Cellular Responses Modulated by FGF-2 Adsorbed on Albumin/Heparin Layer-by-Layer Assemblies

In a typical cell culture system, growth factors immobilized on the cell culture surfaces can serve as a reservoir of bio-signaling molecules, without the need to supplement them additionally into the culture medium. In this paper, we report on the fabrication of albumin/heparin (Alb/Hep) assemblies for controlled binding of basic fibroblast growth factor (FGF-2). The surfaces were constructed by layer-by-layer adsorption of polyelectrolytes albumin and heparin and were subsequently stabilized by covalent crosslinking with glutaraldehyde. An analysis of the surface morphology by atomic force microscopy showed that two Alb/Hep bilayers are required to cover the surface of substrate. The formation of the Alb/Hep assemblies was monitored by the surface plasmon resonance (SPR), the infrared multiinternal reflection spectroscopy (FTIR MIRS) and UV/VIS spectroscopy. The adsorption of FGF-2 on the cross-linked Alb/Hep was followed by SPR. The results revealed that FGF-2 binds to the Alb/Hep assembly in a dose and time-dependent manner up to the surface concentration of 120 ng/cm². The bioactivity of the adsorbed FGF-2 was assessed in experiments in vitro, using calf pulmonary arterial endothelial cells (CPAE). CPAE cells could attach and proliferate on Alb/Hep surfaces. The adsorbed FGF-2 was bioactive and stimulated both the proliferation and the differentiation of CPAE cells. The improvement was more pronounced at a lower FGF-2 surface concentration (30 ng/cm²) than on surfaces with a higher concentration of FGF-2 (120 ng/cm²).     

Arsenic in Agricultural Soils of a Historically Mined and Industrial Region of Southern Serbia and Northern Kosovo: Bioavailability and Uptake by Plants Species Zea mays L. and Solanum tuberosum L.

This article reports the results of a study focused on the presence and bioavailability of arsenic in agricultural soil in the mining and industrial regions of northern Kosovo and southern Serbia, as well as uptake and bioaccumulation of arsenic in two commonly cultivated plant species (Zea mays L. and Solanum tuberosum L.). This area was one of the most important mining districts in Europe. The collected soil samples were subjected to a modified BCR three-step sequential extraction procedure in order to investigate the chemical partitioning of arsenic in the soils. The general distribution of arsenic in various fractions was: exchangeable < reducible < oxidizable fractions. Highest concentrations of total arsenic in soil were found close to industrial facilities and tailing ponds. In addition, fluvisols were significantly more enriched with arsenic than soils at a distance from the river flows. The edible parts of the plant specimen showed different As contents, suggesting that these plant species have different propensities for the uptake and bioaccumulation of arsenic from soil.     

Rac1 Regulates Endometrial Secretory Function to Control Placental Development

During placenta development, a succession of complex molecular and cellular interactions between the maternal endometrium and the developing embryo ensures reproductive success. The precise mechanisms regulating this maternal-fetal crosstalk remain unknown. Our study revealed that the expression of Rac1, a member of the Rho family of GTPases, is markedly elevated in mouse decidua on days 7 and 8 of gestation. To investigate its function in the uterus, we created mice bearing a conditional deletion of the Rac1 gene in uterine stromal cells. Ablation of Rac1 did not affect the formation of the decidua but led to fetal loss in mid gestation accompanied by extensive hemorrhage. To gain insights into the molecular pathways affected by the loss of Rac1, we performed gene expression profiling which revealed that Rac1 signaling regulates the expression of Rab27b, another GTPase that plays a key role in targeting vesicular trafficking. Consequently, the Rac1-null decidual cells failed to secrete vascular endothelial growth factor A, which is a critical regulator of decidual angiogenesis, and insulin-like growth factor binding protein 4, which regulates the bioavailability of insulin-like growth factors that promote proliferation and differentiation of trophoblast cell lineages in the ectoplacental cone. The lack of secretion of these key factors by Rac1-null decidua gave rise to impaired angiogenesis and dysregulated proliferation of trophoblast cells, which in turn results in overexpansion of the trophoblast giant cell lineage and disorganized placenta development. Further experiments revealed that RAC1, the human ortholog of Rac1, regulates the secretory activity of human endometrial stromal cells during decidualization, supporting the concept that this signaling G protein plays a central and conserved role in controlling endometrial secretory function. This study provides unique insights into the molecular mechanisms regulating endometrial secretions that mediate stromal-endothelial and stromal-trophoblast crosstalk critical for placenta development and establishment of pregnancy.     

A Novel Mechanism of Bacterial Toxin Transfer within Host Blood Cell-Derived Microvesicles

Shiga toxin (Stx) is the main virulence factor of enterohemorrhagic Escherichia coli, which are non-invasive strains that can lead to hemolytic uremic syndrome (HUS), associated with renal failure and death. Although bacteremia does not occur, bacterial virulence factors gain access to the circulation and are thereafter presumed to cause target organ damage. Stx was previously shown to circulate bound to blood cells but the mechanism by which it would potentially transfer to target organ cells has not been elucidated. Here we show that blood cell-derived microvesicles, shed during HUS, contain Stx and are found within patient renal cortical cells. The finding was reproduced in mice infected with Stx-producing Escherichia coli exhibiting Stx-containing blood cell-derived microvesicles in the circulation that reached the kidney where they were transferred into glomerular and peritubular capillary endothelial cells and further through their basement membranes followed by podocytes and tubular epithelial cells, respectively. In vitro studies demonstrated that blood cell-derived microvesicles containing Stx undergo endocytosis in glomerular endothelial cells leading to cell death secondary to inhibited protein synthesis. This study demonstrates a novel virulence mechanism whereby bacterial toxin is transferred within host blood cell-derived microvesicles in which it may evade the host immune system.     

Enhanced survival of lethally irradiated adenosine A3 receptor knockout mice. A role for hematopoietic growth factors?

Adenosine A₃ receptor knockout (A₃AR KO) mice and their wild-type (WT) counterparts were compared from the point of view of their abilities to survive exposures to lethal doses of γ-radiation belonging to the range of radiation doses inducing the bone marrow acute radiation syndrome. Parameters of cumulative 30-day survival (experiment using a midlethal radiation dose) or cumulative 11-day survival (experiment using an absolutely lethal radiation dose), and of mean survival time were evaluated. The values of A₃AR KO mice always reflected their higher survival in comparison with WT ones, the P values being above the limit for statistical significance after the midlethal radiation dose and standing for statistical significance after the absolutely lethal radiation dose. This finding was considered surprising, taking into account the previously obtained findings on defects in numbers and functional properties of peripheral blood cells in A₃AR KO mice. Therefore, previous hematological analyses of A₃AR KO mice were supplemented in the present studies with determination of serum levels of the granulocyte colony-stimulating factor, erythropoietin, and thrombopoietin. Though distinct differences in these parameters were observed between A₃AR KO and WT mice, none of them could explain the relatively high postirradiation survival of A₃AR KO mice. Further studies on these mice comprising also those on other than hemopoietic tissues and organs can help to clarify their relative radioresistance.     

Measuring Biomass and Carbon Stock in Resprouting Woody Plants

Resprouting multi-stemmed woody plants form an important component of the woody vegetation in many ecosystems, but a clear methodology for reliable measurement of their size and quick, non-destructive estimation of their woody biomass and carbon stock is lacking. Our goal was to find a minimum number of sprouts, i.e., the most easily obtainable, and sprout parameters that should be measured for accurate sprout biomass and carbon stock estimates. Using data for 5 common temperate woody species, we modelled carbon stock and sprout biomass as a function of an increasing number of sprouts in an interaction with different sprout parameters. The mean basal diameter of only two to five of the thickest sprouts and the basal diameter and DBH of the thickest sprouts per stump proved to be accurate estimators for the total sprout biomass of the individual resprouters and the populations of resprouters, respectively. Carbon stock estimates were strongly correlated with biomass estimates, but relative carbon content varied among species. Our study demonstrated that the size of the resprouters can be easily measured, and their biomass and carbon stock estimated; therefore, resprouters can be simply incorporated into studies of woody vegetation.     

A multiplex biomarker approach for the diagnosis of transitional cell carcinoma from canine urine

Transitional cell carcinoma (TCC), the most common cancer of the urinary bladder in dogs, is usually diagnosed at an advanced disease stage with limited response to chemotherapy. Commercial screening tests lack specificity and current diagnostic procedures are invasive. A proof of concept pilot project for analyzing the canine urinary proteome as a noninvasive diagnostic tool for TCC identification was conducted. Urine was collected from 12 dogs in three cohorts (healthy, urinary tract infection, TCC) and analyzed using liquid chromatography tandem mass spectrometry. The presence of four proteins (macrophage capping protein, peroxiredoxin 5, heterogeneous nuclear ribonucleoproteins A2/B, and apolipoprotein A1) was confirmed via immunoblot. Of the total 379 proteins identified, 96 were unique to the TCC group. A statistical model, designed to evaluate the accuracy of this multiplex biomarker approach for diagnosis of TCC, predicted the presence of disease with 90% accuracy.     

Cysteine peptidases and their inhibitors in breast and genital cancer

Cysteine proteinases and their inhibitors probably play the main role in carcinogenesis and metastasis. The metastasis process need external proteolytic activities that pass several barriers which are membranous structures of the connective tissue which includes, the basement membrane of blood vessels. Activities of the proteinases are regulated by endogenous inhibitors and activators. The imbalance between cysteine proteinases and cystatins seems to be associated with an increase in metastatic potential in some tumors. It has also been reported that proteinase inhibitors, specific antibodies for these enzymes and inhibition of the urokinase receptor may prevent cancer cell invasion. Some proteinase inhibitor could serve as agents for cancer treatment.     

The application of the RT-PCR method for the staging of the prostate cancer progression

Molecular biology seems to bring more convincing markers for the detection of prostate cancer as well as the development of metastases than immunohistochemistry. The main goal of present work was to detect the expression of prostate specific antigen (PSA) and prostate-specific membrane antigen (PSM) genes in the micrometastases by the RT-PCR to assess the progression of prostate cancer. We analyzed 50 patients: 28 patients with clinically localized or locally advanced prostate cancer who underwent radical prostatectomy, 7 patients with clinically proven metastases, 8 patients with benign prostatic hyperplasia, and 7 healthy young men. The results of RT-PCR in the first group of 28 patients varied, however, they were in good correlation with the health status of the patients. Positive results of PSA and notably for PSM were good predictors of beginning metastasing process. Seven patients with metastatic disease had positive RT-PCR results both for PSA and PSM. All of the patients with benign prostatic hyperplasia and healthy young men had negative RT-PCR results for PSA and PSM. The study showed that positive RT-PCR results for PSA and especially for PSM correlated well with the progression of the disease and negative results reflected good health status of the patients.     

Effects of different detachment procedures on viability,nitroxide reduction kinetics and plasma membrane heterogeneity of V‐79 cells

Cell detachment procedures can cause severe damage to cells. Many studies require cells to be detached before measurements; therefore, research on cells that have been grown attached to the bottom of the culture dish and later detached represents a special problem with respect to the experimental results when the properties of cell membranes undergo small changes such as in spectroscopic studies of membrane permeability. We characterized the influence of three different detachment procedures: cell scraping by rubber policeman, trypsinization and a citrate buffer treatment on V‐79 cells in the plateau phase of growth (arrested in G₁). We have measured cell viability by a dye‐exclusion test; nitroxide reduction kinetics and membrane fluidity by EPR (electron paramagnetic resonance) method using the lipophilic spin‐probe MeFASL(10,3) (5‐doxylpalmitoyl‐methylester), which partitions mainly in cell membranes and the hydrophilic spin‐probe TEMPONE (4‐oxo‐2,2,6,6‐tetramethylpiperidine‐1‐oxyl). The resulting cell damage due to the detachment process was observed with SEM (scanning electron microscopy). We found out that cell viability was 91% for trypsin treatment, 85% for citrate treatment and 70% for cell scraping. Though the plasma membrane was mechanically damaged by scraping, the membrane domain structure was not significantly altered compared with other detachment methods. On the other hand, the spin‐probe reduction rate, which depends both on the transport across plasma membrane as well as on metabolic properties of cells, was the highest for trypsin method, suggesting that metabolic rate was the least influenced. Only the reduction rate of trypsin‐treated cells stayed unchanged after 4 h of stirring in suspension. These results suggest that, compared with scraping cells or using citrate buffer, the most suitable detachment method for V‐79 cells is detachment by trypsin and keeping cells in the stirred cell suspension until measurement. This method provides the highest cell viability, less visible damage on SEM micrographs and leaves the metabolic rate of cells unchanged.