Development of a highly sensitive chemiluminescence enzyme immunoassay using enhanced luminol as substrate

In this study, a high sensitivity chemiluminescence enzyme immunoassay (CLEIA) based on novel enhancers was developed. Under optimal conditions, we developed an enhanced chemiluminescence reaction (ECR) catalyzed by horseradish peroxidase (HRP‐C) in the presence of 3‐(10'‐phenothiazinyl) propane‐1‐sulfonate (SPTZ) and 4‐morpholinopyridine (MORP) as enhancers. The limit of detection of the newly prepared chemiluminescent cocktail for HRP was 0.33 pg/well, which is lower than that of commercial Super Signal substrate. The results showed that this novel chemiluminescent cocktail can significantly increase the light output of HRP‐catalyzed ECR, which can be translated into a corresponding improvement in sensitivity. Similar improvements were observed in CLEIA for the determination of chloramphenicol in milk. In addition, the ECR of N‐azoles as secondary enhancer was also presented. Copyright © 2013 John Wiley & Sons, Ltd.     

A highly efficient and economical cell-free protein synthesis system using the S12 extract of <Emphasis Type="Italic">Escherichia coli</Emphasis>

We have developed an economical and simple cell-free protein synthesis system that produces milligram quantities of proteins in a milliliter batch reaction. In this system, the S12 extract, which was prepared from glucose-adapted cells, was employed and glucose alone was successfully used for the efficient and stable regeneration of ATP. The ATP level in the reaction mixture remained stable over a remarkably extended reaction period, which enabled prolonged protein synthesis, and the issues associated with proton accumulation and amino acid depletion were simultaneously addressed. Under the reaction conditions established in this study, protein synthesis continued for 6 h and the amount of the accumulated protein reached 1.8 mg/mL.     

Recycling of the Posttermination Complexes of Mycobacterium smegmatis and Escherichia coli Ribosomes Using Heterologous Factors

In eubacteria, ribosome recycling factor (RRF) and elongation factor G (EFG) function together to dissociate posttermination ribosomal complexes. Earlier studies, using heterologous factors from Mycobacterium tuberculosis in Escherichia coli revealed that specific interactions between RRF and EFG are crucial for their function in ribosome recycling. Here, we used translation factors from E. coli, Mycobacterium smegmatis and M. tuberculosis, and polysomes from E. coli and M. smegmatis, and employed in vivo and in vitro experiments to further understand the role of EFG in ribosome recycling. We show that E. coli EFG (EcoEFG) recycles E. coli ribosomes with E. coli RRF (EcoRRF), but not with mycobacterial RRFs. Also, EcoEFG fails to recycle M. smegmatis ribosomes with either EcoRRF or mycobacterial RRFs. On the other hand, mycobacterial EFGs recycle both E. coli and M. smegmatis ribosomes with either of the RRFs. These observations suggest that EFG establishes distinct interactions with RRF and the ribosome to carry out ribosome recycling. Furthermore, the EFG chimeras generated by swapping domains between mycobacterial EFGs and EcoEFG suggest that while the residues needed to specify the EFG interaction with RRF are located in domains IV and V, those required to specify its interaction with the ribosome are located throughout the molecule.     

Subribosomal particle analysis reveals the stages of bacterial ribosome assembly at which rRNA nucleotides are modified

Modified nucleosides of ribosomal RNA are synthesized during ribosome assembly. In bacteria, each modification is made by a specialized enzyme. In vitro studies have shown that some enzymes need the presence of ribosomal proteins while other enzymes can modify only protein-free rRNA. We have analyzed the addition of modified nucleosides to rRNA during ribosome assembly. Accumulation of incompletely assembled ribosomal particles (25S, 35S, and 45S) was induced by chloramphenicol or erythromycin in an exponentially growing Escherichia coli culture. Incompletely assembled ribosomal particles were isolated from drug-treated and free 30S and 50S subunits and mature 70S ribosomes from untreated cells. Nucleosides of 16S and 23S rRNA were prepared and analyzed by reverse-phase, high-performance liquid chromatography (HPLC). Pseudouridines were identified by the chemical modification/primer extension method. Based on the results, the rRNA modifications were divided into three major groups: early, intermediate, and late assembly specific modifications. Seven out of 11 modified nucleosides of 16S rRNA were late assembly specific. In contrast, 16 out of 25 modified nucleosides of 23S rRNA were made during early steps of ribosome assembly. Free subunits of exponentially growing bacteria contain undermodified rRNA, indicating that a specific set of modifications is synthesized during very late steps of ribosome subunit assembly.     

Lipid-induced up-regulation of human acyl-CoA synthetase 5 promotes hepatocellular apoptosis

In the pathogenesis of nonalcoholic fatty liver disease, accumulation of lipids in hepatocytes and hepatocyte apoptosis are strongly implicated in disease progression from the potentially reversible condition of steatosis to severe acute and chronic liver injury. Acyl-CoA synthetase 5, a member of the ACSL gene family that catalyzes the activation of long-chain fatty acids for lipid biosynthesis, is the only ACSL isoform that is both, located on mitochondria and functionally involved in enterocyte apoptosis. In this study, the regulation of human ACSL5 in hepatocellular fatty acid degeneration and its involvement in hepatocyte apoptosis was investigated using models of in vitro and in vivo steatosis as well as plasmid-mediated stable gene transfer and RNAi-mediated gene silencing. ACSL5 mRNA and protein were strongly increased by uptake of dietary derived fatty acids in primary human hepatocytes, HepG2 cells and human steatotic liver. Over-expression of ACSL5 decreased HepG2 cell viability and increased susceptibility to TRAIL- and TNFα-, but not FAS- induced apoptosis, whereas knock down of ACSL5 reduced apoptosis susceptibility. High ACSL5 activity resulted in enhanced caspase-3/7 activity, but was not accompanied by up-regulation of death receptors, DR4, DR5 or TNF-R1. This study gives evidence that hepatocyte steatosis is associated with ACSL5 up-regulation resulting in increased susceptibility to hepatic cell death. We propose that ACSL5 could play a role in promoting fatty acid-induced lipoapoptosis in hepatocytes as important mechanism in fatty liver-related disorders.     

A New Critical Conformational Determinant of Multidrug Efflux by an MFS Transporter

Secondary multidrug (Mdr) transporters utilize ion concentration gradients to actively remove antibiotics and other toxic compounds from cells. The model Mdr transporter MdfA from Escherichia coli exchanges dissimilar drugs for protons. The transporter should open at the cytoplasmic side to enable access of drugs into the Mdr recognition pocket. Here we show that the cytoplasmic rim around the Mdr recognition pocket represents a previously overlooked important regulatory determinant in MdfA. We demonstrate that increasing the positive charge of the electrically asymmetric rim dramatically inhibits MdfA activity and sometimes even leads to influx of planar, positively charged compounds, resulting in drug sensitivity. Our results suggest that unlike the mutants with the electrically modified rim, the membrane-embedded wild-type MdfA exhibits a significant probability of an inward-closed conformation, which is further increased by drug binding. Since MdfA binds drugs from its inward-facing environment, these results are intriguing and raise the possibility that the transporter has a sensitive, drug-induced conformational switch, which favors an inward-closed state.     

A chromosomal chloramphenicol acetyltransferase determinant from a probiotic strain of Bacillus clausii

The mechanism of resistance to chloramphenicol was studied in four strains of Bacillus clausii included in a probiotic mixture, which is administered to humans for prevention of gastrointestinal side effects due to oral antibiotic therapy. By cloning experiments, a chloramphenicol acetyltransferase (CAT) gene, cat _Bcl , coding for a putative 228-amino acid CAT protein was identified in B. clausii SIN. The deduced amino acid sequence displayed from 31% to 85% identity with 56 CAT proteins from other Gram-positive bacterial strains. The cat _Bcl gene was also detected by PCR in the three other B. clausii strains resistant to chloramphenicol, whereas it was absent in the three control strains susceptible to chloramphenicol. Pulse-field gel electrophoresis of total DNA digested by I-CeuI followed by hybridization with a cat -specific probe as well as unsuccessful repeated attempts of in vitro transfer of chloramphenicol resistance to various recipient cells indicated that cat _Bcl was chromosomally located in all four resistant B. clausii strains.     

Transcriptional regulation of the p73 gene, a member of the p53 family, by early growth response-1 (Egr-1)

To elucidate the regulatory mechanism of p73 gene expression, we analyzed the human p73 promoter and found three putative Egr-1-binding sites located upstream of exon 1 (-1728, -321, and -38). The Egr-1 responsiveness of these sites was analyzed by transient transfection assays using 5'- and 3'-serial truncations of the p73 promoter, subcloned in a CAT reporter vector. The functional significance of the region was further confirmed by an electrophoretic mobility shift assay using the Egr-1 protein synthesized in vitro and a [32P]-labeled middle site sequence, followed by competition with unlabeled wild-type or mutant oligonucleotides and supershift assays using an anti-Egr-1 antibody. When induced by either the nitric oxide donor NOC-18 or the PPARgamma agonist troglitazone, Egr-1 bound to the p73 promoter, as assessed by chromatin immunoprecipitation assays, accompanied by increased expression of p73. MTT assays revealed that cell growth was significantly inhibited on treating the cells with troglitazone. Overall, our results provide direct evidence that Egr-1 positively regulated p73 expression by binding to its promoter in vivo, consistent with Egr-1 and p73 being involved in p53-independent tumor suppression.     

Thyroid hormone responsive genes in the murine hepatocyte cell line AML 12

Thyroid hormone (T3) plays an important role in gene regulation in the liver. Previous studies have been done in complex systems such as animal models, or in transformed malignant hepatic cell lines in which thyroid hormone receptor (TR) was over-expressed by co-transfection. Therefore, the aim of this study was to characterize T3-responsive genes in a simple system, by using a non-transformed hepatic cell line that is able to express sufficient amounts of endogenous TRs. For this purpose we used the murine non-transformed hepatocyte cell line AML 12. We performed analyses using a cDNA microarray containing 15,000 murine genes. We found 12 genes to be up-regulated and 5 genes to be down-regulated in the presence of T3. For some of the genes not previously known to be regulated by T3, we confirmed the regulation by T3 using real-time PCR. Our data in AML 12 cells provide a simple and physiologically relevant system to study T3 action, without the influence of neoplastic transformation or artificial TR over-expression. Furthermore, our data describe novel T3 responsive genes and provide insight into the role of T3 in important processes such as cholesterol metabolism, bile acid secretion, oncogenesis, among others, that can be tested in future experiments in vivo.