The effect of mycophenolate mofetil on podocytes in nephrotoxic serum nephritis.

Mycophenolate mofetil (MMF) is applied in proteinuric kidney diseases, but the exact mechanism of its effect on podocytes is still unknown. Our previous in vitro experiments suggested that MMF can ameliorate podocyte damage via restoration of the Ca2+-actin cytoskeleton axis. The goal of this study was to characterize podocyte biology during MMF treatment in nephrotoxic serum (NTS) nephritis (NTN). NTN was induced in three-week old wild-type mice. On day 3, half of the mice were treated with MMF (100 mg/kgBW/d p.o.) for one week. On day 10, we performed proteomic analysis of glomeruli as well as super-resolution imaging of the slit diaphragm. For multiphoton imaging of Ca2+ concentration ([Ca2+]i), the experimental design was repeated in mice expressing podocyte-specific Ca2+ sensor. MMF ameliorated the proteinuria and crescent formation induced by NTS. We identified significant changes in the abundance of proteins involved in Ca2+ signaling and actin cytoskeleton regulation, which was further confirmed by direct [Ca2+]i imaging in podocytes showing decreased Ca2+ levels after MMF treatment. This was associated with a tendency to restoration of podocyte foot process structure. Here, we provide evidence that MPA has a substantial direct effect on podocytes. MMF contributes to improvement of [Ca2+]i and amelioration of the disorganized actin cytoskeleton in podocytes. These data extend the knowledge of direct effects of immunosuppressants on podocytes that may contribute to a more effective treatment of proteinuric glomerulopathies with the least possible side effects.

Strong hypoxia reduces leptin synthesis in purified primary human trophoblasts.

INTRODUCTION: Oxygen availability severely affects placental function. During placental hypoxia, stabilization of hypoxia inducible factors (HIFs) affects transcription, and leptin gene expression concomitantly increases in vivo and in vitro. However, a causal relationship is uncertain. METHODS: We investigated the effect of oxygen availability on HIF-1 alpha (HIF1A) and leptin regulation in primary human trophoblasts isolated from six normal term placentae cultured at 0.1%, 1%, 3%, and 8% oxygen for 6 h, 24 h and 48 h. Gene expressions of leptin (LEP), leptin receptors (LEPR), HIF1A, insulin receptor (INSR) and further genes relevant in hypoxia (VEGFA, EPO, NOS2) or apoptosis (BCL2, BAX, Tp53) were examined. Leptin, HIF1A, INSR, phospho-AKT/AKT (insulin receptor signaling), caspase 3 and cleaved caspase 3 (apoptosis) proteins were measured. RESULTS: A hypoxic reaction with stabilization of HIF1A protein as well as up-regulation of HIF1A and VEGFA gene expressions, but without any hint for apoptosis, was present at 0.1% and 1% oxygen. However, leptin protein concentration (cell supernatants) peaked at 8% oxygen (normoxia) and was significantly reduced at 0.1% oxygen. There was no significant correlation between leptin and HIF1A, neither on the gene nor on the protein level. DISCUSSION: Elevated leptin gene expression in hypoxic placentas may not originate from trophoblasts, but from other placental cells, or from interaction of trophoblasts with other cells. Not only fetal hyperleptinemia, but also fetal hypoleptinemia under hypoxic conditions is conceivable. Strategies to prevent leptin dysregulation during pregnancy should be elucidated to protect the offspring from fetal programming of leptin resistance and adiposity in later life.

Leptin does not induce an inflammatory response in the murine placenta.

Leptin is described as a pro-inflammatory signal in fat tissue, which is released from adipocytes and in turn activates immune cells. Also, leptin levels are known to be increased in pregnancies complicated with enhanced inflammatory processes in the placenta. Hence, we assumed that increased leptin amounts might contribute to inducing an inflammatory response in the placenta. To test this hypothesis, pregnant mice were continuously infused with recombinant murine leptin s. c. from day g13 to g16, resulting in a 3-fold increase of maternal circulating serum leptin levels. Dissected placentas were examined for the expression of pro-inflammatory cytokines IL-6 and TNF-alpha and the anti-inflammatory cytokine IL-10 using qPCR analysis. No changes were found except for TNF-alpha, which was slightly elevated upon leptin stimulation. However, TNF-alpha protein levels were not significantly higher in placentas from leptin treated mice. Also, leukocyte infiltration in the labyrinth section of placentas was not increased. In summary, our data demonstrate for the first time that elevated leptin levels alone do not induce an inflammatory response in the placenta.

[Procalcitonin as a marker for severe sepsis in an immunosuppressed patient]. / Procalcitonin als Marker der schweren Sepsis bei einer immunsupprimierten Patientin.

A 22 year old female was admitted to the emergency department with high fever up to 41,5 degrees C, tachycardia, and arterial hypotension. Clinically, she presented with bilateral pulmonary coarse crackles. Diagnosis on admission was pneumonia with septic shock. Intriguingly, procalcitonin (PCT) was increased early, reaching up to 435 ng/mL, while C-reactive protein levels were only moderately increased, with several days delay. The sepsis was originated from a multi-resistant pseudomonas aeruginosa pneumonia. Remarkably, the course of PCT levels reflected the severity of septic shock in that it paralleled noradrenaline demand. Ten months previously, the patient had been diagnosed with acute disseminated brainstem encephalitis (ADEM), and had received two cycles of intravenous cyclophosphamide. Our case illustrates that PCT is an early marker for sepsis and it indicates that PCT may also be a valuable marker for the severity of sepsis in immunosuppressed patients.

[The incidence of microlaryngoscopy associated complications]. / Inzidenz spatelbedingter Nebenwirkungen der Mikrolaryngoskopie.

BACKGROUND: The frequency of local and cardiovascular side effects of microlaryngoscopy is generally underestimated. There are no data available in the literature from recent and prospective clinical trials. PATIENTS AND METHODS: We examined 81 patients between 03/1998 and 03/2000 who underwent microlaryngoscopy in our department. This was done following a standard protocol before, during and after surgery. Side effects of endotracheal intubation were avoided by using supraglottic jet-ventilation. RESULTS: In 79% of our cases we encountered side effects due to the microlaryngoscopic procedure.86% of them were reversible lesions,hematomas and edemas of the mucous membranes or mild cardiovascular dysregulations. In two cases there was a dental complication (one fracture, one dislocation), and seven cases of moderate hemodynamic effects were noted. CONCLUSIONS: The incidence of tissue damage caused by microlaryngoscopic endoscopes is much higher than commonly assumed in clinical practice. This has to be explained to the patient when obtaining his written consent to a certain microlaryngoscopic procedure and to be considered during the postoperative follow up.The consequent use of tooth protection and a good control of muscle relaxation and analgesia can be effective in preventing side effects.

Murrastifoline-F: first total synthesis, atropo-enantiomer resolution, and stereoanalysis of an axially chiral N,C-coupled biaryl alkaloid.

The first total synthesis of the Murraya alkaloid murrastifoline-F (3), an unsymmetric, N,C-bonded heterobiarylic biscarbazole, is described. Starting from the likewise naturally occurring-but here synthetically prepared-"monomer" murrayafoline-A (6), lead tetraacetate-mediated oxidative non-phenolic biaryl coupling gives 3 as the main regioisomer. The existence of this natural product as a pair of stable atropo-enantiomers was demonstrated analytically through LC-CD investigations. Preparatively, the racemate resolution succeeded by O-demethylation, derivatization with Mosher's reagent, and chromatographic separation of the resulting diastereomers. The absolute configurations of the atropisomers were assigned by CD spectroscopy in combination with quantum chemical CD calculations at the stage of the alkaloid 3 and by ROESY experiments of the diastereomeric Mosher derivatives. In the root extract of the curry leaf plant Murraya koenigii (Rutaceae), murrastifoline-F (3) was found to exist as a 56:44 mixture in favor of the M-enantiomer, by LC-CD coupling.

A photometric screening method for dimeric naphthylisoquinoline alkaloids and complete on-line structural elucidation of a dimer in crude plant extracts, by the LC-MS/LC-NMR/LC-CD triad.

An efficient evaluation procedure for the chemical screening and on-line structural elucidation of dimeric naphthylisoquinoline alkaloids has been developed. The method is based on the lead tetraacetate oxidation of the central binaphthalene core of the alkaloids. UV spectra of the extracts after addition of the oxidant show, in the presence of naphthylisoquinoline dimers, the appearance of a characteristic long-wavelength absorption indicative of dinaphthoquinones. The efficiency and relevance of the method has been demonstrated in the discovery of a constitutionally and configurationally new dimeric naphthylisoquinoline alkaloid, named ancistrogriffithine A (4), from the previously uninvestigated Asian liana Ancistrocladus griffithii. After verification of this screening result by LC-ESI-MS/MS, the constitution and the relative configuration of the compound were elucidated on line, by LC-NMR and LC-CD on the extract. Using an LC-NMR-WET-ROESY experiment, itwas possible for the first time to determine the relative axial configuration of a natural biaryl compound on line, by observing long-range ROE interactions. Finally, an oxidative degradation right on the extract delivered the absolute configuration of 4, without isolation of the alkaloid. Ancistrogriffithine A is the as yet only dimeric naphthylisoquinoline from an Asian Ancistrocladaceae plant.

Droserone from cell cultures of Triphyophyllum peltatum (Dioncophyllaceae) and its biosynthetic origin.

The growth and droserone content of callus cultures of Triphyophyllum peltatum grown in liquid 1/5 Linsmaier and Skoog medium was studied. During a lag phase in growth, droserone concentrations in the medium reached a value of 2.1 mg g-1 fr. wt. After this maximum value the concentration decreased slightly to 1.8 mg g-1 fr. wt., while the growth of the calli was enhanced (25% increase in fr. wt. within 7 days). Plumbagin and isoshinanolone were likewise present in the medium. By feeding 13C2-labelled acetate to the cultures the biosynthesis of droserone was elucidated. The incorporation of whole C2-units unambiguously shows its acetogenic origin and fits well in the biosynthetic scheme suggested for the structurally--and biogenetically--related naphthylisoquinoline alkaloids.

Observation of exchangeable protons by high-performance liquid chromatography-nuclear magnetic resonance spectroscopy and high-performance liquid chromatography-electrospray ionization mass spectrometry: a useful tool for the hyphenated analysis of natural products.

The first high-performance liquid chromatography-nuclear magnetic resonance (HPLC-NMR) investigation of exchangeable protons of low-molecular-mass natural products is reported. Model alkaloids or crude plant extracts were dissolved in 2H2O-1H2O-MeCN (deuterium oxide-water-acetonitrile) or 2H2O-MeCN and, after direct injection or chromatographic separation, examined in a 60-microl NMR flow probe. Exchangeable amino protons initially detected by HPLC-electrospray ionization mass spectrometry were subsequently identified and investigated by stop-flow 1H-NMR, two-dimensional (2D) total correlation spectroscopy (TOCSY), and 2D nuclear Overhauser effect spectroscopy (NOESY). These experiments extend the applicability of HPLC-NMR for the investigation and structure elucidation of natural products.

Role of fiberoptic bronchoscopy in conjunction with the use of double-lumen tubes for thoracic anesthesia: a prospective study.

BACKGROUND: Fiberoptic bronchoscopy has been recommended to verify the position of double-lumen tubes (DLT), but this remains controversial. The authors studied the role of bronchoscopy for placing and monitoring right- and left-sided DLTs after blind intubation and after positioning the patient. METHODS: Two hundred patients having thoracic surgery requiring DLT insertion were prospectively studied. "Blind" tracheal intubations were done with 163 left-sided and 37 right-sided disposable polyvinyl chloride Robertshaw tubes. Bronchoscopy was performed by a different anesthesiologist after intubation and conventional clinical verification of correct placement and after patient positioning for thoracotomy. A DLT was considered malpositioned when it had to be moved >0.5 cm to correct its position. Critical malpositions were those that might have affected patient safety or influenced the surgical procedure if left uncorrected. RESULTS: After "blind" DLT intubation, clinical evidence of malpositioning was found in 28 patients. This was confirmed by fiberoptic assessment. In 172 patients in whom placement was judged correct by clinical assessment, malpositioning was detected by bronchoscopy in 79 cases, 25 of which were critical. After patient positioning, DLTs were found to be displaced in 93 patients, 48 of which were critical. Right-sided DLTs were significantly more likely to be malpositioned than were left-sided DLTs. Two complications were related to unsatisfactory lung separation in the 200 patients studied. CONCLUSIONS: After blind intubation and patient positioning, more than one third of DLTs required repositioning. Routine bronchoscopy is therefore recommended after intubation and after patient positioning.