Short-term regulation of basolateral organic anion uptake in proximal tubular OK cells: EGF acts via MAPK,PLA(2), and COX1

The organic anion transport system of the kidney is of major importance for the excretion of a variety of endogenous compounds, drugs, and potentially toxic substances. The basolateral uptake into proximal tubular cells is mediated by a tertiary active transport system. Epidermal growth factor (EGF) leads to an increase in the basolateral uptake rate of the model substrate para-aminohippuric acid (PAH) in opossum kidney (OK) cells. This stimulation is mediated by successive activation of the mitogen-activated protein kinases,mitogen-activated/extracellular signal-regulated kinase kinase (MEK) and extracellular regulated kinase isoforms 1 and 2 (ERK1/2). This study investigates the regulatory network of EGF action on PAH uptake downstream ERK1/2 in more detail. EGF stimulation of the basolateral uptake rate of [(14)C]PAH was abolished by the phospholipase A(2) inhibitor AACOCF3.[(14)C]PAH uptake was enhanced by arachidonic acid. Furthermore, EGF led to an increase in arachidonic acid release and to the generation of prostaglandins. AACOCF3 did not influence EGF-induced ERK1/2 activation, indicating that ERK1/2 is upstream of PLA(2). In addition, EGF stimulated the influx of extracellular Ca(2+). However, Ca(2+)-influx was not required for the stimulatory action of EGF on [(14)C]PAH uptake. Inhibitors of COX and lipoxygenases reduced [(14)C]PAH uptake dose-dependently, whereas inhibition of cytochrome P450 did not. In the presence of indomethacin, EGF had no stimulatory effect on [(14)C]PAH uptake. The inhibitory effect of indomethacin was not due to competitive action on PAH uptake. Furthermore, prostaglandin E(2) (PGE(2)) increased basolateral [(14)C]PAH uptake rate dose-dependently, and this increase was also observed in the presence of indomethacin. Selective inhibition of COX2 by indomethacin amid or indomethacin n-heptyl ester did not inhibit [(14)C]PAH uptake, whereas selective inhibition of COX1 dose-dependently inhibited [(14)C]PAH uptake. This and previous data lead to the conclusion that EGF successively activates MEK, ERK1/2, and PLA(2), leading to an increased release of arachidonic acid. Subsequently, arachidonic acid is metabolized to prostaglandins via COX1, which then mediate EGF-induced stimulation of basolateral organic anion uptake rate.     

Isolationsbedingungen für Zellkerne aus Hefeprotoplasten

The structure of isolated nuclei from Saccharomyces fragilis protoplasts depends on the composition of the medium used for isolation. Buffered solutions of polymers as for instance Dextran, Ficoll and Polyvinylpyrrolidon at pH 5.5–6.5 are suitable both for carefully lysing the protoplasts and for the isolation of the nuclei. The structure of isolated nuclei does not differ markedly from nuclei in living cells. Isolated nuclei do not behave like osmometers but are swelling in buffered solution of sugars such as sucrose, mannitol and sorbitol.     

Immune recovery after conventional and non-myeloablative allogeneic stem cell transplantation

The immune recovery of 66 patients undergoing allogeneic stem cell transplantation with either conventional or non-myeloablative conditioning regimen was studied. Infections post-transplant were enumerated and quantitative immunoglobuilins (IgG, IgA, IgM) and lymphocyte sub-sets 3, 6 and 12 months post-transplant were measured. A significant difference was found in the immunologic recovery of non-myeloablative and conventional ASCT in the patient population. The T-helper cell reconstitution was significantly faster after NMA than conventional transplantation and the recovery of B cells was faster after conventional transplantation. Regarding immunoglobulin levels, a faster recovery of IgM levels after NMA-ASCT and a delayed recovery of IgA levels was observed in both groups. These were accompanied by a significant difference in the frequency and severity of infectious episodes.     

Multiparameter analysis of a screen for progesterone receptor ligands: comparing fluorescence lifetime and fluorescence polarization measurements

Direct measurement of the fluorescence lifetime (FLT) of a fluorescent label is an emerging method for high-throughput screening. Changes in the fluorescence lifetime can be correlated to changes in the non-radiative relaxation pathway(s) for the excited state of the label. These pathways can be environmentally sensitive, such as when a labeled analyte is free in solution versus bound to a receptor. Because lifetime is an intrinsic property of a fluorophore, it is not concentration dependent, and therefore has advantages similar to those of ratiometric fluorescent techniques such as fluorescence resonance energy transfer or fluorescence polarization. We have applied the FLT measurement technique to a screen of a small compound library in order to identify compounds that bind to the progesterone receptor, and compared the results to those obtained by performing the assay in fluorescence polarization mode. Each readout modality showed excellent Z'; values, with the FLT readout performing slightly better in this respect. Interfering compounds could be rapidly identified for either assay format by comparing the results between the two formats.     

Quantitative molecular urinary cytology by fluorescence in situ hybridization: a tool for tailoring surveillance of patients with superficial bladder cancer?

OBJECTIVE: To determine whether it is possible to stratify patients with superficial bladder cancer into low- and high-risk groups for tumour recurrence/progression based on the chromosomal pattern detected by fluorescence in situ hybridization (FISH) in one urine cytology specimen used for follow-up testing. PATIENTS AND METHODS: Voided urine samples from 47 consecutive patients with urinary tract neoplasms (13 with no history of urothelial malignancy and 34 under follow-up after complete transurethral resection of superficial urothelial carcinoma of the bladder) were evaluated by liquid-based cytology (ThinPrep(R), CYTYC Corp., Boxborough, MA, USA) and UroVysion FISH (Vysis-Abbott, Downers Grove, IL). RESULTS: Of the 34 patients under surveillance, the UroVysion test was negative in four, 17 had loss of 9p21 sequences either alone or combined with low-frequency trisomy/ies or tetrasomy/ies of chromosomes 3, 7 and 17 in single cells (low-risk FISH), and 13 also had complex aneusomies of the remaining chromosomes (high-risk FISH). One of the four FISH-negative neoplasms, four of the 17 low-risk FISH cases and five of the 11 informative high-risk FISH-positive patients developed recurrence. Progression occurred only in patients with high-risk FISH results, showing high-frequency complex chromosomal polysomies (four of 11). CONCLUSION: The results from this pilot study indicate that the UroVysion FISH test may help to individually assess the clinical behaviour of superficial bladder cancer, based on the chromosomal pattern of exfoliated tumour cells in follow-up urinary cytology. It might be of use to identify those patients likely to progress at earlier and curable stages of disease, and lengthen the surveillance period in those with persistent or recurrent low-risk disease.     

Über die Wirkung von Phenylbutazon,seinen Metaboliten und verwandten Verbindungen auf die Gewebekultur

Zusammenfassung Phenylbutazon, seine Metaboliten und einige verwandte Verbindungen, darunter Sulfinpyrazon, wurden bezüglich ihrer Wirkung auf normale und maligne Zellen in der Gewebekultur geprüft. In den Zellkulturen von tierischen und menschlichen Geweben bewirkten Butazolidin und alle geprüften Derivate bei 1000 /ml Zellzerstörung und Zelltod. — Bei 100 /ml verursachten Phenylbutazon, seine Metaboliten I und II und Sulfinpyrazon in allen untersuchten Geweben deutlich schwächere Zellalterationen, während G 31442 überhaupt nicht die Kulturen angriff. Bei gleicher Konzentration alterierten aber das schwefelhaltige Derivat G 25671 und seine Metaboliten G 33378 und G 32642 besonders stark die Mastocytomkulturen. Die Substanz G 32642 schädigte darüber hinaus auch die HeLa-Zellen stark.Konzentrationen von 10 /ml und 1 /ml Phenylbutazon und aller geprüften Derivate beeinflußten das Zellenwachstum der verschiedenen Gewebearten nicht.Mitosesenkungen, die in einigen Fällen bei Behandlung mit 100 /ml zu beobachten waren, traten in solchen Kulturen auf, die deutlich erkennbare morphologische Schädigungen aufwiesen.Mit 8 Textabbildungen     

Inducible neuronal expression of transgenic TGF-beta1 in vivo: dissection of short-term and long-term effects

Various chronic neurological diseases are associated with increased expression of transforming growth factor-beta1 (TGF-beta1) in the brain. TGF-beta1 has both neuroprotective and neurodegenerative functions, depending on conditions such as duration and the local and temporal pattern of its expression. Previous transgenic approaches did not enable control for these dynamic aspects. To overcome these limitations, we established a transgenic mouse model with inducible neuron-specific expression of TGF-beta1 based on the tetracycline-regulated gene expression system. TGF-beta1 expression was restricted to the brain where it was particularly pronounced in the neocortex, hippocampus and striatum. Transgene expression was highly sensitive to the presence of doxycycline and completely silenced within 6 days after doxycycline application. After long-term expression, perivascular thioflavin-positive depositions, formed by amyloid fibrils, developed. These depositions persisted even after prolonged silencing of the transgene, indicating an irreversible process. Similarly, strong perivascular apolipoprotein E (ApoE) depositions were found after TGF-beta1 expression and these remained despite TGF-beta1 removal. These in vivo observations suggests that the continuous presence of TGF-beta1 as initial trigger is not necessary for the persistence and development of chronic lesions. Neuroprotective effects were observed after short-term expression of TGF-beta1. Death of striatal neurons induced by 3-nitropropionic acid was markedly reduced after induced TGF-beta1 expression.     

Efficacy of psychodynamic short-term psychotherapy for children and adolescents with anxiety disorders

Anxiety disorders can be regarded as one of the most prevalent disorders in children and adolescents. Although psychodynamic psychotherapies are frequently carried out in this field, the evaluation of its efficacy for anxiety disorders is still deficient. Therefore the aim of the study was to evaluate psychodynamic short-term psychotherapy (PSTP) comprising 25 therapy sessions for children and adolescents with anxiety disorders. In a controlled trial PSTP was compared to a waiting list control condition. 26 children and adolescents with anxiety disorders were included in the study. Treatment outcome was measured by the Impairment-Score for Children and Adolescents (IS-CA). Moreover, the Child Behavior Checklist (CBCL) and the Psychic and Social-Communicative Findings Sheet for Children and Adolescents (PSCFS-CA) were administered at the beginning and end of the treatment. The statistical and clinical significance of changes in these measures was evaluated. A significant advantage of the treatment group compared to the waiting control group for the IS-CA was shown. For the IS-CA total score, an effect size of 1.6 was found. Whereas 62% of the patients in the treatment group showed clinically significant and reliable improvement at the end of therapy, this was the case for only 8% of the subjects in the waiting list condition. Effect sizes comparable to the IS-CA were found for the PSCFS-CA. In the CBCL significant improvement could be shown for the treatment and control group. The findings support the evidence that psychodynamic short-term psychotherapy (PSTP) is an effective treatment for children and adolescents with anxiety disorders. However, some of the studied children and adolescents seem to be in need of more intensive treatment.     

Testing of extracorporeal membrane oxygenation circuit related hemolysis using long-term stored packed red cells and fresh frozen plasma

BACKGROUND: The resistance of blood used in these studies to hemolysis differs markedly from that used in neonatal extracorporeal circulation under clinical circumstances. In this study, the possibility of using expired packed red cells to determine hemolysis caused by mechanical and/or environmental factors was investigated. METHODS: Packed red blood cells stored for 42 days were mixed with fresh frozen plasma and the resultant mixture was divided into three groups, two study groups and a control. For the study groups, two different centrifugal pump heads (Medtronic BP 50 and Jostra RF 32) were used in an extracorporeal membrane oxygenation (ECMO) circuit. Free hemoglobin, lactate dehydrogenase, lactic acid, pH, potassium, and glucose were investigated at various time intervals. RESULTS: Hemolysis did not differ between the groups. Free hemoglobin increased in all groups after 12 h. Lactic acid increased linearly in all groups up to 12 h. Glucose and pH decreased steadily in all groups. Hemolysis created during mock ECMO did not differ between the circuits using the two different pump heads noted. CONCLUSION: Human donor blood stored up to its expiration date is a feasible medium for mock circulation tests of up to 12 h duration under the circumstances described.