Initial Reductive Reactions in Aerobic Microbial Metabolism of 2,4,6-Trinitrotoluene

Because of its high electron deficiency, initial microbial transformations of 2,4,6-trinitrotoluene (TNT) are characterized by reductive rather than oxidation reactions. The reduction of the nitro groups seems to be the dominating mechanism, whereas hydrogenation of the aromatic ring, as described for picric acid, appears to be of minor importance. Thus, two bacterial strains enriched with TNT as a sole source of nitrogen under aerobic conditions, a gram-negative strain called TNT-8 and a gram-positive strain called TNT-32, carried out nitro-group reduction. In contrast, both a picric acid-utilizing Rhodococcus erythropolis strain, HL PM-1, and a 4-nitrotoluene-utilizing Mycobacterium sp. strain, HL 4-NT-1, possessed reductive enzyme systems, which catalyze ring hydrogenation, i.e., the addition of a hydride ion to the aromatic ring of TNT. The hydride-Meisenheimer complex thus formed (H⁻-TNT) was further converted to a yellow metabolite, which by electrospray mass and nuclear magnetic resonance spectral analyses was established as the protonated dihydride-Meisenheimer complex of TNT (2H⁻-TNT). Formation of hydride complexes could not be identified with the TNT-enriched strains TNT-8 and TNT-32, or with Pseudomonas sp. clone A (2NT⁻), for which such a mechanism has been proposed. Correspondingly, reductive denitration of TNT did not occur.     

A data-driven model of the generation of human EEG based on a spatially distributed stochastic wave equation

We discuss a model for the dynamics of the primary current density vector field within the grey matter of human brain. The model is based on a linear damped wave equation, driven by a stochastic term. By employing a realistically shaped average brain model and an estimate of the matrix which maps the primary currents distributed over grey matter to the electric potentials at the surface of the head, the model can be put into relation with recordings of the electroencephalogram (EEG). Through this step it becomes possible to employ EEG recordings for the purpose of estimating the primary current density vector field, i.e. finding a solution of the inverse problem of EEG generation. As a technique for inferring the unobserved high-dimensional primary current density field from EEG data of much lower dimension, a linear state space modelling approach is suggested, based on a generalisation of Kalman filtering, in combination with maximum-likelihood parameter estimation. The resulting algorithm for estimating dynamical solutions of the EEG inverse problem is applied to the task of localising the source of an epileptic spike from a clinical EEG data set; for comparison, we apply to the same task also a non-dynamical standard algorithm.     

A validated high-performance liquid chromatographic assay for the simultaneous determination of denaverine and its N-monodemethyl metabolite in human plasma

An isocratic reversed-phase high-performance liquid chromatographic method for the simultaneous determination of denaverine and its N-monodemethyl metabolite (MD 6) in human plasma is described. The assay involves the extraction with an n-heptane–2-propanol mixture (9:1, v/v) followed by back extraction into 12.5% (w/w) phosphoric acid. The analytes of interest and the internal standard were separated on a Superspher RP8 column using a mobile phase of acetonitrile–0.12 M NH₄H₂PO₄–tetrahydrofuran (24:17.2:1, v/v), adjusted to pH 3 with 85% (w/w) phosphoric acid. Ultraviolet detection was used at an operational wavelength of 220 nm. The retention times of MD 6, denaverine and the internal standard were 5.1, 6.3 and 10.2 min, respectively. The assay was validated according to international requirements and was found to be specific, accurate and precise with a linear range of 2.5–150 ng/ml for denaverine and MD 6. Extraction recoveries for denaverine and MD 6 ranged from 44 to 49% and from 42 to 47%, respectively. The stability of denaverine and MD 6 in plasma was demonstrated after 24 h storage at room temperature, after three freeze–thaw cycles and after 7 months frozen storage below −20°C. The stability of processed samples in the autosampler at room temperature was confirmed after 24 h storage. The analytical method has been applied to analyses of plasma samples from a pharmacokinetic study in man.     

ERBB4 Promoter Polymorphism Is Associated with Poor Distant Disease-Free Survival in High-Risk Early Breast Cancer

A number of genetic variants have been linked to increased risk of breast cancer. Little is, however, known about the prognostic significance of hereditary factors. Here, we investigated the frequency and prognostic significance of two ERBB4 promoter region variants, −782G>T (rs62626348) and −815A>T (rs62626347), in a cohort of 1010 breast cancer patients. The frequency of nine previously described somatic ERBB4 kinase domain mutations was also analyzed. Clinical material used in the study consisted of samples from the phase III, adjuvant, FinHer breast cancer trial involving 1010 women. Tumor DNA samples were genotyped for ERBB4 variants and somatic mutations using matrix-assisted laser desorption ionization/time of flight mass spectrometry. Paraffin-embedded tumor sections from all patients were immunohistochemically stained for ErbB4 expression. Association of ERBB4 genotype to distant disease-free survival (DDFS) was assessed using Kaplan-Meier and Cox regression analyses. Genotyping was successful for 91–93% of the 1010 samples. Frequencies observed for the ERBB4 variants were 2.5% and 1.3% for −782G>T and −815A>T, respectively. Variant −815A>T was significantly associated with poor survival (HR  = 2.86 [95% CI 1.15–6.67], P = 0.017). In contrast, variant −782G>T was associated with well-differentiated cancer (P = 0.019). Two (0.2%) ERBB4 kinase domain mutations were found, both of which have previously been shown to be functional and promote cancer cell growth in vitro. These data present the germ-line ERBB4 variant −815A>T as a novel prognostic marker in high-risk early breast cancer and indicate the presence of rare but potentially oncogenic somatic ERBB4 mutations in breast cancer.     

Enantioselective determination of hydroxychloroquine and its major metabolites in urine and the observation of a reversal in the (+)/(−)-hydroxychloroquine ratio

A sequential achiral-chiral high-performance liquid chromatographic system has been developed for the quantitation in urine of the enantiomers of hydroxychloroquine (HCQ), and of its 3 major metabolites, desethylhydroxychloroquine (DHCQ), desethylchloroquine (DCQ), and bisdesethylchloroquine (BDCQ). HCQ and its metabolites were separated and quantified on a cyano-bonded phase, and the enantiomeric ratios were determined using a Chiral-AGP chiral stationary phase. The assay validation and application of this method to a preliminary study in a human volunteer are presented. In this subject, the initial 0-4 h urine contained the 2 HCQ enantiomers in a ratio of (+)-HCQ:(?)-HCQ of 3:2; by the 2,064 h of the study, this ratio had reversed to (+)-HCQ:(?)-HCQ of 3:7. © 1993 Wiley-Liss, Inc.     

Detection of a germline mutation and somatic homozygous loss of the von Hippel-Lindau tumor-suppressor gene in a family with a de novo mutation

von Hippel-Lindau (VHL) disease is a pleioropic disorder featuring a variety of malignant and benign tumors of the eye, central nervous system, kidney, and adrenal gland. Recently the VHL gene has been identified in the chromosomal region 3p25-26. Prognosis and successful management of VHL patients and their descendants depend on unambiguous diagnosis. Due to recurrent hemangioblastomas, a 29-year-old patient without familial history of VHL disease was diagnosed to be at risk for the disease. Histopathological examination of a small renal mass identified a clear cell tumor with a G1 grading. Genetic characterization of the germline and of the renal tumor was performed. Polymerase chain reaction/single strand conformation polymorphism (PCR/SSCP) analysis with primers from the VHL gene identified a deletion of a single nucleotide in exon 2 in the patient's germline and in the tumor, but not in the DNA of his parents. This deletion therefore must be a de novo mutation. Comparative genome hybridization (CGH) and fluorescence in situ hybridization (FISH) analysis of the G1 tumor with differentially labelled yeast artifical chromosome (YAC) clones showed loss of 3p and of the 3p26 signals, respectively. In conclusion, we identified a de novo germline mutation in the VHL gene of a young patient and a somatic chromosome 3p loss at the homologous chromosome 3 in his renal tumor. Our results suggest a recessive mode of inactivation of the VHL gene, providing solid evidence for its tumor-suppressor gene characteristics. Our data show the diagnostic potential of genetic testing, especially in patients without VHL family history. Furthermore, the findings of homozygous inactivation of the VHL gene in a G1 tumor support the notion that the inactivation of the VHL gene is an early event in tumorigenesis of renal cell carcinoma.     

Induced cell fusion inForaminifera

Summary The fusion of cells of the monothalamous foraminiferMyxotheca arenilega (Protozoa, Rhizopoda) is described. The fusion process is induced by the separation of the extracellular organic shell from the cell body. As several cells can fuse either stepwise or simultanously, a giant cell of some millimeters in diameter can be formed.     

Über die Feinstruktur vonLabyrinthula coenocystis Schmoller nach Gefrierätzung

Zusammenfassung Gefrierätz-Untersuchungen an Cysten vonL. coenocystis ergaben, daß jede Zelle von einer gewellt verlaufenden Zellmembran umgeben wird, welche zylindrische Einsenkungen mit einem Durchmesser von 0,3 m aufweist. Diese Einsenkungen stellen die fürLabyrinthula charakteristischen Organellen, die bothrosomes, dar. Sie weisen in ihrem Zentrum 1–3 quergebrochene Erhebungen auf. Diese stellen Poren in der Zellmembran vonL. coenocystis dar. Die Anzahl der bothrosomes beträgt pro Zellhälfte 0–3, so daß die Gesamtzahl pro Zelle auf 5–8 geschätzt wird. Die Verteilung dieser Organellen auf der Zelloberfläche ist nicht regelmäßig, sondern zufällig. Die geringe Anzahl, ihre relativ geringe Größe im Vergleich zur Zelle und ihre unregelmäßige Verteilung auf der Zelloberfläche haben es unwahrscheinlich gemacht, daß die bothrosomes allein für die in ihrem Mechanismus noch völlig ungeklärte kontinuierliche gleitende Bewegung, etwa durch Abscheidung von Substanzen an diesen Organellen nach dem Rückstoßprinzip, verantwortlich gemacht werden können.

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The fine structure ofLabyrinthula coenocystis schmoller as revealed by freeze-etching

Summary Freeze-etched cysts ofL. coenocystis show that each cell is surrounded by a waved cell membrane in which cylindrical invaginations with a diameter of 0.3 m are visible. These invaginations represent the bothrosomes, characteristic organelles inLabyrinthula. Centered in the bothrosomes are 1–3 evaginations, which are cross-fractioned pores in the cell membrane. The number of bothrosomes per half cell is 0–3; so that the total number of these organelles is calculated at 5–8 per cell. The distribution of the bothrosomes on the cell surface is not regular but random. Their low number, their relative small size in comparison to the total cell area, and their random distribution on the cell surface lead us to presume that these organelles alone can not be held responsible for the cell's gliding movement as, for example, by emission of substances from the organelles by the repulsionprincip.

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Wir danken Herrn Prof. Dr. K. G.Grell, Zoologisches Institut der Universität Tübingen, für die Überlassung derLabyrinthula-Kultur und der Deutschen Forschungsgemeinschaft für ihre Unterstützung.