High levels of acute phase proteins and soluble 70 kDa heat shock proteins are independent and additive risk factors for mortality in colorectal cancer

Recently, we reported that high soluble Hsp70 (sHsp70) level was a significant predictor of mortality during an almost 3-year-long follow-up period in patients with colorectal cancer. This association was the strongest in the group of <70-year-old female patients as well as in those who were in a less advanced stage of the disease at baseline. According to these observations, measurement of the serum level of sHsp70 is a useful, stage-independent prognostic marker in colorectal cancer, especially in patients without distant metastasis. Since many literature data indicated that measurement of C-reactive protein (CRP) and other acute phase proteins (APPs) may also be suitable for predicting the mortality of patients with colorectal cancer, it seemed reasonable to study whether the effect of sHsp70 and other APPs are related or independent. In order to answer this question, we measured the concentrations of CRP as well as of other complement-related APPs (C1 inhibitor, C3, and C9) along with that of the MASP-2 complement component in the sera of 175 patients with colorectal cancer and known levels of sHsp70, which have been used in our previous study. High (above median) levels of CRP, C1 esterase inhibitor (C1-INH), and sHsp70 were found to be independently associated with poor patient survival, whereas no such association was observed with the other proteins tested. According to the adjusted Cox proportional hazards analysis, the additive effect of high sHsp70, CRP, and C1-INH levels on the survival of patients exceeded that of high sHsp70 alone, with a hazard ratio (HR) of 2.83 (1.13–70.9). In some subgroups of patients, such as in females [HR 4.80 (1.07–21.60)] or in ≤70-year-old patients [HR 11.53 (2.78–47.70)], even greater differences were obtained. These findings indicate that the clinical mortality–prediction value of combined measurements of sHsp70, CRP, and C1-INH with inexpensive methods can be very high, especially in specific subgroups of patients with colorectal cancer.     

ABC transporters affect the detection of intracellular oxidants by fluorescent probes

Intracellular production of reactive oxygen species (ROS) plays an important role in the control of cell physiology. For the assessment of intracellular ROS production, a plethora of fluorescent probes is commonly used. Interestingly, chemical structures of these probes imply they could be substrates of plasma membrane efflux pumps, called ABC transporters. This study tested whether the determination of intracellular ROS production and mitochondrial membrane potential by selected fluorescent probes is modulated by the expression and activity of ABC transporters. The sub-clones of the HL-60 cell line over-expressing MDR1, MRP1 and BCRP transporters were employed. ROS production measured by luminol- and L-012-enhaced chemiluminescence and cytochrome c reduction assay showed similar levels of ROS production in all the employed cell lines. It was proved that dihydrorhodamine 123, dihexiloxocarbocyanine iodide, hydroethidine, tetrachloro-tetraethylbenzimidazolocarbo-cyanine iodide and tetramethylrhodamine ethyl ester perchlorate are substrates for MDR1; dichlorodihydrofluoresceine, hydroethidine and tetramethylrhodamine ethyl ester perchlorate are substrates for MRP1; dichlorodihydrofluoresceine, dihydrorhodamine 123, hydroethidine and tetrachloro-tetraethylbenzimidazolocarbo-cyanine iodide are substrates for BCRP. Thus, the determination of intracellular ROS and mitochondrial potential by the selected probes is significantly altered by ABC transporter activities. The activity of these transporters must be considered when employing fluorescent probes for the assessment of ROS production or mitochondrial membrane potential.     

Drug localization in different lung cancer phenotypes by MALDI mass spectrometry imaging

Lung cancer is a common cause of cancer mortality in the world, largely due to the risk factor of tobacco smoking. The drug therapy at the molecular level includes targeting the epidermal growth factor receptor (EGFR) tyrosine kinase activity by using inhibitors, such as erlotinib (Tarceva) and gefitinib (Iressa). The heterogeneity of disease phenotypes and the somatic mutations presented in patient populations have a great impact on the efficacy of treatments using targeted personalized medicine. In this study, we report on basic physical and chemical properties of erlotinib and gefitinib in three different lung cancer tumor phenotypes, using MALDI instrumentation in imaging mode, providing spatial localization of drugs without chemical labeling. Erlotinib and gefitinib were analyzed in i) planocellular lung carcinoma, ii) adenocarcinoma and iii) large cell lung carcinoma following their deposition on the tissue surfaces by piezo-dispensing, using a controlled procedure. The importance of high-resolution sampling was crucial in order to accurately localize the EGFR tyrosine kinase inhibitors deposited in heterogeneous cancer tissue compartments. This is the first report on personalized drug characterization with localizations at a lateral resolution of 30μm, which allowed us to map these compounds at attomolar concentrations within the lung tumor tissue microenvironments.     

Construction of a xylanase A variant capable of polymerization

The aim of our work is to furnish enzymes with polymerization ability by creating fusion constructs with the polymerizable protein, flagellin, the main component of bacterial flagellar filaments. The D3 domain of flagellin, exposed on the surface of flagellar filaments, is formed by the hypervariable central portion of the polypeptide chain. D3 is not essential for filament formation. The concept in this project is to replace the D3 domain with suitable monomeric enzymes without adversely affecting polymerization ability, and to assemble these chimeric flagellins into tubular nanostructures. To test the feasibility of this approach, xylanase A (XynA) from B. subtilis was chosen as a model enzyme for insertion into the central part of flagellin. With the help of genetic engineering, a fusion construct was created in which the D3 domain was replaced by XynA. The flagellin-XynA chimera exhibited catalytic activity as well as polymerization ability. These results demonstrate that polymerization ability can be introduced into various proteins, and building blocks for rationally designed assembly of filamentous nanostructures can be created.     

Dysregulation of proinflammatory and regulatory cytokines in HIV infected persons with active tuberculosis

Proinflammatory and regulatory cytokines have been implicated to play important role in immunopathology of HIV and tuberculosis (TB) infection. Capacity of unstimulated and mitogen-stimulated peripheral blood mononuclear cells (PBMCs) to secrete cytokines (interleukin (IL)-2, interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), IL-4, IL-10 and IL-6) was estimated for 15 HIV-TB coinfected patients, 22 HIV seropositives without TB, 32 HIV negative TB patients, and 36 healthy subjects. Dually infected patients had suppression of both Th1 and Th2 cytokine secretion as evidenced by significantly lower production of IL-2, IFN-gamma and TNF-alpha as well as IL-4 and IL-10. Production of IL-2 and TNF-alpha was significantly decreased only in case of HIV infection. Significantly higher IL-6 secretion was found in unstimulated cultures in dually infected patients. The mitogen induced cytokine secretion was generally lower in HIV-TB coinfected patients indicating profound perturbation of both Th1 and Th2 responses.     

Synthesis and evaluation of anti-HIV activity of isatin beta-thiosemicarbazone derivatives

On the basis of pharmacophoric modelling studies of existing NNRTIs, a series of isatin beta-thiosemicarbazone derivatives was synthesized and evaluated for their anti-HIV activity in HTLV-III(B) strain in the CEM cell line. Three compounds showed significant anti-HIV activity, whereupon compound 6 was found to be the most active compound with an EC(50) value of 2.62 microM and a selectivity index of 17.41, while not being cytotoxic to the cell line at a CC(50) value of 44.90 microM. Other tested compounds exhibited marked activity below their toxicity threshold.     

A simple model for the cooperative stabilisation of actin filaments by phalloidin and jasplakinolide

The stabilisation of magnesium actin filaments by phalloidin and jasplakinolide was studied using the method of differential scanning calorimetry. The results showed that actin could adapt three conformations in the presence of drugs. One conformation was adapted in direct interaction with the drug, while another conformation was identical to that observed in the absence of drugs. A third conformation was induced through allosteric inter-protomer interactions. The effect of both drugs propagated cooperatively along the actin filaments. The number of the cooperative units determined by using a quantitative model was larger for jasplakinolide (15 actin protomers) than for phalloidin (7 protomers).