AMPA-induced Ca(2+) influx in cultured rat cortical nonpyramidal neurones: pharmacological characterization using fura-2 microfluorimetry

Immunocytochemical and Co(2+) uptake studies revealed that in primary cultures of rat cortical neurones, the majority of neurones are gamma-aminobutyric acid (GABA) immunopositive and can express Ca(2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors. By fura-2 microfluorimetry, it was shown that the stimulation with the selective agonist (S)-AMPA (0.3-300 microM) induced a concentration-dependent but cell-variable increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) (EC(50) value 7.4 microM) in more than 80% of the medium-sized multipolar neurones studied. The AMPA-induced rise in [Ca(2+)](i) seems to be due to Ca(2+) entry through AMPA receptor channels, because the response was abolished in Ca(2+)-free solution and by AMPA receptor selective antagonists, but was not significantly influenced by cyclopiazonic acid, an inhibitor of the endoplasmatic Ca(2+)-ATPase, by selective N-methyl-D-aspartic acid (NMDA) receptor antagonists, as well as the Na(+) channel blocker tetrodotoxin and the majority of tested Ca(2+) channel blockers. In conclusion, the results indicate that the cerebral cortical neurones in culture represent mostly GABAergic interneurone-like cells and the majority of them possess Ca(2+)-permeable AMPA receptors, important for intracellular signal transduction and neuronal plasticity.     

Highly refractive silicone lens with sharp optic edge (CeeOn Edge,Model 911): one year results of a multicenter clinical trial on performance and efficacy

PURPOSE: To investigate the performance and efficacy of a new high refractive index (1.46) silicone intraocular lens with sharp optic edges. MATERIALS AND METHODS: In this open prospective multicenter study 149 patients underwent cataract surgery and implantation of CeeOn Edge in one eye. Best corrected visual acuity was determined at day 1 - 2, week 1 - 2, month 6 and month 12 postoperatively. Posterior capsule was clinically evaluated according to existence or absence of posterior capsule opacification (PCO). PCO was defined as relevant if the visual acuity loss exceeded at least two lines. The intraoperative handling of the lens was assessed concerning foldability, haptic handling, positioning, unfolding and general assessment. RESULTS: Visual acuity increased from preoperatively 0.3 to 0.9 after one year. PCO with impact on visual acuity (loss of 2 lines or more) was recorded in only two patients, after 2 weeks (YAG) resp. 1 year (no YAG). The handling and implantation was considered to be good to very good. No lens-related complications were seen during the one year study period. CONCLUSIONS: CeeOn Edge, Model 911, restores visual acuity after cataract surgery and shows promising handling results. The low rate of PCO and YAG is most probably related to the sharp edge of the lens.     

Vis-à-tergo: uveal effusion and expulsive bleeding in cataract surgery

Hintergrund: Durch Einführung der selbstschlie?enden Wundkonstruktion k?nnen schwerwiegende Komplikationen, welche durch eine exzessive Drucksteigerung hervorgerufen werden, vermieden werden. Die Differentialdiagnose sowie die Therapie der uvealen Effusion (CE) und der expulsiven Blutung (EB) werden dargestellt. Patienten und Methode: Die H?ufigkeit der Vis-à-tergo bei unseren Patienten mit Kataraktoperation liegt bei 1,7 %. Eine CE wurde in 0,1 % der F?lle festgestellt, zu einer EB kam es einmal (0,03 %) w?hrend der letzten 3000 Kataraktoperationen. Die differentialdiagnostische Unterscheidung erfolgte durch Ultraschalluntersuchung. Ergebnisse: Bei den Patienten mit CE war die Kataraktoperation selbst komplikationslos durchführbar, wobei bei einem Patienten mit CE die abgebrochene Operation am folgenden Tag abgeschlossen wurde. Eine Patientin mit EB hatte eine massive Drucksteigerung unmittelbar nach der Linsenimplantation bei peripherer Kapselruptur. Postoperativ zeigte sich eine ausgedehnte Aderhautblutung in zwei Quadranten einschlie?lich der Makula. Eine Woche postoperativ wurde eine hintere Sklerotomie bei gleichzeitiger Infusion der Vorderkammer durchgeführt. Hierbei kam die Netzhaut nach Drainage des verflüssigten Blutes weitgehend zum Anliegen. Der Visus hat sich schlie?lich auf 0,5 verbessert. Schlu?folgerung: Die Vis-à-tergo in der modernen Kataraktchirurgie deutet nur selten eine CE oder eine EB an. Auch im Falle einer EB stellt sich eine inkomplette Form dar. Durch entsprechende Ma?nahme kann eine gute visuelle Funktion wiederhergestellt werden.      

Stress protein/peptide complexes derived from autologous tumor tissue as tumor vaccines.

Vaccination of inbred mice with tumor-derived stress proteins hsp70, hsp90, and gp96/grp94 elicits a protective immunity to the tumor from which the vaccine was purified. There is now comprehensive experimental evidence that the antigenicity of tumor-derived hsp70, hsp90, and gp96 preparations results from diverse arrays of endogenous peptide antigens complexed with these stress proteins. Vaccination with tumor-derived stress protein/peptide complexes leads to their uptake and processing by professional antigen-presenting cells and to presentation of associated tumor peptide antigens to cytotoxic T cells. This induces a tumor-specific cytotoxic T cell response. The attractiveness of the concept of using tumor-derived stress proteins as vaccines is derived from two observations: (i) tumor stress protein vaccines mirror the individual antigenicity of a tumor, which results from random mutations due to genetic instability; and (ii) stress proteins represent powerful adjuvants for the peptide antigens complexed to them.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-mediated membrane translocation of c-Src protein kinase in liver WB-F344 cells

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widespread environmental contaminant and the most potent agonist of the aryl hydrocarbon receptor (AhR). Persistent activation of the AhR has been shown to be responsible for most TCDD-mediated toxic responses, including liver tumour promotion. However, the mechanisms responsible for these complex toxic reactions are still unknown. TCDD (1 nM) has previously been shown to reduce DNA synthesis of primary hepatocyte cultures and cell contact inhibition of confluent WB-F344 cells. The latter model was used to study early effects of TCDD on protein tyrosine kinase c-Src in confluent WB-F344 cells. It was found that TCDD decreased cytosolic c-Src (protein and tyrosine kinase activity) after 20–60 min, and increased c-Src in the membrane fraction. Membrane translocation of c-Src occurred in the presence of 100 μM cycloheximide and was observed after treatment with 1 nM TCDD or 50 nM 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin. Under these conditions epidermal growth factor (EGF) receptor tyrosine phosphorylation was also studied. As expected, its phosphorylation was low in confluent cells but was significantly enhanced by TCDD treatment. Pretreatment of WB-F344 cells for 1 h with 1 μM geldanamycin, which disrupts cytosolic heat shock protein Hsp90 complexes with AhR and Src, abolished TCDD-mediated Src translocation and TCDD-mediated reduction of cell contact inhibition. The WB-F344 cell model appears to be very useful to study TCDD effects on protein tyrosine kinases and of signaling pathways responsible for modulation of the cell cycle by TCDD. Received: 21 December 1998 / Accepted: 15 February 1999     

Interferon-γ-inducing Factor Gene Transfection into Lewis Lung Carcinoma Cells Reduces Tumorigenicity in vivo

To investigate the immunoregulatory effect of murine mterferon-γ-inducing factor (mIGIF), we transfected Lewis lung carcinoma (IXC) cells with a mammalian expression vector containing the mIGIF complementary DNA. The culture medium of the transfectant cells stimulated interferon-γ (IFN-γ) production by spleen cells in vitro in the presence of anti-CD3 antibody and markedly potentiated the effect of interleukin-12 (IL-12) on IFN-γ production by spleen cells. mIGIF transfectant cells showed reduction of tumorigenicity and induction of an in vivo immimo-protective effect against the parental LLC cells. To examine the combined effect of systemic administration of recomhinant IL-12 (rIL-12) and local mIGIF on the tumorigenicity, mice were challenged with LLC or transfectant cells on day 0, and the tumor-bearing mice were injected with 50 ng of rIL-12 intraperitoneally from day 7 to 11. Systemic rIL-12 showed an anti-tumor effect. However, mIGIF gene expression did not potentiate this effect of systemic rIL-12 in vivo.     

Treatment of advanced pancreatic cancer with 5-fluorouracil, folinic acid and interferon alpha-2A: results of a phase II trial.

Interferon alpha-2a (IFN-alpha) and folinic acid (FA) have been shown to modulate the cytotoxic effects of 5-fluorouracil (5-FU) in the treatment of cancer. A phase II study was initiated to evaluate the effect of a combination of 5-FU/FA/IFN-alpha in patients with advanced pancreatic cancer. Sixty previously untreated patients with advanced adenocarcinoma of the pancreas were treated with 500 mg m-2 FU via an intravenous bolus 1 h after the initiation of a 2 h infusion of 500 mg m-2 FA. Before starting the FA infusion, 6 million units (MU) of IFN-alpha was administered subcutaneously. The treatment was repeated once a week. Of 57 evaluable patients, eight (14%) had a partial response (PR), eight (14%) a minor response (MR) and 28 (49%) no change of disease (NC). Thirteen patients (23%) had progressive disease (PD). The median survival time was 10 months for all patients, 22 months for patients with partial remission and 5 months for patients with progressive disease. Many patients with tumour-related pain whose tumours were affected in terms of PR, MR, NC were free of pain during treatment with this regimen (22/36 patients). The common toxicities observed were fever (56%), nausea (37%) and diarrhoea (33%). These data suggest that biochemical modulation of 5-FU with FA and IFN-alpha has some positive effects in the treatment of pancreatic cancer of moderate toxicity.     

In vivo screening models of cisplatin-resistant human lung cancer cell lines using SCID mice

In vivo screening models of a cisplatin (CDDP)-resistant human small-cell lung cancer cell (SCLC) line, H69/CDDP, and a non-small-cell lung cancer cell (NSCLC) line, PC-14/CDDP, were evaluated. The transplantability of the tumor xenografts to SCID mice was more than 90%. Tumor xenografts of H69/CDDP and PC-14/CDDP showed CDDP resistance during in vivo treatment. The novel anticancer agent 254-S showed only a partial effect on the growth of H69/CDDP and PC-14/CDDP while ormaplatin showed no cross resistance to CDDP. The in vivo results correlated well with the results of the in vitro MTT assay. In this in vivo sensitivity test, H69/CDDP and PC-14/CDDP were more sensitive to ormaplatin than its parental cell lines. In vivo sensitivity testing using SCID mice bearing transplanted CDDP-resistant tumors was shown to be useful for evaluating the effects of new anti-cancer drugs, especially those that might overcome CDDP resistance.Abbreviations SCLC small-cell lung cancer - NSCLC non-small-cell lung cancer - SCID severe combined immunodeficiency - CDDP cisplatin - PBS phosphate-buffered saline - FBS fetal bovine serum This work was supported, in part, by Grants-in-Aid for Cancer Research from the Ministry of Health and Welfare, the Comprehensive Ten-year Strategy for Cancer Control from the Ministry of Education, Science and Culture. Support from the Bristol-Myers Squibb Foundation is also appreciated     

The Lantibiotic Mersacidin Inhibits Peptidoglycan Synthesis by Targeting Lipid II

The lantibiotic mersacidin exerts its bactericidal action by inhibition of peptidoglycan biosynthesis. It interferes with the membrane-associated transglycosylation reaction; during this step the ultimate monomeric peptidoglycan precursor, undecaprenyl-pyrophosphoryl-MurNAc-(pentapeptide)-GlcNAc (lipid II) is converted into polymeric nascent peptidoglycan. In the present study we demonstrate that the molecular basis of this inhibition is the interaction of mersacidin with lipid II. The adsorption of [¹⁴C]mersacidin to growing cells, as well as to isolated membranes capable of in vitro peptidoglycan synthesis, was strictly dependent on the availability of lipid II, and antibiotic inhibitors of lipid II formation strongly interfered with this binding. Direct evidence for the interaction was provided by studies with isolated lipid II. [¹⁴C]mersacidin associated tightly with [¹⁴C]lipid II micelles; the complex was stable even in the presence of 1% sodium dodecyl sulfate. Furthermore, the addition of isolated lipid II to the culture broth efficiently antagonized the bactericidal activity of mersacidin. In contrast to the glycopeptide antibiotics, complex formation does not involve the C-terminal d-alanyl–d-alanine moiety of the lipid intermediate. Thus, the interaction of mersacidin with lipid II apparently occurs via a binding site which is not targeted by any antibiotic currently in use.The family of lantibiotics comprises an increasing number of uniquely modified antibacterial peptides which are produced by a variety of gram-positive species (for a review, see reference 32). They are currently divided into two major groups (19, 32): the elongated, amphipathic, pore-forming type A lantibiotics, such as Pep5 or nisin (26, 31), and the globular peptides of the type B category, which appear to inhibit enzyme reactions (8, 15, 34). Mersacidin and actagardine (formerly “gardimycin”), another lantibiotic employed in this study, are representatives of the latter group. Both peptides contain four intramolecular thioether bridges, formed predominantly by β-methyllanthionine residues, which impose a globular shape and restricted flexibility on the molecules (10, 41). Furthermore, mersacidin and actagardine are of similar sizes (1,825 and 1,890 Da, respectively) and hydrophobicities and contain a conserved sequence motif which comprises one entire ring structure (8).Previous studies on the mode of action indicated that, unlike type A lantibiotics, mersacidin did not impair the overall integrity of the cytoplasmic membrane (7); instead, it selectively blocked peptidoglycan metabolism and caused cell lysis in staphylococci (7, 25). Accumulation of the ultimate cytoplasmic peptidoglycan precursor, UDP-MurNAc-pentapeptide, in mersacidin-treated cells suggested blockage of a membrane-associated biosynthetic step, which was identified as the transglycosylation reaction by using a wall membrane preparation of Bacillus megaterium (8). Similar experiments were conducted with actagardine, and these indicated that its bactericidal activity is also based on inhibition of peptidoglycan synthesis at the level of transglycosylation (8, 34).The aim of the present study was to investigate the molecular mechanism of this inhibition. Binding studies were conducted to determine whether mersacidin interferes with transglycosylation directly as a competitive enzyme inhibitor or whether it forms a complex with the peptidoglycan precursor and thus sterically prevents the action of transglycosylases.