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Abstract Background Corynebacterium diphtheriae, the causative agent of diphtheria, is well-investigated in respect to toxin production, while little is known about C. diphtheriae factors crucial for colonization of the host. In this study, we investigated strain-specific differences in adhesion, invasion and intracellular survival and analyzed formation of pili in different isolates. Results Adhesion of different C. diphtheriae strains to epithelial cells and invasion of these cells are not strictly coupled processes. Using ultrastructure analyses by atomic force microscopy, significant differences in macromolecular surface structures were found between the investigated C. diphtheriae strains in respect to number and length of pili. Interestingly, adhesion and pili formation are not coupled processes and also no correlation between invasion and pili formation was found. Using RNA hybridization and Western blotting experiments, strain-specific pili expression patterns were observed. None of the studied C. diphtheriae strains had a dramatic detrimental effect on host cell viability as indicated by measurements of transepithelial resistance of Detroit 562 cell monolayers and fluorescence microscopy, leading to the assumption that C. diphtheriae strains might use epithelial cells as an environmental niche supplying protection against antibodies and macrophages. Conclusions The results obtained suggest that it is necessary to investigate various isolates on a molecular level to understand and to predict the colonization process of different C. diphtheriae strains.

Abstract Background Corynebacterium diphtheriae, the causative agent of diphtheria, is well-investigated in respect to toxin production, while little is known about C. diphtheriae factors crucial for colonization of the host. In this study, we investigated the function of surface-associated protein DIP1281, previously annotated as hypothetical invasion-associated protein. Results Microscopic inspection of DIP1281 mutant strains revealed an increased size of the single cells in combination with an altered less club-like shape and formation of chains of cells rather than the typical V-like division forms or palisades of growing C. diphtheriae cells. Cell viability was not impaired. Immuno-fluorescence microscopy, SDS-PAGE and 2-D PAGE of surface proteins revealed clear differences of wild-type and mutant protein patterns, which were verified by atomic force microscopy. DIP1281 mutant cells were not only altered in shape and surface structure but completely lack the ability to adhere to host cells and consequently invade these. Conclusions Our data indicate that DIP1281 is predominantly involved in the organization of the outer surface protein layer rather than in the separation of the peptidoglycan cell wall of dividing bacteria. The adhesion- and invasion-negative phenotype of corresponding mutant strains is an effect of rearrangements of the outer surface.

It is thought that cells surviving ionizing radiation exposure repair DNA double-strand breaks (DSBs) and restore their genomes. However, the recent biochemical and genetic characterization of DSB repair pathways reveals that only homologous recombination (HR) can function in an error-free manner and that the non-homologous end joining (NHEJ) pathways canonical NHEJ (c-NHEJ), alternative end joining (alt-EJ), and single-strand annealing (SSA) are error-prone, and potentially leave behind genomic scars and altered genomes. The strong cell cycle restriction of HR to S/G2 phases and the unparalleled efficiency of c-NHEJ throughout the cell cycle, raise the intriguing question as to how far a surviving cell "reaches" after repairing the genome back to its pre-irradiation state. Indeed, there is evidence that the genomes of cells surviving radiation treatment harbor extensive genomic alterations. To directly investigate this possibility, we adopted next-generation sequencing (NGS) technologies and tested a normal human fibroblast cell line, 82-6 hTert, after exposure up to 6 Gy. Cells were irradiated and surviving colonies expanded and the cells frozen. Sequencing analysis using the Illumina sequencing platform and comparison with the unirradiated genome detected frequent genomic alterations in the six investigated radiation survivor clones, including translocations and large deletions. Translocations detected by this analysis and predicted to generate visible cytogenetic alterations were frequently (three out of five) confirmed using mFISH cytogenetic analysis. PCR analysis of selected deletions also confirmed seven of the ten examined. We conclude that cells surviving radiation exposure tolerate and pass to their progeny a wide spectrum of genomic alterations. This recognition needs to be integrated into the interpretation of biological results at all endpoints, as well as in the formulation of mathematical models of radiation action. NGS analysis of irradiated genomes promises to enhance molecular cytogenetics by increasing the spectrum of detectable genomic alterations and advance our understanding of key molecular radiobiological effects and the logic underpinning DSB repair. However, further developments in the technology will be required to harness its full potential., (© 2025 by Radiation Research Society. All rights of reproduction in any form reserved.)

The most important factor that complicates the work of dysmorphologists is the significant phenotypic variability of the human face. Next-Generation Phenotyping (NGP) tools that assist clinicians with recognizing characteristic syndromic patterns are particularly challenged when confronted with patients from populations different from their training data. To that end, we systematically analyzed the impact of genetic ancestry on facial dysmorphism. For that purpose, we established the GestaltMatcher Database (GMDB) as a reference dataset for medical images of patients with rare genetic disorders from around the world. We collected 10,980 frontal facial images - more than a quarter previously unpublished - from 8,346 patients, representing 581 rare disorders. Although the predominant ancestry is still European (67%), data from underrepresented populations have been increased considerably via global collaborations (19% Asian and 7% African). This includes previously unpublished reports for more than 40% of the African patients. The NGP analysis on this diverse dataset revealed characteristic performance differences depending on the composition of training and test sets corresponding to genetic relatedness. For clinical use of NGP, incorporating non-European patients resulted in a profound enhancement of GestaltMatcher performance. The top-5 accuracy rate increased by +11.29%. Importantly, this improvement in delineating the correct disorder from a facial portrait was achieved without decreasing the performance on European patients. By design, GMDB complies with the FAIR principles by rendering the curated medical data findable, accessible, interoperable, and reusable. This means GMDB can also serve as data for training and benchmarking. In summary, our study on facial dysmorphism on a global sample revealed a considerable cross ancestral phenotypic variability confounding NGP that should be counteracted by international efforts for increasing data diversity. GMDB will serve as a vital reference database for clinicians and a transparent training set for advancing NGP technology.

Purpose: To evaluate clinical characteristics, quality of life (QoL) and effectiveness in patients with menstrual cycle disorders (MCDs) including abnormal uterine bleeding, dysmenorrhea and mastodynia/mastalgia related to premenstrual syndrome taking the Vitex agnus-castus (VAC) products Cyclodynon® or Mastodynon® in a real-world setting., Methods: A single-center retrospective longitudinal cohort study (3 ± 1 months), using data obtained from healthcare data archive and telephone interviews. The main study variables were changes in bleeding, menstrual pain, breast tenderness and patients' QoL., Results: Data from 1700 women with a mean age of 30.2 years (± 6.3) were analyzed. The most common MCDs were dysmenorrhea (43.8%) and mastodynia/mastalgia (21.1%). Three-month treatment with VAC extract substantially decreased the percentage of patients with irregular cycle (from 9.1% to 0.1%) and breast tenderness (from 39.9% to 0.8%). Improvement in bleeding intensity, frequency and menstrual pain was experienced by 83.4%, 79.2%, and 85.2% of the patients, respectively. When analyzed by disease category, these parameters improved in almost all dysmenorrhea patients, while they improved to a lesser extent in mastodynia/mastalgia patients. QoL improved in all aspects, but was reported by a higher proportion of dysmenorrhea patients compared to mastodynia/mastalgia patients. Treatment was overall well tolerated with a favorable safety profile., Conclusion: These real-world data demonstrate the effectiveness of the VAC-containing products Cyclodynon® and Mastodynon® in the three-month treatment of MCDs, with a pronounced improvement in key disease symptoms and QoL. Intriguingly, while QoL was generally greatly improved, the response to VAC therapy varied depending on the type of underlying MCD., (© 2024. The Author(s).)

Introduction: Acute lower uncomplicated urinary tract infection (uUTI) affects a large proportion of women. Increased antimicrobial resistance has created an urgent need for novel therapeutics and the phytotherapeutic drug BNO 1045 (Canephron® N) has previously been shown to be noninferior to standard antimicrobial stewardship. This sub-analysis from a randomized, double-blind, controlled phase III noninferiority clinical trial using BNO 1045 versus fosfomycin to treat uUTI aimed to determine how urine cytokine levels are altered by the two different treatments., Methods: Urine samples from a predefined subset of women diagnosed with uUTI (18-70 years) and treated with BNO 1045 (n = 58) or fosfomycin (n = 69) were analyzed for urine levels of IL-6 and IL-8, using analyte-to-creatinine ratios., Results: BNO 1045 treatment showed similar effects to fosfomycin treatment in reducing both urine IL-6 and IL-8 levels. Mean IL-6 and IL-8 levels were markedly reduced in all patients regardless of treatment. BNO 1045 treatment decreased urine IL-8 significantly (p = 0.0142) and showed a trend toward reduction of urine IL-6 (p = 0.0551). Fosfomycin treatment reduced both IL-6 and IL-8 levels significantly (p = 0.0038, <0.0001 respectively)., Conclusion: BNO 1045 is, in addition to reducing symptoms, comparable to fosfomycin treatment in reducing the local inflammatory response associated with uUTI., (© 2023 The Author(s). Published by S. Karger AG, Basel.)

Epithelial surfaces in humans are home to symbiotic microbes (i.e., microbiota) that influence the defensive function against pathogens, depending on the health of the microbiota. Healthy microbiota contribute to the well-being of their host, in general (e.g., via the gut-brain axis), and their respective anatomical site, in particular (e.g., oral, urogenital, skin, or respiratory microbiota). Despite efforts towards a more responsible use of antibiotics, they are often prescribed for uncomplicated, self-limiting infections and can have a substantial negative impact on the gut microbiota. Treatment alternatives, such as non-steroidal anti-inflammatory drugs, may also influence the microbiota; thus, they can have lasting adverse effects. Herbal drugs offer a generally safe treatment option for uncomplicated infections of the urinary or respiratory tract. Additionally, their microbiota preserving properties allow for a more appropriate therapy of uncomplicated infections, without contributing to an increase in antibiotic resistance or disturbing the gut microbiota. Here, herbal treatments may be a more appropriate therapy, with a generally favorable safety profile., Competing Interests: B Nausch, CB Bittner, M Höller, and D Abramov-Sommariva are employees of Bionorica SE. A. Gessner receives consultancy fees from Bionorica SE.

The extreme metal tolerance of up to 130 mM NiSO 4 in Streptomyces mirabilis P16B-1 was investigated. Genome sequencing revealed the presence of a large linear plasmid, pI. To identify plasmid-encoded determinants of metal resistance, a newly established transformation system was used to characterize the predicted plasmid-encoded loci nreB, hoxN , and copYZ . Reintroduction into the plasmid-cured S. mirabilis ΔpI confirmed that the predicted metal transporter gene nreB constitutes a nickel resistance factor, which was further supported by its heterologous expression in Escherichia coli. In contrast, the predicted nickel exporter gene hoxN decreased nickel tolerance, while copper tolerance was enhanced. The predicted copper-dependent transcriptional regulator gene copY did not induce tolerance toward either metal. Since genes for transfer were identified on the plasmid, its conjugational transfer to the metal-sensitive Streptomyces lividans TK24 was checked. This resulted in acquired tolerance toward 30 mM nickel and additionally increased the tolerance toward copper and cobalt, while oxidative stress tolerance remained unchanged. Intracellular nickel concentrations decreased in the transconjugant strain. The high extracellular nickel concentrations allowed for biomineralization. Plasmid transfer could also be confirmed into the co-occurring actinomycete Kribbella spp. in soil microcosms. IMPORTANCE Living in extremely metal-rich environments requires specific adaptations, and often, specific metal tolerance genes are encoded on a transferable plasmid. Here, Streptomyces mirabilis P16B-1, isolated from a former mining area and able to grow with up to 130 mM NiSO 4 , was investigated. The bacterial chromosome, as well as a giant plasmid, was sequenced. The plasmid-borne gene nreB was confirmed to confer metal resistance. A newly established transformation system allowed us to construct a plasmid-cured S. mirabilis as well as an nreB -rescued strain in addition to confirming nreB encoding nickel resistance if heterologously expressed in E. coli. The potential of intra- and interspecific plasmid transfer, together with the presence of metal resistance factors on that plasmid, underlines the importance of plasmids for transfer of resistance factors within a bacterial soil community.

Objective: The goal of the present study was to evaluate treatment with Canephron ® compared to standard antibiotic treatment after diagnosis of acute cystitis or urinary tract infection (UTI), with regard to the risk of sporadic recurrent UTIs, frequent recurrent UTIs, UTI-related sick leave, additional antibiotic prescriptions, and renal complications (pyelonephritis). Methods: This retrospective cohort study was based on data from the IMS ® Disease Analyzer database (IQVIA), and included outpatients in Germany with at least one diagnosis of acute cystitis or UTI with a prescription of either Canephron ® or standard antibiotics between January 2016 and June 2019 and treated in general practitioner (GP), gynecologist, or urologist practices, from which the data were obtained. Multivariable regression models were used to investigate the association between Canephron ® prescription and the amount of sporadic or frequent recurrent UTIs, as well as the duration of UTI-related sick leave, the number of additional antibiotic prescriptions, and cases of pyelonephritis. The effects of Canephron ® were adjusted for age, sex, insurance status, and Charlson comorbidity score (CCI). Results: 2320 Canephron ® patients and 158,592 antibiotic patients were available for analysis. Compared to antibiotic prescription, Canephron ® prescription was significantly associated with fewer sporadic recurrences of UTI infections 30-365 days after the index date (odds ratio (OR): 0.66; 95%, confidence interval (CI): 0.58-0.72), as well as less frequent recurrences of UTI infections (OR: 0.61; 95% CI: 0.49-0.88), and also with reduced additional antibiotic prescription within 31-365 days (OR: 0.57; 95% CI: 0.52-0.63). No significant differences were observed between the Canephron ® and antibiotic cohorts with regard to the likelihood of sick leave (OR: 0.99; 95% CI: 0.86-1.14), new antibiotic prescription within 1-30 days (OR: 1.01; 95% CI: 0.87-1.16), or occurrence of pyelonephritis (Hazard Ratio (HR): 1.00; 95% CI: 0.67-1.48). Conclusion: These real-world data show that Canephron ® is an effective, safe symptomatic treatment for acute cystitis or UTI. It should be considered as an alternative treatment, particularly to also strengthen antimicrobial stewardship strategies.

Purpose: To investigate the influence of connector diameter on the mechanical load to fracture in monolithic three-unit lithium disilicate fixed partial dentures (FPDs)., Materials and Methods: A total of 24 FPDs were designed and manufactured using computer-aided design/computer-assisted manufacturing (CAD/CAM) with connector diameters of 16 mm2, 12 mm 2 , or 9 mm 2 (Groups A, B, and C, respectively; n = 8 for each group). After thermal and mechanical aging, the FPDs were subjected to mechanical load-to-fracture assessment., Results: Fracture loads of Groups B (834 ± 105 N) and C (796 ± 41 N) were significantly lower compared to Group A (990 ± 65 N)., Conclusion: Connector dimensions proved to be crucial for fracture resistance of monolithic lithium disilicate FPDs.

Introduction: This randomized, controlled, Phase III non-inferiority clinical trial aimed to determine whether herbal therapy with Canephron® N (BNO 1045) is non-inferior to fosfomycin trometamol (FT) in treating acute lower uncomplicated urinary tract infections (uUTIs)., Materials and Methods: Women aged 18-70 years with typical symptoms of newly diagnosed acute lower uUTIs were randomized to BNO 1045 (n = 325) or FT (n = 334), with corresponding matched placebo. The primary endpoint was the proportion of patients who received additional antibiotics (ABs) to treat uUTIs between Days 1 and 38 ±3., Results: Between Days 1 and 38, 238 (83.5%) patients in the BNO 1045 group and 272 (89.8%) patients in the FT group received no additional ABs. At a 15% non-inferiority margin, BNO 1045 was non-inferior to FT in treating uUTIs (non-AB rate difference: -6.26%; 95% CI -11.99 to -0.53%; 2-sided p = 0.0014). Adverse event rates were similar between groups, with higher rates of gastrointestinal disorders in the FT group and pyelonephritis in the BNO 1045 group. During the trial, no patient died or discontinued due to a treatment-related adverse event., Conclusions: BNO 1045 has the potential to reduce outpatient use of ABs for uUTIs and thus may have a significant impact on antimicrobial stewardship strategies., Trial Registration: NCT02639520, EudraCT number 2013-004529-99., (The Author(s). Published by S. Karger AG, Basel.)

Line integral convolution (LIC) is used as a texture-based technique in computer graphics for flow field visualization. In diffusion tensor imaging (DTI), LIC bridges the gap between local approaches, for example directionally encoded fractional anisotropy mapping and techniques analyzing global relationships between brain regions, such as streamline tracking. In this paper an advancement of a previously published multikernel LIC approach for high angular resolution diffusion imaging visualization is proposed: a novel sampling scheme is developed to generate anisotropic glyph samples that can be used as an input pattern to the LIC algorithm. Multicylindrical glyph samples, derived from fiber orientation distribution (FOD) functions, are used, which provide a method for anisotropic packing along integrated fiber lines controlled by a uniform random algorithm. This allows two- and three-dimensional LIC maps to be generated, depicting fiber structures with excellent contrast, even in regions of crossing and branching fibers. Furthermore, a color-coding model for the fused visualization of slices from T1 datasets together with directionally encoded LIC maps is proposed. The methodology is evaluated by a simulation study with a synthetic dataset, representing crossing and bending fibers. In addition, results from in vivo studies with a healthy volunteer and a brain tumor patient are presented to demonstrate the method's practicality.

Fluorescence fluctuation imaging is a powerful means to investigate dynamics, interactions, and stoichiometry of proteins inside living cells. Pulsed interleaved excitation (PIE) is the method of nanosecond alternating excitation with time-resolved detection and allows accurate, independent, and quasi-simultaneous determination of fluorescence intensities and lifetimes of different fluorophores. In this work, we combine pulsed interleaved excitation with fluctuation imaging methods (PIE-FI) such as raster image correlation spectroscopy (RICS) or number and brightness analysis (N&B). More specifically, we show that quantitative measurements of diffusion and molecular brightness of Venus fluorescent protein (FP) can be performed in solution with PIE-RICS and compare PIE-RICS with single-point PIE-FCS measurements. We discuss the advantages of cross-talk free dual-color PIE-RICS and illustrate its proficiency by quantitatively comparing two commonly used FP pairs for dual-color microscopy, eGFP/mCherry and mVenus/mCherry. For N&B analysis, we implement dead-time correction to the PIE-FI data analysis to allow accurate molecular brightness determination with PIE-NB. We then use PIE-NB to investigate the effect of eGFP tandem oligomerization on the intracellular maturation efficiency of the fluorophore. Finally, we explore the possibilities of using the available fluorescence lifetime information in PIE-FI experiments. We perform lifetime-based weighting of confocal images, allowing us to quantitatively determine molecular concentrations from 100 nM down to <30 pM with PIE-raster lifetime image correlation spectroscopy (RLICS). We use the fluorescence lifetime information to perform a robust dual-color lifetime-based FRET analysis of tandem fluorescent protein dimers. Lastly, we investigate the use of dual-color RLICS to resolve codiffusing FRET species from non-FRET species in cells. The enhanced capabilities and quantitative results provided by PIE-FI make it a powerful method that is broadly applicable to a large number of interesting biophysical studies., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Corynebacterium diphtheriae, the causative agent of diphtheria, has been thoroughly studied with respect to toxin production and pili formation, while knowledge on host responses to C. diphtheriae infection is limited. In this study, we studied adhesion to and invasion of epithelial cells by different C. diphtheriae isolates. When NFκ-B reporter cell lines were used to monitor the effect of C. diphtheriae infection on human cells, strain-specific differences were observed. While adhesion to host cells had no effect, a correlation of invasion rate with NFκ-B induction was found, which indicates that internalization of bacteria is crucial for NFκ-B induction. Immunofluorescence microscopy experiments used to support the reporter assays showed that translocation of p65, as a hallmark of NFκ-B induction, was only observed in association with cell invasion by C. diphtheriae. Our data indicate that the response of epithelial cells to C. diphtheriae infection is determined by internalization of bacteria and that invasion of these cells is an active process; tetracycline-treated C. diphtheriae was still able to attach to host cells, but lost its ability to invade the cytoplasm. Recognition of pathogen-associated molecular patterns such as pili subunits by membrane-bound receptors facing the outside of the cell is not sufficient for NFκ-B induction.

The endoplasmic reticulum is the site of folding, assembly and quality control for proteins of the secretory pathway. The ATP-regulated Hsp70 chaperone BiP (heavy chain-binding protein), together with cochaperones, has important roles in all of these processes. The functional cycle of Hsp70s is determined by conformational transitions that are required for substrate binding and release. Here, we used the intrinsically disordered C(H)1 domain of antibodies as an authentic substrate protein and analyzed the conformational cycle of BiP by single-molecule and ensemble Förster resonance energy transfer (FRET) measurements. Nucleotide binding resulted in concerted domain movements of BiP. Conformational transitions of the lid domain allowed BiP to discriminate between peptide and protein substrates. A major BiP cochaperone in antibody folding, ERdj3, modulated the conformational space of BiP in a nucleotide-dependent manner, placing the lid subdomain in an open, protein-accepting state.

The effect of nitrogen regulation on the level of transcriptional control has been investigated in a variety of bacteria, such as Bacillus subtilis, Corynebacterium glutamicum, Escherichia coli, and Streptomyces coelicolor; however, until now there have been no data for mycobacteria. In this study, we found that the OmpR-type regulator protein GlnR controls nitrogen-dependent transcription regulation in Mycobacterium smegmatis. Based on RNA hybridization experiments with a wild-type strain and a corresponding mutant strain, real-time reverse transcription-PCR analyses, and DNA binding studies using cell extract and purified protein, the glnA (msmeg&#95;4290) gene, which codes for glutamine synthetase, and the amtB (msmeg&#95;2425) and amt1 (msmeg&#95;6259) genes, which encode ammonium permeases, are controlled by GlnR. Furthermore, since glnK (msmeg&#95;2426), encoding a PII-type signal transduction protein, and glnD (msmeg&#95;2427), coding for a putative uridylyltransferase, are in an operon together with amtB, these genes are part of the GlnR regulon as well. The GlnR protein binds specifically to the corresponding promoter sequences and functions as an activator of transcription when cells are subjected to nitrogen starvation.

An emerging number of micro- and nanoelectronics-based biosensors have been developed for non-invasive recordings of physiological cellular activity. The interface between the biological system and the electronic devices strongly influences the signal transfer between these systems. Little is known about the nanoscopic structure of the cell-sensor interface that is essential for a detailed interpretation of the recordings. Therefore, we analysed the interface between the sensor surface and attached cells using transmission electron microscopy (TEM). The maximum possible resolution of our TEM study, however, was restricted by the quality of the interface preparation. Therefore, we complemented our studies with imaging ellipsometry. We cultured HEK293 cells on substrates, which had been precoated with different types of proteins. We found that contact geometry between attached cell membrane and substrate was dependent on the type of protein coating used. In the presence of polylysine, the average distance of the membrane-substrate interface was in the range of 35-40 nm. However, the cell membrane was highly protruded in the presence of other proteins like fibronectin, laminin or concanavalin-A. The presented method allows the nanoscopic characterization of the cell-sensor interface.

We describe the potential of anisotropic diffusion weighted imaging to visualize the course of large cerebral fiber tracts. Five healthy volunteers were investigated at a field strength of 1.5 Tesla, employing a spin-echo diffusion weighted sequence with gradient sensitivity in six non-collinear directions to visualize the course of the pyramidal tracts. The pyramidal tracts were segmented and reconstructed for three-dimensional visualization. Reconstruction results together with a fusioned high resolution 3D T1 weighted image data set were available in a customized neuronavigation system. Origination in the primary motor cortex, convergence in the centrum semiovale, the posterior limb of the internal capsule, the cerebral peduncles, the splitting at the level of the pons, and the pyramidal decussation were identified in all subjects. Fiber tract maps might have the prospect of guiding neurosurgical interventions, especially when being linked to a neuronavigation system. Other potential applications include the demonstration of the anatomical substrate of functional connectivity in the human brain.

The radioimmunological determination (RIA) of primary prostaglandins (PGs) in serum and plasma was evaluated with gas chromatography/mass spectrometry (GC/MS). Human blood was stimulated in vitro in the presence or absence of the specific thromboxane synthase inhibitor ridogrel. TxB2, 6-keto-PGF1 alpha, PGE2, PGD2 and PGF2 alpha were determined with RIA and GC/MS on the same samples. For GC/MS analysis, prostanoids were extracted and derivatized to their methoximepentafluorobenzyl-esters-trimethylsilyl-ethers. An excellent correlation was observed between levels of all eicosanoids determined by RIA or GC/MS (r = 0.996-0.999) when plasma was spiked with known amounts of PGs and TxB2 (2-500 ng/ml). In stimulated blood, with or without inhibition of thromboxane synthase, the correlation between RIA and GC/MS values remained good, except for 6-keto-PGF1 alpha. RIA largely overestimated the levels of 6-keto-PGF1 alpha and no significant correlation was found with levels detected by GC/MS. The results demonstrate the importance of corroborating the reliability of RIA with GC/MS.

A method for quantification of 6-keto-PGF1 alpha, 2,3-dinor-6-keto-PGF1 alpha TXB2, 2,3-dinor TXB2, PGE2, PGD2 and PGF2 alpha in human urine samples, using gas chromatography-negative ion chemical ionization mass spectrometry, is described. Deuterated analogues were used as internal standards. Methoximation was carried out in urine samples which were subsequently applied to phenylboronic acid cartridges, reversed-phase cartridges and thin-layer chromatography. The eluents were further derivatized to pentafluorobenzyl ester trimethylsilyl ethers for final quantification by gas chromatography-mass spectrometry. The overall recovery was 77% for tritiated 6-keto-PGF1 alpha and 55% for tritiated TXB2. Urinary levels of prostanoids were determined in a group of six volunteers before and after intake of the thromboxane synthase inhibitor Ridogrel, and related to creatinine clearance.

Human glioblastoma cells secrete a factor termed glioblastoma derived T cell suppressor factor (G-TsF) or transforming growth factor beta 2 (TGF-beta 2) which inhibits the response of T cells to mitogenic or antigenic stimulation. In the present study we isolated the promoter region of the G-TsF/TGF-beta 2 gene. The promoter region shares no homology to the promoter of the TGF-beta 1 or the 5' region of the TGF-beta 3 gene and harbours several familiar DNA motifs, including the cytokine-1 region, an octamer-like sequence, Sp1- and AP-2-like elements and a putative NF-kappa B site. In contrast to the TGF-beta 1 gene, the G-TsF/TGF-beta 2 gene contains three TATA-like sequences but lacks an AP-1 site. To understand the cell type specificity of expression of G-TsF/TGF-beta 2, the individual contribution of the DNA elements detected in the promoter has to be analysed in further studies.

The influence of fluorocarbon FC 43 on the metabolism of oestrogens in the isolated perfused rat liver was investigated. In comparative once-through perfusions with FC 43 emulsion or albumin solution as perfusion medium, the clearance rates of oestrone and its metabolites were determined. In perfusions with FC 43, the clearance of oestrone was lower, and the metabolites formed in the liver were more concentrated in the outflowing medium than in perfusions without fluorocarbon. This can be explained by the high affinity, even of conjugated oestrogens, to FC 43, which is established by equilibrium dialysis and partition coefficient. The results presented here show that the fluorocarbon has a strong influence of its own on the metabolism of steroids in the isolated perfused liver. Therefore, this solvent should be avoided as medium when the metabolism of steroids is studied in perfusion experiments.

Primidone and phenobarbital (each 85 nmoles/ml were separately perfused through the isolated brain of the rat. After 5 min of perfusion similar amounts of primidone and phenobarbital were taken up into the brain; for both drugs the concentration ratio between brain and perfusion medium was about 0.2. However, after 2 hr of perfusion the mean concentration ratio for primidone was about 0.55; for phenobarbital it was about 0.9 thus indicating a better uptake of phenobarbital. In two regions (hypophysis, mesencephalon) the concentration of phenobarbital was significantly higher than in perfusion medium. During 2 hr of perfusion of primidone, substantial quantities of phenobarbital and PEMA were formed amounting to 1400 pmoles for each metabolite. The highest concentration of the metabolites was found in septum, hypothalamus, hypophysis and mesencephalon. The in situ metabolism of primidone in the intact brain was demonstrated for the first time.

Equimolar concentrations of [4-14C]oestriol and [6,9-3H]oestriol 16 alpha-monoglucuronide were simultaneously perfused through isolated rat livers. Oestriol was hydroxylated to 2-hydroxyoestriol and 6 xi-hydroxyoestriol; 2-hydroxyoestriol was further methylated to 2-methoxyoestriol. Oxidoreduction of oestriol led to the formation of 16 alpha-hydroxyoestrone, 16-0xooestradio-17 beta and 16-epioestriol. In addition, two dehydroxylation products, namely oestrone and oestradiol-17 beta were found. The metabolites formed from oestriol were partly conjugated to monoglucuronides, monosulphates and sulphoglucuronides. About 80% of the oestriol perfused was hydroxylated at C-atom 2. Most of the 2-hydroxyoestriol formed was either methylated (about 37%) or sulphated (about 55%). Only small amounts (less than 2%) of the catecholoestrogens formed were methylated as well as sulphated. The 2-hydroxyoestriol monosulphates accumulated in the liver. After their conjugation with glucuronic acid, the double conjugates formed were immediately excreted into the bile. In fact, 2-hydroxyoestriol 16 alpha-monoglucuronide 2(3?)-monosulphate comprised by far the main biliary metabolite of [4-14C]oestriol, followed by oestriol 16 alpha-monoglucuronide and 2-methoxyoestriol 16 alpha-monoglucuronide. No triated sulphoglucuronides were detected, thus indicating that the monosulphates are the immediate precursors of the double conjugates. [6,9-3H2]Oestriol 16 alpha-monoglucuronide was metabolised only to a small extent. After its uptake into the liver more than 90% of this conjugate was secreted unchanged into the bile. The remaining part was hydrolysed; the oestriol liberated followed the same metabolic reactions as those found for [4-14C]oestriol. This indicates that the 16 alpha-glucuronide of oestriol is not metabolised to any appreciable extent.

A method for the perfusion of the isolated brain of the rat with synthetic fluorocarbon emulsion is described. The functional states of the brains were investigated using biochemical, biophysical, and histological methods. After 4.5 h of perfusion, all brains were in excellent condition and comparable to the in vivo state. The influence of perfusion conditions on the viability of the brains was studied. Deep hypothermia was well tolerated; at 13 degrees the EEG exhibited a mean frequency of about 8 Hz, while the mean amplitude was about 40% lower than at 28 degrees or 36 degrees. Constant-pressure perfusion did not affect brain function, provided that the pressure amplitude was very slowly decreased. During application of pentylenetetrazole (PTZ) it was found that the threshold concentration of PTZ in the perfusion medium to induce epileptic seizure discharges was 0.13 mg/ml. However, after repeated application of PTZ, the threshold concentration decreased exponentially, indicating a kindling phenomenon. It could be shown that not PTZ as such, but the spike potentials provoked by PTZ, induced the kindling effect. This was inhibited by application of primidone or phenobarbital.