Integrating macrophages into organotypic co-cultures: a 3D in vitro model to study tumor-associated macrophages

Tumor progression is controlled by signals from cellular and extra-cellular microenvironment including stromal cells and the extracellular matrix. Consequently, three-dimensional in vitro tumor models are essential to study the interaction of tumor cells with their microenvironment appropriately in a biologically relevant manner. We have previously used organotypic co-cultures to analyze the malignant growth of human squamous cell carcinoma (SCC) cell lines on a stromal equivalent in vitro. In this model, SCC cell lines are grown on a collagen-I gel containing fibroblasts. Since macrophages play a critical role in the progression of many tumor types, we now have expanded this model by integrating macrophages into the collagen gel of these organotypic tumor co-cultures. This model was established as a murine and a human system of skin SCCs. The effect of macrophages on tumor progression depends on their polarization. We demonstrate that macrophage polarization in organotypic co-cultures can be modulated towards and M1 or an M2 phenotype by adding recombinant IFN-γ and LPS or IL-4 respectively to the growth medium. IL-4 stimulation of macrophage-containing cultures resulted in enhanced tumor cell invasion evidenced by degradation of the basement membrane, enhanced collagenolytic activity and increased MMP-2 and MMP-9. Interestingly, extended co-culture with tumor cells for three weeks resulted in spontaneous M2 polarization of macrophages without IL-4 treatment. Thus, we demonstrate that macrophages can be successfully integrated into organotypic co-cultures of murine or human skin SCCs and that this model can be exploited to analyze macrophage activation towards a tumor supporting phenotype.     

Expression and Characterization of Recombinant Ecarin

The snake venom protease ecarin from Echis carinatus was expressed in stable transfected CHO-S cells grown in animal component free cell culture medium. Recombinant ecarin (r-ecarin) was secreted from the suspension adapted Chinese Hamster Ovary (CHO-S) host cells as a pro-protein and activation to the mature form of r-ecarin occurred spontaneously during continued incubation of the cell culture at 37?°C after death of the host cells. Maximal ecarin activity was reached 7?days or more after cell culture viability had dropped to zero. The best producing CHO-S clone obtained produced up to 7,000 EU ecarin/litre in lab scale shaker cultures. The conversion of different concentrations of both prothrombin and prethrombin-2 as substrates for native and r-ecarin were examined with a chromogenic thrombin substrate. At low concentrations both these proteins were converted into thrombin by the two ecarin preparations with comparable rates. However, with prothrombin concentrations above 250?nM r-ecarin apparently had a two times higher turnover than native ecarin, consistent with the observed rapid complete conversion of prothrombin into thrombin by r-ecarin. With r-ecarin a K _m value of 0.4???M prethrombin-2 was determined but only a rough estimate could be made of the K _m for prothrombin of 0.9???M. In conclusion, r-ecarin was identified as a promising candidate for replacement of native ecarin in assays utilizing conversion of prothrombin to thrombin.     

Differences in the frequency of micronucleated erythrocytes in humans in relation to consumption of fried carbohydrate-rich food

The aim of this study was to investigate if consumption of ordinary carbohydrate-rich food prepared in different ways has an impact on chromosome stability, i.e., on the formation of micronucleated young erythrocytes in humans. Twenty-four persons, divided into two groups, participated during 4 days in a semi-controlled food-consumption study. One group (low-heated-food-group, LowHF-group) consumed only food boiled in water (max 100 degrees C) and the other group (high-heated-food-group, HighHF-group) consumed preferentially strongly heated (fried) food. From each of the subjects, blood samples were drawn, before and after 4 days. The frequency (f) of micronucleated (MN) very young erythrocytes (transferrin-positive reticulocytes, Trf-Ret), fMNTrf-Ret, was determined, and the difference in the frequency, before and after the eating period, was calculated. The obtained mean differences for the two groups were compared. As an indicator of highly heated food the acrylamide (AA) content in part of the consumed foodstuffs was analysed by use of LC/MS-MS and the AA intake estimated. In the blood samples the hemoglobin-adduct levels from AA were analysed as a measure of the internal AA dose. The differences between the mean fMNTrf-Ret, before and after the eating period, were -0.15 per thousand for the LowHF-group and +0.17 per thousand for the HighHF-group, p<0.005 (t-test, one-tailed). The mean total AA intake in the HighHF-group during 4 days was estimated to about 3000+/-450microg per person. For the LowHF-group, the mean AA intake was low, 20+/-10microg per person. The lowest dose of AA that caused a significant increase of micronucleated erythrocytes in mice is more than a hundred times higher than the AA level in this study. Thus, it is unlikely that the exposure to AA is the major cause behind the observed difference. The answer is probably to be found in other compounds produced at the same time during heating of the food.     

Proteomic analysis of highly purified prolamellar bodies reveals their significance in chloroplast development

The prolamellar body (PLB) proteome of dark-grown wheat leaves was characterized. PLBs are formed not only in etioplasts but also in chloroplasts in young developing leaves during the night, yet their function is not fully understood. Highly purified PLBs were prepared from 7-day-old dark-grown leaves and identified by their spectral properties as revealed by low-temperature fluorescence spectroscopy. The PLB preparation had no contamination of extra-plastidal proteins, and only two envelope proteins were found. The PLB proteome was analysed by a combination of 1-D SDS-PAGE and nano-LC FTICR MS. The identification of chlorophyll synthase in the PLB fraction is the first time this enzyme protein was found in extracts of dark-grown plants. This finding is in agreement with its previous localization to PLBs using activity studies. NADPH:protochlorophyllide oxidoreductase A (PORA), which catalyses the reduction of protochlorophyllide to chlorophyllide, dominates the proteome of PLBs. Besides the identification of the PORA protein, the PORB protein was identified for the first time in dark-grown wheat. Altogether 64 unique proteins, representing pigment biosynthesis, photosynthetic light reaction, Calvin cycle proteins, chaperones and protein synthesis, were identified. The in number of proteins’ largest group was the one involved in photosynthetic light reactions. This fact strengthens the assumption that the PLB membranes are precursors to the thylakoids and used for the formation of the photosynthetic membranes during greening. The present work is important to enhance our understanding of the significance of PLBs in chloroplast development.     

A Role for Spectrin in Dynactin‐dependent Melanosome Transport in Xenopus laevis Melanophores

The bi‐directional movement of pigment granules in frog melanophores involves the microtubule‐based motors cytoplasmic dynein, which is responsible for aggregation, and kinesin  II and myosin  V, which are required for dispersion of pigment. It was recently shown that dynactin acts as a link between dynein and kinesin  II and melanosomes, but it is not fully understood how this is regulated and if more proteins are involved. Here, we suggest that spectrin, which is known to be associated with Golgi vesicles as well as synaptic vesicles in a number of cells, is of importance for melanosome movements in Xenopus laevis melanophores. Large amounts of spectrin were found on melanosomes isolated from both aggregated and dispersed melanophores. Spectrin and two components of the oligomeric dynactin complex, p150^glued and Arp1/centractin, co‐localized with melanosomes during aggregation and dispersion, and the proteins were found to interact as determined by co‐immunoprecipitation. Spectrin has been suggested as an important link between cargoes and motor proteins in other cell types, and our new data indicate that spectrin has a role in the specialized melanosome transport processes in frog melanophores, in addition to a more general vesicle transport.     

A theoretical study of the mechanism for peptide hydrolysis by thermolysin

The catalytic mechanism for peptide hydrolysis by thermolysin has been investigated using the B3LYP hybrid density functional method. The starting structure for the calculations was based on the X-ray crystal structure of the enzyme inhibited with the ZF (p)LA phosphonamidate transition-state analogue. Besides the three Zn ligands His142, His146 and Glu166, a few additional residues were also included in the model. Following the order of importance, the outer-sphere ligands Glu143, His231 and Asp226 were shown to play significant catalytic roles, well correlated with results from site-directed mutagenesis experiments. A single-step reaction mechanism was obtained starting from the initial enzyme-substrate complex with a pentacoordinated metal center and proceeding to the enzyme-carboxylate complex as a final product, following a proposal by Matthews and co-workers. The transition state combines a nucleophilic water oxygen attack on the peptide carbon and a proton transfer from the water to the peptide nitrogen, mediated by the Glu143 carboxylate. A free activation energy of 15.2 kcal/mol was obtained, compared to the experimental 12.4-16.3 kcal/mol range for various peptide substrates. An interesting aspect of the present single-step mechanism is that the Glu143 carboxylate moves a significant distance of ~1.0 A. Different chemical models were examined, both related to the system size and proper side-chain modeling. The significance of the protein frame rigidity around the active site was estimated by fixing and subsequently releasing the edge atom positions. Finally, alternative mechanistic proposals are briefly summarized.     

A theoretical study of myoglobin working as a nitric oxide scavenger

The mechanism for the reaction between nitric oxide (NO) and O₂ bound to the heme iron of myoglobin (Mb), including the following isomerization to nitrate, has been investigated using hybrid density functional theory (B3LYP). Myoglobin working as a NO scavenger could be of importance, since NO reversibly inhibits the terminal enzyme in the respiration chain, cytochrome c oxidase. The concentration of NO in the cell will thus affect the respiration and thereby the synthesis of ATP. The calculations show that the reaction between NO and the heme-bound O₂ gives a peroxynitrite intermediate whose O–O bond undergoes a homolytic cleavage, forming a NO₂ radical and myoglobin in the oxo-ferryl state. The NO₂ radical then recombines with the oxo-ferryl, forming heme-bound nitrate. Nine different models have been used in the present study to examine the effect on the reaction both by the presence and the protonation state of the distal His64, and by the surroundings of the proximal His93. The barriers going from the oxy-Mb and nitric oxide reactant to the peroxynitrite intermediate and further to the oxo-ferryl and NO₂ radical are around 10 and 7 kcal/mol, respectively. Forming the product, nitrate bound to the heme iron has a barrier of less than ~7 kcal/mol. The overall reaction going from a free nitric oxide and oxy-Mb to the heme bound nitrate is exergonic by more than 30 kcal/mol.     

Intestinal lymphoid tissue changes in the cortisone-treated Wistar rat

This paper continues the previous investigation of the Department on the lymphoid tissue of central and peripheral lymphoid organs under different experimental conditions. The morphological reactional modalities of the intestinal lymphoid tissue in the male Wistar rat were followed up under endocrine imbalance conditions following cortisone administration. Seven days after administration cortisone induced a hyperplasia of the intestinal lymphoid tissue in parallel with a depletion of the lymph node parenchyma and a hypercellularity of bone marrow. After a 6-week postcortisone interval, the lymphoid tissue showed changes corresponding to a cellular depletion in parallel with the restoration of the lymph node parenchyma and a normocellular bone marrow.     

Identification of archaeobotanical Pistacia L. fruit remains: implications for our knowledge on past distribution and use in prehistoric Cyprus

Vegetation History and Archaeobotany - Pistacia spp. remains are common finds among archaeobotanical assemblages in prehistoric sites in Southwest Asia, both in the form of endocarps and charcoal...