into the stroma. Also some villi have been infiltrated which might be caused by inflammatory stimuli. The perfusion system might be useful to test substances for their capacity to influence homing of lymphocytes or other cells.

Assessment of caspase-4 released free AFC by RP-HPLC and fluorescence detection.

A simple RP-HPLC method based on fluorescence detection was developed for the quantitation of 7-amino-4-trifluoro methylcoumarin (AFC) in cell lysates from JEG-3 choriocarcinoma cells for determination of caspase-4 activity. In contrast to the established methods of AFC detection using a fluorescence microplate reader or using a fluorescence photometer, the separation of AFC-signals from interfering fluorescence signals by a reversed phase column affords more precise quantitation of released AFC. This can be important for analyses of cell lysates with low caspase activity or experimental series with marginal differences among samples. By applying this new method, a linear dynamic range of 40pmol/mL to 3nmol/mL with a correlation coefficient of 0.9996 was achieved. Due to the short retention time ( approximately 7min), the determination of AFC by RP-HPLC under isocratic conditions requires small amounts of samples (50microL injection volume), and allows increased sample throughput. This method should be easily applied with little or no modification to other caspase assays by using the same fluorophore.

Impact of PUFA on early immune and fetal development.

It has recently been reported that the increased prevalence in childhood allergy may be linked to deviations in fetal immune development. One reason may be impaired nutrient supply. Hence, a well-differentiated placenta together with an optimal fetal nutrition via the mother are important prerequisites for the establishment of a functional immune system with normal immune responses. Fatty acids and their derivatives can influence both the early immune development and immune maturation by regulating numerous metabolic processes and the gene expression of important proteins such as enzymes and cytokines. The present review summarises the impact of nutritional fatty acids on the development of the immune system as well as the fetal development. It describes the mechanisms of action of PUFA, trans fatty acids and conjugated linoleic acids in programming the fetus with regard to its risk of acquiring atopic diseases in childhood.

Lessons from reproductive immunology for other fields of immunology and clinical approaches.

Reproduction is indispensable to evolution and, thus, life. Nonetheless, it overcomes common rules known to established life. Immunology of reproduction, and especially the tolerance of two genetically distinct organisms and their fruitful symbiosis, is one of the most imposing paradox of life. Mechanisms, which are physiologically used for induction of said tolerance, are frequently abused by pathogens or tumors intending to escape the host's immune response. Understanding the regulation of immune responses in pregnancy and the invasion of allogeneic fetus-derived trophoblast cells into the decidua may lead to new therapeutic concepts. In transplantation, knowledge concerning local physiological immunotolerance may be useful for the development of new therapies, which do not require a general immune suppression of the patient. In immunological disorders, such as autoimmune diseases or allergies, immune deviations occur which are either prevented during pregnancy or have parallels to pregnancy. Vice versa, lessons from other fields of immunology may also offer new notions for the comprehension of reproductive immunology and may lead to new therapies for the treatment of pregnancy-related problems.