Erythropoietin Receptor Expression Is a Potential Prognostic Factor in Human Lung Adenocarcinoma

Recombinant human erythropoietins (rHuEPOs) are used to treat cancer-related anemia. Recent preclinical studies and clinical trials, however, have raised concerns about the potential tumor-promoting effects of these drugs. Because the clinical significance of erythropoietin receptor (EPOR) signaling in human non-small cell lung cancer (NSCLC) also remains controversial, our aim was to study whether EPO treatment modifies tumor growth and if EPOR expression has an impact on the clinical behavior of this malignancy. A total of 43 patients with stage III–IV adenocarcinoma (ADC) and complete clinicopathological data were included. EPOR expression in human ADC samples and cell lines was measured by quantitative real-time polymerase chain reaction. Effects of exogenous rHuEPOα were studied on human lung ADC cell lines in vitro. In vivo growth of human ADC xenografts treated with rHuEPOα with or without chemotherapy was also assessed. In vivo tumor and endothelial cell (EC) proliferation was determined by 5-bromo-2’-deoxy-uridine (BrdU) incorporation and immunofluorescent labeling. Although EPOR mRNA was expressed in all of the three investigated ADC cell lines, rHuEPOα treatment (either alone or in combination with gemcitabine) did not alter ADC cell proliferation in vitro. However, rHuEPOα significantly decreased tumor cell proliferation and growth of human H1975 lung ADC xenografts. At the same time, rHuEPOα treatment of H1975 tumors resulted in accelerated tumor endothelial cell proliferation. Moreover, in patients with advanced stage lung ADC, high intratumoral EPOR mRNA levels were associated with significantly increased overall survival. This study reveals high EPOR level as a potential novel positive prognostic marker in human lung ADC.     

Accelerated Activation of SOCE Current in Myotubes from Two Mouse Models of Anesthetic- and Heat-Induced Sudden Death

Store-operated calcium entry (SOCE) channels play an important role in Ca²⁺ signaling. Recently, excessive SOCE was proposed to play a central role in the pathogenesis of malignant hyperthermia (MH), a pharmacogenic disorder of skeletal muscle. We tested this hypothesis by characterizing SOCE current (I_SkCRAC) magnitude, voltage dependence, and rate of activation in myotubes derived from two mouse models of anesthetic- and heat-induced sudden death: 1) type 1 ryanodine receptor (RyR1) knock-in mice (Y524S/+) and 2) calsequestrin 1 and 2 double knock-out (dCasq-null) mice. I_SkCRAC voltage dependence and magnitude at -80 mV were not significantly different in myotubes derived from wild type (WT), Y524S/+ and dCasq-null mice. However, the rate of I_SkCRAC activation upon repetitive depolarization was significantly faster at room temperature in myotubes from Y524S/+ and dCasq-null mice. In addition, the maximum rate of I_SkCRAC activation in dCasq-null myotubes was also faster than WT at more physiological temperatures (35-37°C). Azumolene (50 µM), a more water-soluble analog of dantrolene that is used to reverse MH crises, failed to alter I_SkCRAC density or rate of activation. Together, these results indicate that while an increased rate of I_SkCRAC activation is a common characteristic of myotubes derived from Y524S/+ and dCasq-null mice and that the protective effects of azumolene are not due to a direct inhibition of SOCE channels.     

Membrane protein shaving with thermolysin can be used to evaluate topology predictors

Topology analysis of membrane proteins can be obtained by enzymatic shaving in combination with MS identification of peptides. Ideally, such analysis could provide quite detailed information about the membrane spanning regions. Here, we examine the ability of some shaving enzymes to provide large‐scale analysis of membrane proteome topologies. To compare different shaving enzymes, we first analyzed the detected peptides from two over‐expressed proteins. Second, we analyzed the peptides from non‐over‐expressed Escherichia coli membrane proteins with known structure to evaluate the shaving methods. Finally, the identified peptides were used to test the accuracy of a number of topology predictors. At the end we suggest that the usage of thermolysin, an enzyme working at the natural pH of the cell for membrane shaving, is superior because: (i) we detect a similar number of peptides and proteins using thermolysin and trypsin; (ii) thermolysin shaving can be run at a natural pH and (iii) the incubation time is quite short. (iv) Fewer detected peptides from thermolysin shaving originate from the transmembrane regions. Using thermolysin shaving we can also provide a clear separation between the best and the less accurate topology predictors, indicating that using data from shaving can provide valuable information when developing new topology predictors.     

Highly Sensitive,Highly Specific Whole-Cell Bioreporters for the Detection of Chromate in Environmental Samples

Microbial bioreporters offer excellent potentialities for the detection of the bioavailable portion of pollutants in contaminated environments, which currently cannot be easily measured. This paper describes the construction and evaluation of two microbial bioreporters designed to detect the bioavailable chromate in contaminated water samples. The developed bioreporters are based on the expression of gfp under the control of the chr promoter and the chrB regulator gene of TnOtChr determinant from Ochrobactrum tritici 5bvl1. pCHRGFP1 Escherichia coli reporter proved to be specific and sensitive, with minimum detectable concentration of 100 nM chromate and did not react with other heavy metals or chemical compounds analysed. In order to have a bioreporter able to be used under different environmental toxics, O. tritici type strain was also engineered to fluoresce in the presence of micromolar levels of chromate and showed to be as specific as the first reporter. Their applicability on environmental samples (spiked Portuguese river water) was also demonstrated using either freshly grown or cryo-preserved cells, a treatment which constitutes an operational advantage. These reporter strains can provide on-demand usability in the field and in a near future may become a powerful tool in identification of chromate-contaminated sites.     

The Natural Product Magnolol as a Lead Structure for the Development of Potent Cannabinoid Receptor Agonists

Magnolol (4-allyl-2-(5-allyl-2-hydroxyphenyl)phenol), the main bioactive constituent of the medicinal plant Magnolia officinalis, and its main metabolite tetrahydromagnolol were recently found to activate cannabinoid (CB) receptors. We now investigated the structure-activity relationships of (tetrahydro)magnolol analogs with variations of the alkyl chains and the phenolic groups and could considerably improve potency. Among the most potent compounds were the dual CB₁/CB₂ full agonist 2-(2-methoxy-5-propyl-phenyl)-4-hexylphenol (61a, K _i CB₁∶0.00957 µM; K _i CB₂∶0.0238 µM), and the CB₂-selective partial agonist 2-(2-hydroxy-5-propylphenyl)-4-pentylphenol (60, K _i CB₁∶0.362 µM; K _i CB₂∶0.0371 µM), which showed high selectivity versus GPR18 and GPR55. Compound 61b, an isomer of 61a, was the most potent GPR55 antagonist with an IC₅₀ value of 3.25 µM but was non-selective. The relatively simple structures, which possess no stereocenters, are easily accessible in a four- to five-step synthetic procedure from common starting materials. The central reaction step is the well-elaborated Suzuki-Miyaura cross-coupling reaction, which is suitable for a combinatorial chemistry approach. The scaffold is versatile and may be fine-tuned to obtain a broad range of receptor affinities, selectivities and efficacies.     

Pristine C60 Fullerene Nanoparticles Ameliorate Hyperglycemia-Induced Disturbances via Modulation of Apoptosis and Autophagy Flux

Neurochemical Research - Diabetes mellitus is a prevalent metabolic disorder associated with multiple complications including neuropathy, memory loss and cognitive decline. Despite a long history...     

Host genetic risk factors for community-acquired pneumonia

This study was conducted to establish the contribution of genetic host factors in the susceptibility to community acquired pneumonia (CAP) in the Russian population. Patients with CAP (n = 334), volunteers without a previous history of CAP, constantly exposed to infectious agents, control A group (n = 141) and a second control group B consisted of healthy persons (n = 314) were included in the study. All subjects were genotyped for 13 polymorphic variants in the genes of xenobiotics detoxification CYP1A1 (rs2606345, rs4646903, and rs1048943), GSTM1 (Ins/del), GSTT1 (Ins/del), ABCB1 rs1045642); immune and inflammation response IL-6 (rs1800795), TNF-a (rs1800629), MBL2 (rs7096206), CCR5 (rs333), NOS3 (rs1799983), angiotensin-converting enzyme ACE (rs4340), and occlusive vascular disease/hyperhomocysteinemia MTHFR (rs1801133). Seven polymorphic variants in genes CYP1A1, GSTM1, ABCB1, NOS3, IL6, CCR5 and ACE were associated with CAP. For two genes CYP1A1 and GSTM1 associations remained significant after correction for multiple comparisons. Multiple analysis by the number of all risk genotypes showed a highly significant association with CAP (P = 2.4 × 10^− 7, OR = 3.03, 95% CI 1.98–4.64) with the threshold for three risk genotypes. Using the ROC-analysis, the AUC value for multi-locus model was estimated as 68.38.     

Melting of Cross-Linked DNA V. Cross-Linking Effect Caused by Local Stabilization of the Double Helix

Abstract

DNA interstrand cross-links are usually formed due to bidentate covalent or coordination binding of a cross-linking agent to nucleotides of different strands. However interstrand linkages can be also caused by any type of chemical modification that gives rise to a strong local stabilization of the double helix. These stabilized sites conserve their helical structure and prevent local and total strand separation at temperatures above the melting of ordinary AT and GC base pairs. This local stabilization makes DNA melting fully reversible and independent of strand concentration like ordinary covalent interstrand cross-links. The stabilization can be caused by all the types of chemical modifications (interstrand cross-links, intrastrand cross-links or monofunctional adducts) if they give rise to a strong enough local stabilization of the double helix. Our calculation demonstrates that an increase in stability by 25 to 30 kcal in the free energy of a single base pair of the double helix is sufficient for this “cross-linking effect” (i.e. conserving the helicity of this base pair and preventing strand separation after melting of ordinary base pairs). For the situation where there is more then one stabilized site in a DNA duplex (e.g., 1 stabilized site per 1000 bp), a lower stabilization per site is sufficient for the “cross-linking effect” (18–20 kcal). A substantial increase in DNA stability was found in various experimental studies for some metal-based anti-tumor compounds. These compounds may give rise to the effect described above. If ligand induced stabilization is distributed among several neighboring base pairs, a much lower minimum increase per stabilized base pair is sufficient to produce the cross-linking effect (1 bp- 24.4 kcal; 5 bp- 5.3 kcal; 10 bp- 2.9 kcal, 25 bp- 1.4 kcal; 50 bp- 1.0 kcal). The relatively weak non-covalent binding of histones or protamines that cover long regions of DNA (20–40 bp) can also cause this effect if the salt concentration of the solution is sufficiently low to cause strong local stabilization of the double helix. Stretches of GC pairs more than 25 bp in length inserted into poly(AT) DNA also exhibit properties of stabilizing interstrand cross-links.