Supplement file Fichera et al.
Gel chromatographic separation of peptidoglycan fragments
The lyophilized residue of peptidoglycan fragments was dissolved in 10 ml of dist. water, and 1-ml portions of this solution were applied to a 2.5 x 80 cm Sephadex G50 coarse column (Amersham Pharmacia Biotech, Freiburg, Germany). The chromatograms were developed at 4°C with dist. water (pH 7.2) at a flow rate of 80 ml/h, collecting 6-ml fractions and measuring the absorbance at 220 nm. After pooling and lyophilization of the fractions of peak II of the individual chromatographies (Fig. 1A), the residue was taken up in 10 ml of dist. water and 1-ml portions of this solution were subjected to chromatography on a 2.5 x 80 cm Bio-Gel P2 column (Bio-Rad Laboratories GmbH, Munich, Germany) in dist. water at a flow rate of 60 ml/h and collecting 4.5-ml fractions. The fractions of peak IV of the individual chromatographies (Fig. 1B) were again pooled and lyophilized. The residue dissolved in 10 ml of dist. water was rechromatographed in 1-ml portions on the same Bio-Gel P2 column, this time at a flow rate of 30 ml/h and collecting 1-ml fractions. Fractions 75 of these final chromatographies (Fig. 1C) were combined and lyophilized. The residue taken up in 3 ml of dist. water was divided into aliquots and kept at –20°C until further use.
Fig. 1.
Fig. 1. Gel chromatographic separation of peptidoglycan fragments isolated from the supernatant of a Penicillin G-treated L. casei cell culture. (A) Chromatography on a Sephadex G-50 column; peak I corresponds to the void volume V0, peak II contains the bulk of peptidoglycan fragments. (B) Chromatography of the peak II material of (A) on a Bio-Gel P2 column at a flow rate of 60 ml/h and collecting 4.5-ml fractions. (C) Re-chromatography of peak IV (fraction “40”) material of (B) on the same Bio-Gel P2 column at a flow rate of 30 ml/h and collecting 1-ml fractions.Through this procedurefragments in the molecular weight range between 100 kd and 0,2 kd are identified (Fig. 1B), of which fraction IV exhibited the highest cytotoxic activity when tested, with comparable weight/concentration, on Yac-1 lymphoma cells
Determination of mitochondrial succinate dehydrogenase (SDH) activity
The method employed (reagent kit from Roche Molecular Biochemicals, Mannheim, Germany) is based on the cleavage of the tetrazolium salt XTT (sodium 3’-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis-(4-methoxy-6-nitro) benzene sulfonic acid hydrate) which is metabolically reduced in viable cells by succinate dehydrogenase to an orange-colored, water-soluble formazan product. This reagent allows direct absorbance readings, thereby avoiding a solubilization step and shortening the micro-culture growth assay. In the presence of N-methyl dibenzopyrazine methyl sulfate (PMS), the XTT reagent yielded usable absorbance values for growth and drug sensitivity evaluations with a variety of cell lines. Briefly, after a short adaptation time in the wells of a microtiter plate, 2.5 x 104 cells/well were treated with usually 80 µg of peptidoglycan fragments or amino acids in a volume of 110 µl for 3 h at 37°C in a humidified atmosphere containing 5% CO2. 50µl of the XTT labeling mixture (prepared by mixing 5 ml of XTT labeling reagent with 0.1 ml of electron-coupling reagent, as described in the manufacturer’s instructions) where then added to each well and the microtiter plate was kept for an appropriate experimental time (12 to 18 h) in a humidified atmosphere containing 5% CO2 at 37°C. At the end of the incubation period, the absorbance was measured spectrophotometrically using an ELISA reader at 450-500 nm and a reference wavelength of 650 nm. In some experiments, the peptidoglycan fragments and amino acids were allowed to react with hexokinase (see Figures in Results section) in a volume of 22 µl of cell culture medium for 30 min at room temperature, before the compounds were added to the cells. An increase in the number of living cells or in cell viability resulted in an increase in the formazan yield. To emphasize the morphological evidence of the formazan production or inhibition in the cells, MTT [ (1) Determination of succinate dehydrogenase)] that yields water-insoluble formazan, was used instead of XTT.
MALDI-TOF mass spectrometry
MALDI-TOF analysis was performed as described [14] using a Kratos Maldi III instrument (Shimadzu Deutschland, Duisburg, Germany) in either linear, positive, high mass mode or reflectron, positive, high mass mode. The peptide sample was applied to the sample slide using a sandwich technique with α-cyano-4-hydroxy cinnamic acid as matrix and masses were calibrated using near external standards, as described [15].
Gas-chromatographic amino acid analysis
The gas chromatography assays were performed using a Hewlett-Packard HP-5890 gas chromatograph. The samples to be analysed were hydrolized with 22% HCl in the presence of 10 % 2-mercaptoethanol by refluxing for 24 h. The solution was then dried in a rotary evaporator and a known quantity of cainic acid solution as internal standard was added to the residue. The solution was again dried and the residue allowed to sit overnight in a desiccator. The residue was transferred to a stoppered tube, treated with 3N HCl in butanol at 110°C for 1 h and dried under nitrogen. It was dissolved in dichloromethane and trifluoroacetic anhydride and incubated for 15 minutes at 110°C. The resulting solution, containing amino acids as N-trifluoroacetyl butylester derivatives, was directly injected into the gas chromatograph. A capillary column HP 17 (25 meters) was used and the flow rate was 1 ml/min. The column temperature was increased from 80 to 250°C with a gradient of 8°C/min. An amino acid mixture of known composition was analysed for reference.
Fluorescence analysis of mitochondria
Rat C6 glioma cells were kindly provided by Dr. Wolfgang Bohn (Heinrich-Pette-Institute for Experimental Virology and Immunology, Hamburg, Germany). Cells were grown in monolayer culture in minimum essential Eagle medium (MEMS; ICN Biomedicals GmbH) supplemented with 10% FCS (Invitrogen GmbH, Karlsruhe, Germany). For the assessment of the mitochondrial membrane potential, 5, 5’, 6, 6’-tetrachloro-1, 1’, 3, 3’-tetraethylbenzimidazolo-carbocyanine iodide (JC-1; Molecular Probes Europe BV, Leiden, The Netherlands) was used. 2 x 105 cells in DMEM supplemented with 10% FCS were seeded in 5 cm dishes on 4.2 cm circular glass coverslips (Menzel, via neoLab Migge Laborbedarf-Vertriebs GmbH, Heidelberg, Germany) and incubated for 2 days. The cells were washed with warm, phenol-red-free RPMI 1640 medium (Gibco-BRL), incubated at 37°C for 10 min in the presence of 2 µM JC-1 (stock solution: 100 µM JC-1 in DMSO) and 0.094 µM peptide in RPMI 1640 and washed briefly with RPMI 1640. Control cells were incubated only with JC-1. The coverslips were then mounted in a temperature-controlled POC chamber (LaCon, Staig, Germany) fitted to a Leica DM RBE fluorescence microscope (Leica Microsystems, Mannheim, Germany). Confocal fluorescence images of the JC-1-labeled cells were obtained using a PLANAPO 63x, 1.32 NA oil immersion objective and the 488 nm and 568 nm excitation lines of an argon/krypton laser in combination with a 488/568 nm double-dichroic beam splitter. Emitted light was further separated by a RSP 580 nm reflection short pass filter before being filtered at 525/550 nm and 590 nm, the wavelengths of the fluorescence maxima of the JC-1 dye in its monomeric and aggregated form, respectively. To minimize photobleaching, the excitation energy was reduced to 10% by means of an acusto-optical tunable filter (AOTF). Confocal pinhole aperture, laser excitation energy and voltage to the photomultiplier tubes of each channel were held constant throughout all experiments. Confocal image data obtained with confocal laser scanning microscopy (CLSM) software (Leica Microsystems) were processed with Adobe Photoshop 7.0 software (Adobe Systems Inc. Mountain View, CA, USA).