Thromboxane synthase suppression induces lung cancer cell apoptosis via inhibiting NF-κB

Accumulating evidence shows that the inhibition of thromboxane synthase (TXS) induced apoptosis in cancer cells. TXS inhibitor 1-Benzylimidzole (1-BI) can trigger apoptosis in lung cancer cells but the mechanism is not fully defined. In this study, lung cancer cells were treated with 1-BI. In this study, the level of reactive oxygen species (ROS) was measured and NF-κB activity was determined in human lung cancer cells. The roles of ROS and NF-κB in 1-BI-mediated cell death were analyzed. The results showed that 1-BI induced ROS generation but decreased the activity of NF-κB by reducing phosphorylated IκBα (p-IκBα) and inhibiting the translocation of p65 into the nucleus. In contrast to 1-BI, antioxidant N-acetyl cysteine (NAC) stimulated cell proliferation and significantly protected the cells from 1-BI-mediated cell death by neutralizing ROS. Collectively, apoptosis induced by 1-BI is associated with the over-production of ROS and the reduction of NF-κB. Antioxidants can significantly block the inhibitory effect of 1-BI.     

Genome sequences of the ethanol-tolerant Lactobacillus vini strains LMG 23202T and JP7.8.9

We report on the genome sequences of Lactobacillus vini type strain LMG 23202(T) (DSM 20605) (isolated from fermenting grape musts in Spain) and the industrial strain L. vini JP7.8.9 (isolated from a bioethanol plant in northeast Brazil). All contigs were assembled using gsAssembler, and genes were predicted and annotated using Rapid Annotation using Subsystem Technology (RAST). The identified genome sequence of LMG 23202(T) had 2.201.333 bp, 37.6% G+C, and 1,833 genes, whereas the identified genome sequence of JP7.8.9 had 2.301.037 bp, 37.8% G+C, and 1,739 genes. The gene repertoire of the species L. vini offers promising opportunities for biotechnological applications.     

Enhancing Integrin α1 Inserted (I) Domain Affinity to Ligand Potentiates Integrin α1β1-mediated Down-regulation of Collagen Synthesis

Integrin α1β1 binding to collagen IV, which is mediated by the α1-inserted (I) domain, down-regulates collagen synthesis. When unligated, a salt bridge between Arg²⁸⁷ and Glu³¹⁷ is thought to keep this domain in a low affinity conformation. Ligand binding opens the salt bridge leading to a high-affinity conformation. How modulating integrin α1β1 affinity alters collagen homeostasis is unknown. To address this question, we utilized a thermolysin-derived product of the α1α2α1 network of collagen IV (α1α2α1(IV) truncated protomer) that selectively binds integrin α1β1. We show that an E317A substitution enhanced binding to the truncated protomer, consistent with a previous finding that this substitution eliminates the salt bridge. Surprisingly, we show that an R287A substitution did not alter binding, whereas R287E/E317R substitutions enhanced binding to the truncated protomer. NMR spectroscopy and molecular modeling suggested that eliminating the Glu³¹⁷ negative charge is sufficient to induce a conformational change toward the open state. Thus, the role played by Glu³¹⁷ is largely independent of the salt bridge. We further show that cells expressing E317A or R287E/E317R substitutions have enhanced down-regulation of collagen IV synthesis, which is mediated by the ERK/MAPK pathway. In conclusion, we have demonstrated that modulating the affinity of the extracellular α1 I domain to collagen IV enhances outside-in signaling by potentiating ERK activation and enhancing the down-regulation of collagen synthesis.     

Rapid detection of avian influenza H5N1 virus using impedance measurement of immuno-reaction coupled with RBC amplification

Avian influenza virus (AIV) subtype H5N1 was first discovered in the 1990 s and since then its emergence has become a likely source of a global pandemic and economic loss. Currently accepted gold standard methods of influenza detection, viral culture and rRT-PCR, are time consuming, expensive and require special training and laboratory facilities. A rapid, sensitive, and specific screening method is needed for in-field or bedside testing of AI virus to effectively implement quarantines and medications. Therefore, the objective of this study was to improve the specificity and sensitivity of an impedance biosensor that has been developed for the screening of AIV H5. Three major components of the developed biosensor are immunomagnetic nanoparticles for the separation of AI virus, a microfluidic chip for sample control and an interdigitated microelectrode for impedance measurement. In this study polyclonal antibody against N1 subtype was immobilized on the surface of the microelectrode to specifically bind AIV H5N1 to generate more specific impedance signal and chicken red blood cells (RBC) were used as biolabels to attach to AIV H5N1 captured on the microelectrode to amplify impedance signal. RBC amplification was shown to increase the impedance signal change by more than 100% compared to the protocol without RBC biolabels, and was necessary for forming a linear calibration curve for the biosensor. The use of a second antibody against N1 offered much greater specificity and reliability than the previous biosensor protocol. The biosensor was able to detect AIV H5N1 at concentrations down to 10(3) EID(50)ml(-1) in less than 2h.     

Mucosal reactive oxygen species decrease virulence by disrupting Campylobacter jejuni phosphotyrosine signaling

Reactive oxygen species (ROS) play key roles in mucosal defense, yet how they are induced and the consequences for pathogens are unclear. We report that ROS generated by epithelial NADPH oxidases (Nox1/Duox2) during Campylobacter jejuni infection impair bacterial capsule formation and virulence by altering bacterial signal transduction. Upon C. jejuni invasion, ROS released from the intestinal mucosa inhibit the bacterial phosphotyrosine network that is regulated by the outer-membrane tyrosine kinase Cjtk (Cj1170/OMP50). ROS-mediated Cjtk inactivation results in an overall decrease in the phosphorylation of C. jejuni outer-membrane/periplasmic proteins, including UDP-GlcNAc/Glc 4-epimerase (Gne), an enzyme required for N-glycosylation and capsule formation. Cjtk positively regulates Gne by phosphorylating an active site tyrosine, while loss of Cjtk or ROS treatment inhibits Gne activity, causing altered polysaccharide synthesis. Thus, epithelial NADPH oxidases are an early antibacterial defense system in the intestinal mucosa that modifies virulence by disrupting bacterial signaling.     

The consequences of Lactobacillus vini and Dekkera bruxellensis as contaminants of the sugarcane-based ethanol fermentation

This work describes the effects of the presence of the yeast Dekkera bruxellensis and the bacterium Lactobacillus vini on the industrial production of ethanol from sugarcane fermentation. Both contaminants were quantified in industrial samples, and their presence was correlated to a decrease in ethanol concentration and accumulation of sugar. Then, laboratory mixed-cell fermentations were carried out to evaluate the effects of these presumed contaminants on the viability of Saccharomyces cerevisiae and the overall ethanol yield. The results showed that high residual sugar seemed the most significant factor arising from the presence of D. bruxellensis in the industrial process when compared to pure S. cerevisiae cultures. Moreover, when L. vini was added to S. cerevisiae cultures it did not appear to affect the yeast cells by any kind of antagonistic effect under stable fermentations. In addition, when L. vini was added to D. bruxellensis cultures, it showed signs of being able to stimulate the fermentative activity of the yeast cells in a way that led to an increase in the ethanol yield.     

Southwestern blotting in investigating transcriptional regulation

Southwestern blotting is used to investigate DNA-protein interactions. The advantage of this technique over other related methods such as electrophoretic mobility shift assay (EMSA) and DNA footprinting is that it provides information regarding the molecular weight of unknown protein factor. This method combines the features of Southern and Western blotting techniques; a denaturing SDS-PAGE is first employed to separate proteins electrophoretically based on size, and after transferring the proteins to a membrane support, the membrane-bound proteins are renatured and incubated with a (32)P-labeled double-stranded oligonucleotide probe of specific DNA sequence. The interaction of the probe with the protein(s) is later visualized by autoradiography. This technique could be combined with database searching (TransFac, http://www.gene-regulation.com/pub/databases.html#transfac), prediction of potential protein factors binding onto a target motif (e.g., Patch search), in vitro supershift EMSA and in vivo chromatin immunoprecipitation (ChIP) assays for effective identification of protein factors. The whole Southwestern blotting procedure takes approximately 4 d to complete. In this article, a commonly used protocol and expected results are described and discussed.     

Inhibition of lipopolysaccharide-induced interleukin-1beta and tumor necrosis factor-alpha production by menthone through nuclear factor-kappaB signaling pathway in HaCat cells

Menthone, the Chinese old remedy extracted from genus Mentha, has been widely used as a cooling agent, a counterirritant for pain relief, and for the treatment of pruritus. However, its detail mechanisms for interfering inflammatory reaction remain unknown. In this study, we found that menthone can suppress the lipopolysaccharide (LPS)-induced proinflammatory cytokines, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), as well as nuclear factor kappaB (NF-kappaB) activity induced by LPS and other inflammatory agents, including 12-O-tetradecanoylphorbol-13-acetate, hydrogen peroxide, okadaic acid, and ceramide. Furthermore, our data also demonstrated that the translocation of NF-kappaB activated by LPS into the nucleus was suppressed by menthone, and I-kappaB and beta-transducin repeat containing protein (beta-TrCP) were both involved in this suppression. To sum up, this study has provided molecular evidence for menthone effect on the LPS-induced cytokine production, NF-kappaB activation, and the involvement of I-kappaB and beta-TrCP.