Genes encoding novel murine cysteine peptidases of the papain family C1A and related genes were cloned and mapped to mouse chromosome 13, colocalizing with the previously assigned cathepsin J gene. We constructed a <460-kb phage artificial chromosome (PAC) contig and characterized a dense cluster comprising eight C1A cysteine peptidase genes, cathepsins J, M, Q, R, -1, -2, -3, and -6; three pseudogenes of cathepsins M, -1, and -2; and four genes encoding putative cysteine peptidase inhibitors related to the proregion of C1A peptidases (trophoblast-specific proteins alpha and beta and cytotoxic T-lymphocyte-associated proteins 2alpha and -beta). Because of sequence homologies of 61.9-72.0% between cathepsin J and the other seven putative cysteine peptidases of the cluster, these peptidases are classified as "cathepsin J-like". The absence of cathepsin J-like peptidases and related genes from the human genome suggests that the cathepsin J cluster arose by partial and complete gene duplication events after the divergence of primate and rodent lineages. The expression of cathepsin J-like peptidases and related genes in the cluster is restricted to the placenta only. Clustered genes are induced at specific time points, and their expression increases toward the end of gestation. The specific expression pattern and high expression level suggest an essential role of cathepsin J-like peptidases and related genes in formation and development of the murine placenta.

Although not widely practiced by oncologists, in vitro tumor chemosensitivity assays (TCA) have proved to increase the lifetime of tumor patients in prospective clinical trials. By individualizing cancer therapy, they can support the clinician's decision which is usually based on empirically retrieved data and thereby prevent inadequate chemotherapy. We present the first results of a new sensor-chip-based technology which might be useful for a multiparametric TCA. In particular, the aspect of dynamic on-line data generation on intact cellular specimens is a major difference to alternative assays. A series of experiments has been performed on cell lines and human tumor explants. Cell cultures and tumor tissue explants were placed on miniaturized silicon and glass sensor chips. The sensor data currently analyze metabolic profiles (rates of extracellular acidification and cellular oxygen consumption) and changes in cell morphology (monitoring of electric impedance). With the cell lines, drug-associated cellular signals have been detected with all three parameters, while primary explants so far caused metabolic responses only. In particular, cellular respiration or mitochondrial activity seems to be a most sensitive indicator of acute cytotoxic effects. The experimental results were achieved using different test versions. Besides giving a status report, the theoretical potential and current problems of sensor chip technology in TCA is discussed.

The pH in the cellular microenvironment (pHM) is an important regulator of cell-to-cell and cell-to-host interactions. Additionally the extracellular acidification rate of a cell culture is an important indicator of global cellular metabolism. In a new approach a biocompatible ion-sensitive field effect transistor (ISFET)-array was developed to measure the pHM close to a surface and the global extracellular acidification rate at the same time. This ISFET-array is part of a new multiparametric microsensor chip. The paper highlights some basic applications of this method for in-vitro measurements. Using a fluid perfusion system for cell culture media, it is possible to measure the pHM of few (five to ten) adherent tumor cells in a distance of 10–100 nm from the cell plasma membrane. Experiments showed a pHM-value of 6.68±0.06 pH. Further experiments suggest that both the low pH, and the extracellular acidification rate of the examined tumor cell line are mainly built up by glycolysis.

Living cells can be considered as complex biochemical plants. Biochemical and biophysical processes enable a cell to maintain itself, to grow, to reproduce and to communicate with the environment. Getting more information about the multifunctional cellular processing of input- and output-signals in different cellular plants is essential for basic research as well as for various fields of biomedical applications. For in-vitro investigations on living cells, the cellular environment differs from the native environment found in vivo. As a first approach for on-line monitoring of cellular reactions under well controlled experimental conditions we have developed the so called Cell Monitoring System (CMS®). It allows parallel and non-invasive measurement of different parameters from cellular systems by the use of microsensors. Microelectronic sensors are the adequate choice for the non-invasive measurement of environmental—as well as in- and output—parameters of cells. In this paper we present a measurement system with pH-sensitive ISFETs (ionsensitive fieldeffect transistors) for the measurement of extracellular pH-related signals on cells and tissues.

Microsensors provide instruments particularly suited for the rapid, noninvasive and on-line analysis of cell and tissue cultures. The microsensor system presented in this paper is a modular arrangement of various planar and nonplanar sensor elements for the measurement of physiological parameters of cell cultures. An optic access to the cultures (e.g. for light microscopy and spectrophotometric techniques) is also provided for a parallel and comparative data acquisition. The system was originally designed for biomedical research in chemotherapy (predicative chemotherapy assays) and pharmacology but it turned out to be also an effective tool for toxicological and environmental research.

Critical parameters to be assessed in cell culture are the number of viable cells and cell viability. Growth, product formation, toxicity effects and the overall success of cell culture can depend largely on these. With interdigitated electrode structures (IDES) adherent cells are cultured directly on a pair of interdigitated electrodes, and the impedance of the system gives insight into the adhesive behaviour of the cells. The signal is influenced by the changes in number, growth and morphological behaviour of adherently growing cells, mainly owing to the insulating effects of the cell membranes. Five different cell lines are used, and their divergent behaviour is monitored over a period of four days, from inoculation of the cells to killing of the cells at the end of the experiments. Even when the cells form close monolayers, great fluctuations in the impedance signal can be observed. Nevertheless, for a more complete description of cellular systems, other parameters, such as acidification and respiration, have to be included in the measuring system.

Article Cell meets silicon - Biomedizinische Sensorik für Diagnostik und Therapie was published on January 1, 1998 in the journal Biomedical Engineering / Biomedizinische Technik (volume 43, issue s1).

A new method for on-line and real-time monitoring of concentration, growth and physiological state of cells in culture is described. This biosensor is based on impedance measurements of adherently growing cells on interdigitated electrode structures (IDES). The measurements can be performed for several days as there is no detectable electrical influence on the cells. The versatility of this new sensor is shown with some exemplary experiments. Cell density, growth and long-term behaviour of cells on the electrodes clearly change the impedance of the IDES. Both, the global influence of serum components (deprivation of foetal bovine serum) and the toxic effects of heavy metal ions (cadmium) result in changes of the sensor signal and can be visualized.

sThe development of an integrated sensor system, called the physiocontrol microsystem, is presented. It is suited for microscopy, and works with both adherent cell types and cultures growing in suspension, as well as with tissue biopsies. The central part, a miniaturized culture chamber equipped with differently constructed microsensors, allows continuous observation of important physiological parameters even in the course of long-lasting experiments. Besides a description of the physical components, the study provides a summary of selected applications of the physio-control microsystem in basic cellular research and biomedical diagnostics.

The development of an integrated sensor system, called the physiocontrol microsystem, is presented. It is suited for microscopy, and works with both adherent cell types and cultures growing in suspension, as well as with tissue biopsies. The central part, a miniaturized culture chamber equipped with differently constructed microsensors, allows continuous observation of important physiological parameters even in the course of long-lasting experiments. Besides a description of the physical components, the study provides a summary of selected applications of the physiocontrol microsystem in basic cellular research and biomedical diagnostics.

Analytical electron microscopy is an ideal tool for holistic data acquisition on biological systems. The use of analytical electron microscopy for both,the investigation of micropharmacokinetic problems and metabolic studies, is becoming more and more important. Depending on the mode of investigation, it is possible to localize drugs and xenobiotics precisely in situ under optical control or to quantify their uptake and distribution in the corresponding target cells without disintegrating the cell or tissue material. In this paper, we present instructive examples for the application of analytical electron energy loss spectroscopy in transmission electron microscopy in order to investigate the cellular uptake and distribution of cisplatin and cyclophosphamide and the metabolic changes induced by an alteration in the extracellular calcium concentration in a holistic manner.

Synthetic lipopeptides, structurally derived from the N-terminal part of bacterial lipoprotein, constitute macrophage and B-lymphocyte activators. The molecular mechanism of macrophage activation by lipopeptides still remains unclear. The purpose of our study was to determine the route and kinetics of lipopeptide distribution in bone marrow-derived macrophages. The intracellular localization of the C-terminally biotinylated lipodipeptide Pam3Cys-Ser was investigated on ultrathin cryosections using the biotinstreptavidin-gold system. Our findings indicate that the lipopeptide penetrates the plasma membrane and can already be found within the cytoplasm, the nuclear membrane, and within the nucleus after 2 min of stimulation. The pattern of lipopeptide distribution obtained 2 min after stimulation resembles that obtained after longer incubation times (8 and 20 min). Correlating distribution patterns were observed when using the method of electron energy loss spectroscopy (EELS). These findings are a clear indication for the rapid uptake of lipopeptides into eukaryotic cells, and are of importance for further studies of the immunostimulating properties of the bacterial lipopeptides and vaccines derived therefrom.

Investigations on uptake and distribution of small molecules and drugs in cells and cells from solide tissues are very important for the evaluation of their physiological behaviour and/or therapeutical potency. In electron microscopy localizations of molecules are usually performed by immunocytochemical techniques using antibodies coupled to electron dense markers (e.g. ferritin, gold). Problems concerning labelling precision and the production of highly specific antibodies to small molecules resulted in the evaluation of a more sensitive and more physical technique.We applied analytical electron energy loss spectroscopy (EELS) on ultrathin cryosections of different cells incubated with various substances (lipopeptide, 5’fluoro-uracil, cis-platinum, etc.) to follow up the route of these molecules from the plasma membrane to ultrastructural target regions. Using cryosections, investigations on almost native material, without the use of disintegrative methods, are possible. The main advantage of this procedure is the exclusion of metabolic interferences after the incubation period.Here, we demonstrate analytical energy loss spectroscopy as a possibility to analyze the distribution of small molecules within cells directly without the use of labelled antibodies.

Premalignant and malignant lesions from human lung tumours have been investigated by ultrastructural stereological methods and compared to normal bronchial tissue. It was possible to develop characteristic quantitative ultrastructural criteria for the different lesions. These criteria are the basic of a data bank system. After verification by further investigations they may be applicable to the development of a diagnosis supporting system.

In a previous publication, we described new devices and results for diagnostic histopathological transmission electron microscopy on large, ultrathin cryosections from several human tumours. As reported, cryo-ultramicrotomy is gaining in significance and use as technical improvements are made. In comparison with epoxy resin procedures, cryo-preparations are more rapid, there is negligible loss of material and thus antigenicity is well preserved. This work contains some newly developed details for a new type of cryo-ultramicrotome, especially adapted for histopathological investigations on large ultrathin cryosections. It is a software-controlled ultramicrotome advance drive system with feedback regulation and various advancement sensors. Some of these developments could also be applied to existing microtome systems.

The ultraimmunohistochemical localization of carcinoembryonic antigen (CEA) in breast, gastric and lung tumours at various stages of differentiation showed that CEA is mainly located on well differentiated tumour cells, the subcellular morphometric data of which resembled more those of benign lesions rather than those of malignant tumours. All these cells were characterized by raised surface to volume ratios as compared to normal cells. The CEA labels were mainly found on the cell membranes, the outer nuclear membrane and the rough endoplasmatic reticulum (rER). Mono- and poly-clonal antibodies produced a similar labelling pattern, but the former appeared to have less affinity for glycocalix-associated structures than the latter.

A new, more direct method for reliable localization of CEA on human tumours has been applied at the ultrastructural level. Freshly prepared biopsy material from breast and gastric tumours, as well as xenografted colon tumours from mice, and a colon tumour cell line (LS 174 T), were briefly fixed in glutaraldehyde and frozen in liquid propane. Ultrathin cryosections were labelled with monospecific CEA antibodies by applying gold-coupled second antibodies. Variations in the labelling patterns were found when comparing breast, colon, and gastric cancers. Apart from breast tumours, all other samples reveal significant extracellular CEA positivity on the cell membrane. Intracellular localization has been found on the endoplasmic reticulum, nuclear membrane, and, in breast tumours, on electron dense bodies. These investigations confirm our previous experimental results using preembedding labelling techniques before Epon inclusion.