Eberhard Spiess

Two-photon fluorescence absorption and emission spectra of dyes relevant for cell imaging

Two‐photon absorption and emission spectra for fluorophores relevant in cell imaging were measured using a 45 fs Ti:sapphire laser, a continuously tuneable optical parametric amplifier for the excitation range 580–1150 nm and an optical multichannel analyser. The measurements included DNA stains, fluorescent dyes coupled to antibodies as well as organelle trackers, e.g. Alexa and Bodipy dyes, Cy2, Cy3, DAPI, Hoechst 33342, propidium iodide, FITC and rhodamine. In accordance with the two‐photon excitation theory, the majority of the investigated fluorochromes did not reveal significant discrepancies between the two‐photon and the one‐photon emission spectra. However, a blue‐shift of the absorption maxima ranging from a few nanometres up to considerably differing courses of the spectrum was found for most fluorochromes. The potential of non‐linear laser scanning fluorescence microscopy is demonstrated here by visualizing multiple intracellular structures in living cells. Combined with 3D reconstruction techniques, this approach gives a deeper insight into the spatial relationships of subcellular organelles.

Cathepsin B activity in human lung tumor cell lines: ultrastructural localization, pH sensitivity, and inhibitor status at the cellular level.

We investigated the appearance and activity of the cysteine proteinase cathepsin B and its physiological inhibitors, stefins A and B, at the cellular level in human tumor cell lines HS-24, derived from a primary lung tumor (squamous cell), and SB-3, derived from a metastasis (lung adenocarcinoma). In addition to cathepsin B, these tumor cells also expressed the immunologically and functionally related cathepsin L, but not cathepsin H. Stefin A was found in HS-24 but not in SB-3 cells; stefin B was found in both cell types. Using a specific fluorogenic cytochemical assay, the intracellular activity of the enzyme was localized and quantified. Thus, the cellular cathepsin B kinetics for the synthetic substrates Z-Arg-Arg-4M beta NA and Z-Val-Lys-Lys-Arg-4M beta NA, its pH dependence and inhibition by E64, stefins A and B, and cystatin C could be determined. From these measurements it appeared that the enzyme exhibited different cleavage rates for these substrates in the different cell types, showed considerable cleavage activity at neutral pH, which was stable under these conditions for extended time periods, and was highly sensitive to the inhibitors E64 and cystatin C but was considerably less sensitive to stefins, particularly stefin A. By conventional light microscopy, confocal laser scanning microscopy, and electron microscopy the enzymatic activity was localized in lysosomes, as expected, but also in the endoplasmic reticulum, nuclear membrane, and plasma membrane. The endoplasmic reticulum is a site at which only pre-mature enzyme forms exist, which are usually not active. The appearance of enzymatic activity at the plasma membrane confirms earlier biochemical and immunofluorescence microscopic investigations. The different sites of localization within the cells make it likely that different forms of the enzyme are expressed simultaneously, which follow alternate ways of processing and sorting. Taken together, the results support an involvement of the enzyme under extracellular conditions in degradative processes.

Two-photon excitation and emission spectra of the green fluorescent protein variants ECFP, EGFP and EYFP

Two‐photon (TP) excitation (820–1150 nm) and emission (280–700 nm) spectra for the fluorescent proteins (FPs) ECFP 3 , EGFP 3 and EYFP 3 produced in human tumour cells were recorded. TP excitation spectra of pure and highly enriched samples were found to be more differentiated in comparison with their one‐photon (OP) spectra. They exhibited more pronounced main and local maxima, which coincided among different purity grades within small limits. TP and OP emission spectra of pure and enriched samples were identical. However, in crude samples, excitation was slightly blue‐shifted and emission red‐shifted. The data indicate that both OP and TP excitation routes led to the same excited states of these molecules. The emission intensity is dependent on the pH of the environment for both types of excitation; the emission intensity maximum can be recorded in the alkaline range. Reconstitution of emission intensity after pH quenching was incomplete, albeit that the respective spectral profiles were identical to those prequenching. When emission data were averaged over the whole range of excitation, the resulting emission profile and maximum coincided with the data generated by optimal excitation. Therefore, out‐of‐maximum excitation, common practice in TP excitation microscopy, can be used for routine application.

Expression of cysteine proteinase inhibitors stefin A, stefin B, and cystatin C in human lung tumor tissue

In human lung tumor tissue specimen (n = 73) concentrations of stefins A and B were found to be increased 2.0-fold (p < 0.01) and 1.3-fold (p < 0.01), respectively, as compared to matched normal tissue. Stefin A and B concentrations were higher in primary tumors than in secondary tumors, i.e. metastases from other organs to the lung (p < 0.01; p < 0.05, respectively). Cystatin C concentrations were rather low and did not differ between tumor and normal tissue. Both concentrations of stefins did not correlate with TNM stages. Stefin A was higher in squamous cell carcinoma than in adenocarcinoma (p < 0.01), while stefin B did not show such a difference. At investigation of a relationship between survival probability of patients with primary tumors it was found that increased stefin B concentrations and total cysteine-protease-inhibitory activities but not stefin A concentrations were positively correlated with survival probability. It is concluded that stefins A and B are major contributors to the cysteine protease inhibitory activity in primary lung tumors. Stefin B proved to be a prognostic factor, especially in squamous cell carcinoma.

Localization of cathepsin B in two human lung cancer cell lines.

We demonstrated the cysteine proteinase cathepsin B in two human lung tumor cell lines by cytochemical and immunocytochemical methods. The cell lines were derived from a squamous cell carcinoma of the lung (HS-24) and a metastasis to the adrenal gland from an adenocarcinoma of the lung (SB-3). For comparison and control, normal human lung fibroblasts cells (Wi-38) were also investigated. Intracellular cathepsin B activity was detected in all three cell lines. SB-3 and the normal fibroblast cells showed almost equal cathepsin B activity, which was considerably stronger than that in the HS-24 cells. Specific inhibitors for cathepsin B (E64, leupeptin, antipain) suppressed its activity completely. Stefin A, the physiological cathepsin B inhibitor, was less effective; this might depend on its limited penetrability into living cells. Localization of the cathepsin B was performed by conventional immunofluorescence microscopy and laser scanning microscopy. With specific anti-cathepsin B antibodies, the enzyme was localized in HS-24, SB-3, and Wi-38 fibroblast cells within perinuclear granules representing the lysosomal compartment. In the SB-3 cells, we additionally localized a minor fraction of the enzyme bound to the plasma membrane in a speckled distribution, accessible to the antibodies from the outside. This direct demonstration of cathepsin B distribution supports biochemical data about the dual localization of the enzyme in tumor cells. It also supports the possibility of a direct involvement of cathepsin B in the degradation of the extracellular matrix, and thus a contribution of the enzyme in invasion and metastasis.

Immunochemical analysis of cathepsin B in lung tumours: an independent prognostic factor for squamous cell carcinoma patients

SummaryIn order to evaluate the possible role of the proteolytic enzyme cathepsin B (cath B) in human non-small cell lung cancer (NSCLC) we examined cath B concentrations (cath Bc) and activities (cath BA) in homogenates of 127 pairs of lung tumour tissues and corresponding non-tumourous lung parenchyma. Total cath B activity (cath BAT) and enzymatic activity of the fraction of cath B, which is stable and active at pH 7.5 (cath BA7.5) were determined by a fluorogenic assay using synthetic substrate Z-Arg-Arg-AMC. The immunostaining pattern of cath B was determined in 239 lung tumour tissue sections, showing the presence of the enzyme in tumour cells (cath BT-I) and in tumour-associated histiocytes (cath BH-I). The median levels of cath BAT, cath BA7.5 and cath BC were 5.6-, 3.2- and 9.1-fold higher (P < 0.001), respectively, in tumour tissue than in non-tumourous lung parenchyma. Out of 131 tissue sections from patients with squamous cell carcinoma (SCC), 59.5% immunostained positively for cath B, while among the 108 adenocarcinoma (AC) patients 48.2% of tumours showed a positive reaction. There was a strong relationship between the levels of cath BAT, cath BA7.5, cath BC and cath BT-I in the primary tumours and the presence of lymph node metastases. Significant correlation with overall survival was observed for cath BT-I and cath BA7.5 (P < 0.01 and P < 0.05, respectively) in patients suffering from SCC. In these patients positive cath B in tumour cells (cath BT-I) and negative cath B in histiocytes (cath BH-I) indicated significantly shorter survival rate compared with patients with negative cath BT-I and positive cath BH-I (P < 0.0001). In contrast, in AC patients, both, positive cath BT-I and positive cath BH-I, indicated poor survival probability (P < 0.014). From these results we conclude that the proteolytic enzyme cath B is an independent prognostic factor for overall survival of patients suffering from SCC of the lung.

Cathepsin B in infiltrated lymph nodes is of prognostic significance for patients with nonsmall cell lung carcinoma.

Tumor cells require specific proteolytic enzymes for invasion and metastasis, including lysosomal peptidases—cathepsins. Cathepsin B is a lysosomal cysteine peptidase, which appears to play a major role in invasion and metastasis of human tumors. In this study, the authors focused on the possible role of cathepsin B in lymphogenic metastasis by investigating the enzyme localization and its activity in lung tumors and corresponding tumor‐infiltrated lymph nodes.

Biochemical Characterization and Mass Spectrometric Disulfide Bond Mapping of Periplasmic α-Amylase MalS of Escherichia coli

Periplasmic α-amylase of Escherichia coli, the malS gene product, hydrolyzes linear maltodextrins. The purified enzyme exhibited a K m of 49 μm and a V max of 0.36 μmol of p-nitrophenylhexaoside hydrolyzed per min per mg of protein. Amylase activity was optimal at pH 8 and was dependent on divalent cations such as Ca2+. MalS exhibited altered migration on SDS-polyacrylamide gel electrophoresis under nonreducing conditions. Analytical ultracentrifugation and electrospray mass spectrometry indicated that MalS is monomeric. The four cysteine residues are involved in intramolecular disulfide bonds. To map disulfide bonds, MalS was proteolytically digested. The resulting peptides were separated by reverse phase-high performance liquid chromatography, and matrix-assisted laser desorption/ionization mass spectrometry analysis indicated the presence of two disulfide bonds,i.e. Cys40–58 and Cys104–520. The disulfide bond at Cys40–58 is located in an N-terminal extension of about 160 amino acids which has no homology to other amylases but to the proposed peptide binding domain of GroEL, the Hsp60 of E. coli. The N-terminal extension is linked to the C-terminal amylase domain via disulfide bond Cys104–520. Reduction of disulfide bonds by dithiothreitol treatment led to aggregation suggesting that the N terminus of MalS may represent an internal chaperone domain.

Fluorometric microassays for the determination of cathepsin L and cathepsin S activities in tissue extracts

Abstract We established a continuous semi-microassay, and for large-scale studies both a stopped and a continuous microtiter plate assay for the fluorometric determination of cathepsin L and cathepsin S activities in body fluids, tissues or cell extracts in the presence of cathepsin B. For the detection of enzymatic activities we used the synthetic substrate Z-Phe-Arg-AMC, and for discrimination between cathepsin L, S and cathepsin B the specific inhibitor CA-074 for blocking interfering cathepsin B activities was applied. Furthermore, we took advantage of the stability of cathepsin S at pH 7.5 for further differentiation between cathepsin L and cathepsin S activities. The kinetic assays were characterized in terms of imprecision, analytical sensitivity, accuracy and substrate concentration. The within-run coefficients of variation were found to be 4.9%–7.2% for the continuous semi-microassay, 10.3%–11.7% for the stopped, and 4.5%–11.8% for the continuous microtiter plate assay. The between-days coefficients of variation for the continuous semimicroassay were 8.1%–8.9%, while for the stopped and continuous microtiter plate assays the coefficients were 11.2%–13.5% and 5.8%–12.2%, respectively. Compared to the continuous semi-microassay, the stopped and the continuous microtiter plate assays showed 3-fold and 11-fold higher sensitivity, respectively. Comparison between the continuous enzyme activity assays at substrate concentrations of 40 μM and 200μM demonstrated a significant correlation of r = 0.97 and r = 0.99, respectively. The newly developed microtiter plate assay will allow efficient, sensitive and high precision determination of cathepsin L and cathepsin S activities in large-scale studies of cysteine-cathepsin dependent diseases.

Cathepsin B fraction active at physiological pH of 7.5 is of prognostic significance in squamous cell carcinoma of human lung

In this study we examined both the pH dependence of cathepsin B (cath B) activity and its stability at physiological pH of 7.5 in lung tumours and normal lung tissue by means of fluorogenic assays with Z-Arg-Arg-AMC as specific substrate. Specificity was verified with the cath B blocking inhibitors E-64 and CA-074. With respect to pH dependence of activity, we found a deviation from a normal-shaped pH- activity curve. Besides the typical activity peak at pH 6.0, there were shoulders at pH 4.5-5.5 and at pH 7.0-7.5. This heterogeneity was found in both tumour and normal tissue. To test the stability of cath B at physiological pH of 7.5, homogenates were kept at pH 7.5 for 60 min. Altogether, 82-100% of residual cath B activity was found at pH 5.0-5.5, whereas activity in the range between 5.5 and 7.4 dropped drastically to 26-42%. At pH 7.5, there was still 20-34% residual cath B activity detectable. To test the hypothesis whether the cath B fraction active at pH 7.5 is more abundant in tumour tissues compared with the normal counterparts, we determined this fraction in 91 pairs of lung tumour and normal lung tissue. We found a 2.3-fold increase of median cath B fraction active at pH 7.5 in tumour tissue, although this fraction represented only a small part (about 16%) of the native, acidic (pH 6.0) cath B activity. However, in contrast to native cath B at 6.0, the cath B fraction active at pH 7.5 was related to post-operative probability of survival in curatively operated patients, since activity values higher than 292 (muEU mg(-1) protein) were significantly associated with poor prognosis in patients with squamous cell carcinomas (n = 33, P= 0.04). It is concluded that in lung tumour and in normal lung tissue, cath B activity can be divided into at least three fractions with stability optima at different pH values, indicating various forms of cath B. The cath B fraction active at pH 7.5 provides prognostic information in patients with squamous cell carcinoma.