Journal of Cellular Biotechnology

 The signal role in the cellular reactions of gaseous transmitters (NO and H₂S) is known. However there are only a few studies on the effect of NO on red blood cell aggregation (RBCA) and deformability (RBCD). However it remains virtually unexplored role of hydrogen sulfide as a signaling molecule in the analysis of RBC microrheology changes. The purpose of the present study was to investigate changes of red blood cell (RBC) microrheological properties under the influence of some gasotransmitter donors, based on the use of several red blood cell models. RBC microrheology was recorded after cell incubation with: 1) NO donor – sodium nitroprusside (SNP, 10, 50, 100 μM); 2) hydrogen sulphide donor – sodium hydrosulfide (NaHS, 10, 50, 100 μM). Cells were incubated for 30 min at 37°C. RBC suspension prepared in drug-free solution was used as a control sample. It was found that an exposure of RBCs both types of the gasotransmitter (GT) donors led to significant positive changes in the RBC microrheological properties. To study the dose-dependent effect of GT donors, RBCs were incubated in a medium with different GT donor concentrations. The results of the study showed that SNP was more effective at a concentration of 100 μM, while NaHS – at a concentration of 50 μM. In the study of the RBC ghost microrheology, it was found that SNP increased their deformability by 7% (p < 0.01), whereas NaHS much more, by 12%. Using a cellular model of RBC age fractions, it was found that GT donors moderately and positively influenced the deformability of all three age populations. However, the old erythrocytes more significantly responded to GTs by the increase in their deformability. Taken together obtained data allow us to conclude that both gasotransmitter donors positively affect RBC microrheology: significantly reduce their aggregation and moderately but statistically significant increase the cell deformability.

 EphA2 receptor tyrosine kinase fulfils various functions in the development of cancers. Here we analyzed how regulation of EphA2 receptor influences metastatic properties in human melanoma cells in vitro and lung metastasis in vivo. Further, we investigated whether the effects are mediated by Src kinase/focal adhesion kinase (FAK) signaling downstream of EphA2. Therefore, as model Mel-Juso and A375 melanoma cell lines showing different intrinsic EphA2 expression levels were used. To regulate EphA2 expression and activity, we used RNA interference, transgenic EphA2 overexpression, and stimulation of EphA2 activity by adding EphrinA1. Adhesion to fibronectin was increased in EphA2-silenced cells and decreased in EphA2-overexpressing cells. Migration and planar motility were unaffected in Mel-Juso cells, but increased in EphA2-silenced A375 cells and decreased in EphA2-overexpressing A375 cells. Adhesion and migration were unaffected by EphrinA1-stimulation, indicating ligand-independent mechanisms. In vivo we detected increased lung metastasis in mice inoculated with EphA2-overexpressing Mel-Juso cells, substantiating the pro-metastatic effects of EphA2 in melanoma. Activity of Src kinase and FAK were unaffected in EphA2-silenced cells and in response to EphrinA1-stimulation. However, in EphA2-overexpressing A375 cells Src phosphorylation was increased, indicating enhanced Src activity. Together, these data suggest that EphA2 receptor promotes malignancy ligand-independently by mechanisms different from Src kinase/FAK signaling.

Spirulina platensis, a multicelluar, photosynthetic prokaryote (algae) contains a high amount of proteins, vitamins and minerals superior to many foods as e.g. soybeans. Thus, Spirulina platensis was recognized as nutritious food by the United Nations World Food Conference. Due to the high amount of nutritive ingredients Spirulina has a long history as dietary supplement. In addition, spirulina platensis is also efficiently used as forage with known effects on flesh, egg and plumage color, milk yield and fertility. The versatile utilization of the alga can be explained on the one hand with the nutrient levels and on the other hand with recognized effects as anti-viral, anti-bacterial, anti-oxidant, anti-diabetic, anti-cancer and anti-inflammatory substance. Therefore, this alga is named as “superfood”. Beyond, these algae convert carbon dioxide into organic substances and produce oxygen during their growth in alkaline and saline water thereby not wasting fresh water allowing the production in barren areas.

Despite this diverse use of Spirulina platensis due to its beneficial properties, many basic mechanisms on a molecular and cellular level are not well understood and should be explored in future studies.

The liver is the place of biotransformation, where drugs or other substances are metabolized. Cytochrome P450 oxidoreductases (CYPs) play a prominent role in these processes and thus sufficient CYP expression levels are the prerequisite for physiologically relevant liver metabolism or toxicity studies. Human primary hepatocytes, the most popular in vitro liver model for such studies, have several limiting properties: poor availability, rapid dedifferentiation, substantial donor variability and restricted proliferation capacity in vitro. This prompted many research groups to develop alternative models for the investigation of biotransformation-related questions. The hepatoblastoma-derived HepG2 cell line is a highly proliferative and easy to handle in vitro model, but has the disadvantage that expression levels of relevant CYP enzymes are dramatically downregulated. The generation of CYP-overexpressing HepG2 cells is a way to overcome this disadvantage and such cells were used as in vitro alternative to primary hepatocytes. Coding sequences of various CYP isoforms were transiently or stably introduced into HepG2 cells by using viral transduction or transfection reagents. With the frequently used adenoviral transduction, the level of recombinant enzyme activity usually is high within a time window of several days and simultaneous expression of several CYP enzymes is possible. High expression levels can also be achieved with lentiviral transduction which is stable upon virus integration into the host genome. Transient and stable CYP-expressing HepG2 cells serve as a convenient tool for toxicity studies and risk assessment of drugs or other substances undergoing biotransformation, clearance and drug-drug interaction. Furthermore, they can be used for rapid identification of CYP enzymes relevant to a specific reaction or screening for CYP enzyme inhibitors. The use of CYP-overexpressing HepG2 systems also have some disadvantages, such as the cancerous cell origin und their lack of other liver specific functions.

The broad spectrum of possible applications of these CYP-expressing HepG2 cells, especially in the early phase of drug development, can quickly and easily provide important information about drug metabolism in the liver and toxicity behaviour of potential metabolites. In this way, unsuitable drug candidates can be excluded at an early stage of pharmacological studies in order to safe costs and to reduce in vivo animal trials.

In static or low-flow conditions erythrocytes form linear or three-dimensional aggregates with characteristic face-to-face morphology, similar to a stack of coins, often called rouleaux formation. This aggregation is reversible and shear dependent (i.e. dispersed at high shear and reformed at low shear or stasis) and caused by a variety of macromolecules present in the blood plasma. The plasma protein fibrinogen is the major plasma component promoting red blood cell (RBC) aggregation in blood, with an almost linear relationship between aggregate size and plasma fibrinogen concentration. However, other plasma proteins are also reported to increase RBC aggregation, e.g. α2-macroglobulin, immunoglobulin M or G. In addition, there is evidence, that plasma lipids like cholesterol or triglyceride may influence the aggregation of erythrocytes.

In this study we evaluated whether there is an independent influence of proteins and lipids on the RBC aggregation. Using a regression analysis, we analyzed the correlation between the fibrinogen-, α2-macrogobulin-, immunoglobulin M-, Antithrombin III-, Protein C-, Factor VIII-, total cholesterol- and triglyceride concentration with RBC aggregation in blood samples from 2717 apparently healthy subjects or patients.

An univariate analysis showed, that the only variable which correlates on a biologically relevant level is fibrinogen (r = 0.46). The multiple correlation coefficient corresponded to r_mult = 0.589 what indicated that nearly 59% of the variation of the erythrocyte aggregation can be explained by the influencing factors used in this model. This clearly showed that there are additional factors which are involved in the process of erythrocyte aggregation and still are under discussion.