Quercetin acts via the G3BP1/YWHAZ axis to inhibit glycolysis and proliferation in oral squamous cell carcinoma.

There is increasing evidence that the GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) plays important roles in the formation of various tumors. However, the biological functions and mechanism of G3BP1 in promoting the progression of oral squamous cell carcinoma remain largely unknown. The impacts of quercetin on glycolysis and proliferation of the CAL27 oral squamous cell carcinoma line were investigated, and the mediating role of the G3BP1/YWHAZ pathway was explored. CAL27 cells stably over- or underexpressing G3BP1 were treated with quercetin, and then cell proliferation was assayed together with the expression of proteins involved in glucose uptake, glycolysis, and lactate production, as well as the activity of hexokinase, pyruvate kinase, and lactate dehydrogenase. CAL27 cells expressed G3BP1 and YWHAZ at significantly higher levels than normal oral squamous cells. CAL27 cells showed the highest expression of both proteins among the three carcinoma lines (TSCCA, SCC15, 42 CAL27). Overexpressing G3BP1 in CAL27 cells markedly induced glucose uptake, glycolysis, cell proliferation, and YWHAZ expression. Knocking down G3BP1 or YWHAZ exerted the opposite effects, which were similar to the effects of inhibiting glycolysis. Quercetin repressed glucose uptake, glycolysis, cell proliferation, and G3BP1/YWHAZ signaling in a dose-dependent way, and these effects were antagonized by G3BP1 overexpression. Quercetin can inhibit glycolysis and cell proliferation of oral squamous cell carcinoma, apparently by inhibiting the G3BP1/YWHAZ axis.

Cytochrome P450 3A5 polymorphism affects the metabolism of sorafenib and its toxicity for hepatocellular carcinoma cells in vitro.

OBJECTIVE: Cytochrome P450 3A5 (CYP3A5) is a highly polymorphic gene and the encoded protein variants differ in catalytic activity, leading to inter-individual variation in metabolic ability. The aim of the current study was to investigate the effects of seven allelic variants on the ability of CYP3A5 to metabolize sorafenib in vitro and further explore the impacts of CYP3A5 polymorphism on the proliferation and apoptosis of hepatocellular carcinoma cell line (HepG2) induced by sorafenib. METHODS: Wild-type and variant CYP3A5 enzymes were expressed in Spodoptera frugiperda insect cells using a baculovirus dual-expression system, and protein expression was checked by western blot. The enzymes were incubated with sorafenib at 37°C for 30 min, and formation of the major metabolite sorafenib N-oxide was assayed using ultra-performance liquid chromatography and tandem mass spectrometry. Intrinsic clearance values (Vmax/Km) were calculated for each enzyme. Additionally, recombinant HepG2 cells transfecting with CYP3A5 variants were used to investigate the effects of sorafenib on the proliferation of HepG2 cells. RESULTS: Intrinsic clearance of the six variants CYP3A5*2, CYP3A5*3A, CYP3A5*3C, CYP3A5*4, CYP3A5*5, and CYP3A5*7 was 26.41-71.04% of the wild-type (CYP3A5*1) value. In contrast, the clearance value of the variant CYP3A5*6 was significantly higher (174.74%). Additionally, the decreased ATP levels and cell viability and the increased cell apoptosis in HepG2 cells transfected with CYP3A5*2, CYP3A5*3A, CYP3A5*3C, CYP3A5*4, CYP3A5*5, and CYP3A5*7 were observed, whereas, the increased ATP levels and cell viability and the reduced cell apoptosis in HepG2 cells transfected with CYP3A5*6 were also investigated when compared to CYP3A5*1. CONCLUSION: Our results suggest that CYP3A5 polymorphism influences sorafenib metabolism and pharmacotherapeutic effect in hepatic carcinomas. These data may help explain differential response to drug therapy for hepatocellular carcinoma, and they support the need for individualized treatment.

Cytochrome P450 3A5 polymorphism affects the metabolism of sorafenib and its toxicity for hepatocellular carcinoma cells in vitro.

OBJECTIVE: Cytochrome P450 3A5 (CYP3A5) is a highly polymorphic gene and the encoded protein variants differ in catalytic activity, leading to inter-individual variation in metabolic ability. The aim of the current study was to investigate the effects of seven allelic variants on the ability of CYP3A5 to metabolize sorafenib in vitro and further explore the impacts of CYP3A5 polymorphism on the proliferation and apoptosis of hepatocellular carcinoma cell line (HepG2) induced by sorafenib. METHODS: Wild-type and variant CYP3A5 enzymes were expressed in Spodoptera frugiperda insect cells using a baculovirus dual-expression system, and protein expression was checked by western blot. The enzymes were incubated with sorafenib at 37°C for 30 min, and formation of the major metabolite sorafenib N-oxide was assayed using ultra-performance liquid chromatography and tandem mass spectrometry. Intrinsic clearance values (Vmax/Km) were calculated for each enzyme. Additionally, recombinant HepG2 cells transfecting with CYP3A5 variants were used to investigate the effects of sorafenib on the proliferation of HepG2 cells. RESULTS: Intrinsic clearance of the six variants CYP3A5*2, CYP3A5*3A, CYP3A5*3C, CYP3A5*4, CYP3A5*5, and CYP3A5*7 was 26.41-71.04% of the wild-type (CYP3A5*1) value. In contrast, the clearance value of the variant CYP3A5*6 was significantly higher (174.74%). Additionally, the decreased ATP levels and cell viability and the increased cell apoptosis in HepG2 cells transfected with CYP3A5*2, CYP3A5*3A, CYP3A5*3C, CYP3A5*4, CYP3A5*5, and CYP3A5*7 were observed, whereas, the increased ATP levels and cell viability and the reduced cell apoptosis in HepG2 cells transfected with CYP3A5*6 were also investigated when compared to CYP3A5*1. CONCLUSION: Our results suggest that CYP3A5 polymorphism influences sorafenib metabolism and pharmacotherapeutic effect in hepatic carcinomas. These data may help explain differential response to drug therapy for hepatocellular carcinoma, and they support the need for individualized treatment.

Pathological and ultrastructural observations and liver function analysis of Eimeria stiedai-infected rabbits.

To study the pathogenicity of Eimeria stiedai, sporulated oocysts were given orally to coccidian-free two-month-old New Zealand rabbits(1000±20g). After 30days, blood samples from the rabbit hearts were collected for routine blood tests, liver functions and four characteristics of blood coagulation. Additionally, specimens of the liver, bile duct and duodenum were collected to observe the changes in pathology and ultrastructure. E. stiedai severely restricted the growth and development of rabbits. Blood tests showed that glutamine transferase (GGT) and serum cholinesterase (ChE) were significantly different from the non-infected controls. Other extremely significant differences were observed in the biochemical indices of routine blood tests, liver function and four blood coagulation characteristics, indicating that the liver functions were significantly affected. Staining showed that, compared with the negative control group, the liver, bile duct and duodenum contained significant numbers of lesions, and organs and cell structures suffered severe damage in ultrastructure, which greatly affecting bodily functions. E. stiedai-infected rabbits model was successfully established, which might provide a theoretical basis for research on the pathogenesis of rabbit coccidia, and the diagnosis and prevention of coccidiosis in rabbits.

Surface Plasmon Resonance Study of Cooperative Interactions of Estrogen Receptor α and Specificity Protein 1 with Composite DNA Elements.

Estrogen receptor α (ERα) and Specificity protein 1 (Sp1) are transcription factors (TF) that are involved in regulating progesterone receptor (PR) gene expression through cooperative interactions with DNA. The natural composite DNA +571 ERE/Sp1 site in promoter A of the progesterone receptor contains a half-site of estrogen response elements (½ERE) upstream of two Sp1 binding sites (the proximal Sp1 (Sp1/P) and distal Sp1 (Sp1/D)) with a 4 bp spacer. Here, we have developed a protocol for studying the cooperative interaction of Sp1 and ERα with the composite DNA of +571 ERE/Sp1 site using Biacore T200, a high sensitivity surface plasmon resonance spectroscopy. With this protocol, we have concluded that Sp1 binding enhances the overall ERα binding to the composite DNA. We have also determined the optimal spacer distance between the ½ERE and Sp1/D for the best cooperative protein binding. This study is pivotal in guiding the bioinformatics simulation to yield an exact model of the spacer dependency of the transcription factor/cofactor-DNA interactions, which is important for understanding the nuclear receptor regulating activity through other coactivators.

The effect of SIRT1 gene polymorphisms on ambulatory blood pressure of hypertensive patients in the Kazakh population.

OBJECTIVE: To investigate the effect of silent information regulator 1 (SIRT1) gene polymorphisms on ambulatory blood pressure in hypertensive patients. METHODS: Three hundred forty hypertensive patients were recruited from January 2013 to January 2015. SIRT1 Tag single-nucleotide polymorphisms (SNPs; rs2273773, rs4746720, and rs7896005) were genotyped using a PCR-direct sequencing method, and the association between the SIRT1 gene SNPs and ambulatory blood pressure was analyzed. RESULTS: After adjusting for confounding factors, patients with the rs2273773/CT+CC genotypes had lower 24-h systolic and diastolic blood pressures; there were no associations between rs4746720 and rs7896005 genotypes and blood pressure. CONCLUSION: The SIRT1 gene polymorphism (rs2273773) is significantly associated with ambulatory blood pressure level in Han Chinese patients with hypertension.

Identification of causative pathogens in mouse eyes with bacterial keratitis by sequence analysis of 16S rDNA libraries.

The clone library method using PCR amplification of the 16S ribosomal RNA (rRNA) gene was used to identify pathogens from corneal scrapings of C57BL/6-corneal opacity (B6-Co) mice with bacterial keratitis. All 10 samples from the eyes with bacterial keratitis showed positive PCR results. All 10 samples from the normal cornea showed negative PCR results. In all 10 PCR-positive samples, the predominant and second most predominant species accounted for 20.9 to 40.6% and 14.7 to 26.1%, respectively, of each clone library. The predominant species were Staphylococcus lentus, Pseudomonas aeruginosa, and Staphylococcus epidermidis. The microbiota analysis detected a diverse group of microbiota in the eyes of B6-Co mice with bacterial keratitis and showed that the causative pathogens could be determined based on percentages of bacterial species in the clone libraries. The bacterial species detected in this study were mostly in accordance with results of studies on clinical bacterial keratitis in human eyes. Based on the results of our previous studies and this study, the B6-Co mouse should be considered a favorable model for studying bacterial keratitis.

Imprinted gold 2D nanoarray for highly sensitive and convenient PSA detection via plasmon excited quantum dots.

We designed and fabricated two new nanostructured biosensing chips, with which the sensitive detection of prostate specific antigen (PSA) as low as 100 pg ml(-1) can be achieved, by measuring the plasmon enhanced fluorescence through a conventional dark field microscope. The gold nanostructure arrays, one with gold nanopillars of 140 nm, the other with gold nanoholes of 140 nm, were fabricated via nanoimprinting onto glass substrate, as localized surface plasmon resonance (LSPR) generators to enhance the fluorescent emission of fluorophore, e.g. quantum dot (QD). A sandwich bioassay of capture anti-PSA antibody (cAb)/PSA/detection anti-PSA (dAb) labeled by QD-655 was established on the nanostructures, and the perfect LSPR excitation distance (10-15 nm) between the nanostructure and QD-655 was simulated and controlled by a cleft cAb fragment and streptavidin modified QD. QD was chosen in this study due to its photo stability, broad Stokes shift, and long lifetime. As far as we know, this is the first time that QD is applied for PSA detection on the uniform nanostructured sensing chips based on the LSPR enhanced fluorescence. Due to the miniaturized nanoarray sensing chip (1.8 mm × 1.8 mm), the convenience and specificity for the detection of PSA via the sandwich assay, and the high optical detection sensitivity, the platform has great potential for the development of a portable point-of-care (POC) system for outpatient diagnosis and treatment monitoring.

Transient absorption spectroscopy on spiropyran monolayers using nanosecond pump-probe Brewster angle reflectometry.

Self-assembled monolayers of 11-(3',3'-dimethyl-6,8-dinitrospiro[chromene-2,2'-indoline]-1'-yl) undecanoic acid (amphiphilic spiropyran) at the air-water interface are studied using Brewster angle reflectometry. Transient kinetics of the spiropyran to merocyanine conversion are recorded in a UV-pump, VIS-probe configuration. By varying the probe wavelength using an optical parametric oscillator, we are able to reconstruct absorption spectra of intermediate states with a time-resolution of 10 nanoseconds, limited by the temporal convolution of the two laser pulses. After UV irradiation, spiropyran converts to merocyanine in two stages. The first occurs within a timescale of several tens of nanoseconds and is heavily convoluted with the system response time, whereas the second stage occurs over a few hundred nanoseconds. During the rise time there is a small red shift in the transient absorption spectrum of ~20 nm. We assign the red shift and the slower kinetics to the isomerization of a merocyanine isomer cis about the central methine bond to those that are trans about the same bond.

Study sequence rules of estrogen receptor α-DNA interactions using dual polarization interferometry and computational modeling.

Estrogen receptor α (ERα) is a ligand-activated transcription factor. In a classical model, ERα regulates gene expression by binding to DNA sequences called estrogen response elements (EREs). A perfect ERE contains a palindromic consensus sequence of 5'-GGTCAnnnTGACC-3'. A slight variation in ERE sequence alters ERα binding affinity and, thus, the gene transcription activity. In this study, all possible singly mutated EREs of 15 sequences (three possible base substitutions at each of one to five positions of one half-site) were created. Dual polarization interferometry (DPI) was used to measure the receptor binding to generate an in vitro binding energy model. A motif discovery algorithm, Thermodynamic Modeling of ChIP-seq (TherMos), was used to compute the binding energy model from in vivo genome-wide ERα binding data. The in vitro affinity model measured by DPI correlates very well with the TherMos prediction (in vivo model), with a rank correlation coefficient of 0.91, which indicates that the DPI-determined model is reliable and powerful in understanding of ERα binding in vivo in the whole genome. This is the first report of DPI study of protein-double-stranded DNA (dsDNA) interactions. The assay protocols developed are efficient for screening a large quantity of DNA sequences with single base variation sensitivity.

Comparative study of random and oriented antibody immobilization as measured by dual polarization interferometry and surface plasmon resonance spectroscopy.

Dual polarization interferometry (DPI) is used for a detailed study of antibody immobilization with and without orientation control, using prostate specific antigen (PSA) and its antibody as model. Thiol modified DPI chips were activated by a heterobifunctional cross-linker (sulfo-GMBS). PSA antibody was either directly immobilized via covalent binding or coupled via the Fc-fragment to protein G covalently attached to the activated chip. The direct covalent binding leads to a random antibody orientation and the coupling through protein G leads to an end-on orientation. Ethanolamine (ETH) was used to block remaining active sites following the direct antibody immobilization and protein G immobilization. A homobifunctional cross-linker (BS3) was used to stabilize the antibody layer coupled on protein G. DPI provides a real-time measurement of the stepwise molecular binding processes and gives detailed geometrical and structural values of each layer, i.e., thickness, mass, and density. These values evidence the end-on orientation of closely packed antibody on protein G layer and reveal structural effects of ETH blocking/deactivation and BS3 stabilization. With the end-on immobilized antibody, PSA at 10 pg/mL can be detected by DPI through a sandwich complex that satisfies the clinical requirement (assuming <30 pg/mL as clinically safe). However, the randomly immobilized antibody failed to detect PSA at 1 ng/mL. In a parallel study using surface plasmon resonance (SPR) spectroscopy, random and end-on antibody immobilization on streptavidin-modified gold surface was evaluated to further validate the importance of antibody orientation control. With the closely packed antibody layer on protein G surface, SPR can also detect PSA at 10 pg/mL.

Electrophysiological effects of haloperidol on isolated rabbit Purkinje fibers and guinea pigs papillary muscles under normal and simulated ischemia.

AIM: Overdoses of haloperidol are associated with major ventricular arrhythmias, cardiac conduction block, and sudden death. The aim of this experiment was to study the effect of haloperidol on the action potentials in cardiac Purkinje fibers and papillary muscles under normal and simulated ischemia conditions in rabbits and guinea pigs. METHODS: Using the standard intracellular microelectrode technique, we examined the effects of haloperidol on the action potential parameters [action potential amplitude (APA), phase 0 maximum upstroke velocity (V(max)), action potential amplitude at 90% of repolarization (APD(90)), and effective refractory period (ERP)] in rabbit cardiac Purkinje fibers and guinea pig cardiac papillary cells, in which both tissues were under simulated ischemic conditions. RESULTS: Under ischemic conditions, different concentrations of haloperidol depressed APA and prolonged APD(90) in a concentration-dependent manner in rabbit Purkinje fibers. Haloperidol (3 micromol/L) significantly depressed APA and prolonged APD(90), and from 1 micromol/L, haloperidol showed significant depression on V(max); ERP was not significantly affected. In guinea pig cardiac papillary muscles, the thresholds of significant reduction in APA, V(max), EPR, and APD(90) were 10, 0.3, 1, and 1 mumol/L, respectively, for haloperidol. CONCLUSION: Compared with cardiac conductive tissues, papillary muscles were more sensitive to ischemic conditions. Under ischemia, haloperidol prolonged ERP and APD(90) in a concentration-dependent manner and precipitated the decrease in V(max) induced by ischemia. The shortening of ERP and APD(90) in papillary muscle action potentials may be inhibited by haloperidol.

SIRT1 modulating compounds from high-throughput screening as anti-inflammatory and insulin-sensitizing agents.

The nicotinamide adenine dinucleotide (NAD(+))-dependent protein deacetylase SIRT1 has been linked to fatty acid metabolism via suppression of peroxysome proliferator-activated receptor gamma (PPAR-gamma) and to inflammatory processes by deacetylating the transcription factor NF-kappaB. First, modulation of SIRT1 activity affects lipid accumulation in adipocytes, which has an impact on the etiology of a variety of human metabolic diseases such as obesity and insulin-resistant diabetes. Second, activation of SIRT1 suppresses inflammation via regulation of cytokine expression. Using high-throughput screening, the authors identified compounds with SIRT1 activating and inhibiting potential. The biological activity of these SIRT1-modulating compounds was confirmed in cell-based assays using mouse adipocytes, as well as human THP-1 monocytes. SIRT1 activators were found to be potent lipolytic agents, reducing the overall lipid content of fully differentiated NIH L1 adipocytes. In addition, the same compounds have anti-inflammatory properties, as became evident by the reduction of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). In contrast, a SIRT1 inhibitory compound showed a stimulatory activity on the differentiation of adipocytes, a feature often linked to insulin sensitization.

Nickel-catalyzed homoallylation reaction of aldehydes with 1,3-dienes: stereochemical and mechanistic studies.

[reaction: see text] Contrary to what was reported, the coupling reaction of nickel-catalyzed cyclic diene such as cyclohexadiene with carbonyl compounds in the presence of diethyl zinc afforded gamma,delta-alkenyl alcohols in good yields.