Impairment of Hypoxia-Induced CA IX by Beta-Blocker Propranolol—Impact on Progression and Metastatic Potential of Colorectal Cancer Cells

The coexistence of cancer and other concomitant diseases is very frequent and has substantial implications for treatment decisions and outcomes. Beta-blockers, agents that block the beta-adrenergic receptors, have been related also to cancers. In the model of multicellular spheroids formed by colorectal cancer cells we described a crosstalk between beta-blockade by propranolol and tumour microenvironment. Non-selective beta-blocker propranolol decreased ability of tumour cells to adapt to hypoxia by reducing levels of HIF1α and carbonic anhydrase IX in 3D spheroids. We indicated a double action of propranolol in the tumour microenvironment by inhibiting the stability of HIF1α, thus mediating decrease of CA IX expression and, at the same time, by its possible effect on CA IX activity by decreasing the activity of protein kinase A (PKA). Moreover, the inhibition of β-adrenoreceptors by propranolol enhanced apoptosis, decreased number of mitochondria and lowered the amount of proteins involved in oxidative phosphorylation (V-ATP5A, IV-COX2, III-UQCRC2, II-SDHB, I-NDUFB8). Propranolol reduced metastatic potential, viability and proliferation of colorectal cancer cells cultivated in multicellular spheroids. To choose the right treatment strategy, it is extremely important to know how the treatment of concomitant diseases affects the superior microenvironment that is directly related to the efficiency of anti-cancer therapy     

CRKL,AIFM3, AIF,BCL2, and UBASH3A during Human Kidney Development

We aimed to investigate the spatio-temporal expression of possible CAKUT candidate genes CRKL, AIFM3, and UBASH3A, as well as AIF and BCL2 during human kidney development. Human fetal kidney tissue was stained with antibodies and analyzed by fluorescence microscopy and RT-PCR. Quantification of positive cells was assessed by calculation of area percentage and counting cells in nephron structures. Results showed statistically significant differences in the temporal expression patterns of the examined markers, depending on the investigated developmental stage. Limited but strong expression of CRKL was seen in developing kidneys, with increasing expression up to the period where the majority of nephrons are formed. Results also lead us to conclude that AIFM3 and AIF are important for promoting cell survival, but only AIFM3 is considered a CAKUT candidate gene due to the lack of AIF in nephron developmental structures. Our findings imply great importance of AIFM3 in energy production in nephrogenesis and tubular maturation. UBASH3A raw scores showed greater immunoreactivity in developing structures than mature ones which would point to a meaningful role in nephrogenesis. The fact that mRNA and proteins of CRKL, UBASH3A, and AIFM3 were detected in all phases of kidney development implies their role as renal development control genes.     

Synthesis of starch benzoate in aqueous media

Summary The results of investigating starch benzoate (SB) synthesis in aqueous media are presented in this study. Starch esterification with benzoyl chloride was performed in two steps, the first step being the alkalization of starch and the second step esterification. The influence of the concentration of reactants, reaction medium composition, temperature and time of synthesis on the degree of substitution (DS), degree of swelling in water and thermal stability of the synthesized starch benzoate was investigated. The optimal conditions for the synthesis of starch benzoate with degree of substitution from 0.23 to 1.76 were determined. It was shown that starch benzoate with a DS of 1.76 practically did not swell in water. However, starch benzoate is less thermally stable than native starch probably due to a change in the supermolecular order induced by the esterification reaction. Received: 18 March 2002/Revised version: 18 October 2002/ Accepted: 28 October 2002 Correspondence to Katarina Jeremić     

Immunohistochemical Expression Pattern of FGFR1, FGFR2, RIP5, and HIP2 in Developing and Postnatal Kidneys of Dab1−/− (yotari) Mice

This study aimed to explore how Dab1 gene functional silencing influences the spatial and temporal expression patterns of fibroblast growth factor receptor 1 (FGFR1), fibroblast growth factor receptor 2 (FGFR2), receptor-interacting protein kinase 5 (RIP5), and huntingtin-interacting protein 2 (HIP2) in the developing and postnatal kidneys of the yotari mice as potential determinants of normal kidney formation and function. Dab1^−/− animal kidneys exhibit diminished FGFR1/FGFR2 expression in all examined developmental stages, whereas RIP5 cell immunoreactivity demonstrated negligible variation. The HIP2 expression revealed a discernible difference during the postnatal period, where we noted a significant decrease in almost all the observed kidney structures of yotari animals. An extracellular signal-regulated kinase (Erk1/2) and mammalian target of rapamycin (mTOR) expression in yotari kidneys decreased in embryonic and postnatal developmental phases for which we can hypothesize that the Erk1/2 signaling pathway in the yotari mice kidneys is dependent on Reelin with Dab1 only partially implicated in Reelin-mediated MEK/Erk1/2 activation. The impairment of FGFR1 and FGFR2 expression suggests the involvement of the observed markers in generating the CAKUT phenotype resulting in renal hypoplasia. Our study demonstrates the critical role of HIP2 in reducing cell death throughout nephrogenesis and maturation in wild-type mice and indicates a possible connection between decreased HIP2 expression in postnatal kidney structures and observed podocyte injury in yotari. Our results emphasize the crucial function of the examined markers throughout normal kidney development and their potential participation in kidney pathology and diagnostics, where they might serve as biomarkers and therapeutic targets.     

Alternative DNA amplification methods to PCR and their application in GMO detection: a review

Nucleic acids, especially DNA, are targets of qualitative and quantitative diagnostics for genetically modified organisms (GMO) in seeds, food- and feedstuff. The amplification of the nucleic acid is an essential step for further analyses of the target sequence. The PCR has been the method of choice for DNA amplification in most laboratories, and its real-time version (qPCR) also enables quantitative analysis of target contents. Despite its numerous advantages, PCR technology has some limitations such as the lack of true multiplexing properties. To alleviate the drawbacks linked to PCR technology, alternative nucleic acid amplification methods with promising characteristics are being developed fast. These methods, their advantages, and the inconveniences, which are not yet resolved are summarized in the paper. Special focus is given to the possibilities of using these alternative methods for GMO detection in future, when expansion of GMOs both in diversity and frequencies will make current GMO detection systems difficult to operate.     

Science ethics: A study of eminent scientists' professional values

A questionnaire study of eminent scientists' professional values was carried out within more extensive research of professional ethics. The structure of scientists' professional values is composed of five factors: collegial and professorial responsibility, the protection of respondents or patients, wider social and professional responsibility, scientific precision and originality and scientific objectivity. The core of the scientists' professional code consists of the values with the highest ratings. These are: cognitive standards which define the research role, explicit expectations of the scientists' social responsibility, and requirements for excellence of scientific institutions and personnel. At the same time, significant differences have been found among the observed scientific fields. The largest discriminative power has been shown in the importance of precise measurements and then also in the protection of respondents and patients. These results question the traditional unitary concept of science, but also the concept of intellectual and social atomisation of scientific disciplines and fields.     

Hypoxia Marker Carbonic Anhydrase IX Is Present in Abdominal Aortic Aneurysm Tissue and Plasma

Abdominal aortic aneurysms (AAA) are a significant cause of premature deaths worldwide. Since there is no specific treatment for reducing AAA progression, it is crucial to understand the pathogenesis leading to aneurysm wall weakening/remodeling and identify new proteins involved in this process which could subsequently serve as novel therapeutic targets. In this study, we analyzed the presence of the hypoxia-related proteins carbonic anhydrase IX (CA IX), hypoxia-inducible factor 1α (HIF-1α), and AKT as the key molecule in the phosphoinositide-3-kinase pathway in the AAA wall. Additionally, we used a blood-based assay to examine soluble CA IX (s-CA IX) levels in the plasma of AAA patients. Using western blotting, we detected CA IX protein in 12 out of 15 AAA tissue samples. Immunohistochemistry staining proved CA IX expression in the media of the aneurysmal wall. Evaluation of phosphorylated (p-AKT) and total AKT showed elevated levels of both forms in AAA compared to normal aorta. Using ELISA, we determined the concentration of s-CA IX >20 pg/mL in 13 out of 15 AAA patients. Results obtained from in silico analysis of CA9 and aneurysm-associated genes suggest a role for CA IX in aneurysmal wall remodeling. Our results prove the presence of hypoxia-related CA IX in AAA tissues and indicate a possible role of CA IX in hypoxia-associated cardiovascular diseases.     

Fatty acid methyl ester as reactive diluent in thermally cured solvent-borne coil-coatings—The effect of fatty acid pattern on the curing performance and final properties

Four different fatty acid methyl esters (FAMEs): rape seed methyl ester (RME), tall oil methyl ester (TOME), and two types of linseed oil methyl ester (Linutin) have been studied as reactive diluents in thermally cured solvent-borne coil-coatings. The purpose was to evaluate the effect of fatty acid methyl ester structure on the curing performance and final properties of the coating. The permanent incorporation of the reactive diluent via transesterification reaction has been followed with ¹H NMR analysis of model systems. Dynamic mechanical analysis (DMA) measurements, of free-standing films, show that the glass transition temperature (T_g) decreases upon addition of the reactive diluent. Both the amount of incorporated reactive diluent and the final film properties are affected by the number and placement of alkene-bonds in the FAME.     

Application of Dynamic and Static Light Scattering for Size and Shape Characterization of Small Extracellular Nanoparticles in Plasma and Ascites of Ovarian Cancer Patients

In parallel to medical treatment of ovarian cancer, methods for the early detection of cancer tumors are being sought. In this contribution, the use of non-invasive static (SLS) and dynamic light scattering (DLS) for the characterization of extracellular nanoparticles (ENPs) in body fluids of advanced serous ovarian cancer (OC) and benign gynecological pathology (BP) patients is demonstrated and critically evaluated. Samples of plasma and ascites (OC patients) or plasma, peritoneal fluid, and peritoneal washing (BP patients) were analyzed. The hydrodynamic radius (R_h) and the radius of gyration (R_g) of ENPs were calculated from the angular dependency of LS intensity for two ENP subpopulations. R_h and R_g of the predominant ENP population of OC patients were in the range 20–30 nm (diameter 40–60 nm). In thawed samples, larger particles (R_h mostly above 100 nm) were detected as well. The shape parameter ρ of both particle populations was around 1, which is typical for spherical particles with mass concentrated on the rim, as in vesicles. The R_h and R_g of ENPs in BP patients were larger than in OC patients, with ρ ≈ 1.1–2, implying a more elongated/distorted shape. These results show that SLS and DLS are promising methods for the analysis of morphological features of ENPs and have the potential to discriminate between OC and BP patients. However, further development of the methodology is required.