A Dual Read-Out Assay to Evaluate the Potency of Compounds Active against Mycobacterium tuberculosis

Tuberculosis is a serious global health problem caused by the bacterium Mycobacterium tuberculosis. There is an urgent need for discovery and development of new treatments, but this can only be accomplished through rapid and reproducible M. tuberculosis assays designed to identify potent inhibitors. We developed an automated 96-well assay utilizing a recombinant strain of M. tuberculosis expressing a far-red fluorescent reporter to determine the activity of novel compounds; this allowed us to measure growth by monitoring both optical density and fluorescence. We determined that optical density and fluorescence were correlated with cell number during logarithmic phase growth. Fluorescence was stably maintained without antibiotic selection over 5 days, during which time cells remained actively growing. We optimized parameters for the assay, with the final format being 5 days’ growth in 96-well plates in the presence of 2% w/v DMSO. We confirmed reproducibility using rifampicin and other antibiotics. The dual detection method allows for a reproducible calculation of the minimum inhibitory concentration (MIC), at the same time detecting artefacts such as fluorescence quenching or compound precipitation. We used our assay to confirm anti-tubercular activity and establish the structure activity relationship (SAR) around the imidazo[1,2-a]pyridine-3-carboxamides, a promising series of M. tuberculosis inhibitors.     

Stimulation of Muscarinic Receptors Induces Expression of Individual fos and jun Genes Through Different Transduction Pathways

Abstract: The transduction pathways coupling muscarinic receptors to induction of fos and jun genes were investigated in neuroblastoma SH-SY5Y cells. Stimulation with carbachol induced expression of c- fos , fosB , c- jun , junB , and junD . This effect was abolished by pretreatment with atropine, indicating an involvement of muscarinic receptors. These genes were also induced by activation of protein kinase C with phorbol ester or by elevating the intracellular Ca²⁺ concentration with a Ca²⁺ ionophore. The Ca²⁺ effect was inhibited by KN-62, suggesting an induction through Ca²⁺/calmodulin-dependent kinase II. Inhibition of protein kinase C with GF109203X suppressed the carbachol-stimulated increase in mRNA levels of c- fos , fosB , and junB by ∼70% but had only minor effects on the expression of c- jun and junD . On the other hand, preincubation with KN-62 attenuated the carbachol-induced increase in c- jun and junD expression by 70% but had no effect on c- fos , fosB , and junB mRNA levels. Simultaneous inhibition of both protein kinase C and Ca²⁺/calmodulin-dependent kinase II completely abolished the carbachol-stimulated expression of c- jun and junD , but c- fos , fosB , and junB were still expressed to a certain extent under this condition. Comparison of the inhibitory effects of GF109203X and Gö 6976 suggests the involvement of classical protein kinase C isozymes in muscarinic receptor-stimulated expression of fos and jun genes. These results demonstrate that the muscarinic receptor-induced expression of individual fos and jun genes is regulated via different pathways, primarily protein kinase C or Ca²⁺/calmodulin-dependent kinase II.     

p14ARF Post-Transcriptional Regulation of Nuclear Cyclin D1 in MCF-7 Breast Cancer Cells: Discrimination between a Good and Bad Prognosis?

As part of a cell's inherent protection against carcinogenesis, p14ARF is upregulated in response to hyperproliferative signalling to induce cell cycle arrest. This property makes p14ARF a leading candidate for cancer therapy. This study explores the consequences of reactivating p14ARF in breast cancer and the potential of targeting p14ARF in breast cancer treatment. Our results show that activation of the p14ARF-p53-p21-Rb pathway in the estrogen sensitive MCF-7 breast cancer cells induces many hallmarks of senescence including a large flat cell morphology, multinucleation, senescence-associated-β-gal staining, and rapid G1 and G2/M phase cell cycle arrest. P14ARF also induces the expression of the proto-oncogene cyclin D1, which is most often associated with a transition from G1-S phase and is highly expressed in breast cancers with poor clinical prognosis. In this study, siRNA knockdown of cyclin D1, p21 and p53 show p21 plays a pivotal role in the maintenance of high cyclin D1 expression, cell cycle and growth arrest post-p14ARF induction. High p53 and p14ARF expression and low p21/cyclin D1 did not cause cell-cycle arrest. Knockdown of cyclin D1 stops proliferation but does not reverse senescence-associated cell growth. Furthermore, cyclin D1 accumulation in the nucleus post-p14ARF activation correlated with a rapid loss of nucleolar Ki-67 protein and inhibition of DNA synthesis. Latent effects of the p14ARF-induced cellular processes resulting from high nuclear cyclin D1 accumulation included a redistribution of Ki-67 into the nucleoli, aberrant nuclear growth (multinucleation), and cell proliferation. Lastly, downregulation of cyclin D1 through inhibition of ER abrogated latent recurrence. The mediation of these latent effects by continuous expression of p14ARF further suggests a novel mechanism whereby dysregulation of cyclin D1 could have a double-edged effect. Our results suggest that p14ARF induced-senescence is related to late-onset breast cancer in estrogen responsive breast cancers and/or the recurrence of more aggressive breast cancer post-therapy.     

The 5-hydroxytryptamine stimulated formation of inositol phosphate is inhibited in platelets from alcoholics

The accumulation of inositol monophosphate (IP1) was measured after stimulation of 5-hydroxytryptamine2 (5-HT2) receptors on platelets from alcoholics and healthy controls. In controls, 5-HT induced a dose-dependent response with an EC50 = 2 x 10(-6) M and a maximal response at 10(-5) M. Ritanserin, a selective 5-HT2 antagonist, markedly reduced the accumulation. The IP1 formation after stimulation by 10(-5) M 5-HT was significantly impaired in platelets from alcoholics as compared to controls. This study indicates that the 5-HT2 receptor function is inhibited in alcoholics. It also illustrates the possibility of using IP1 formation in peripheral cells as a mean of studying receptor function in disease.     

Ethanol potentiates the uptake of [14C]Serine into phosphatidylserine by base-exchange reaction in NG 108-15 cells

Phospholipid base-exchange enzymes catalyze the incorporation of nitrogenous bases into phosphoglycerides by a calcium-dependent mechanism. In this study, we describe the effect of ethanol on the incorporation of radioactive serine, choline and ethanolamine into their respective phospholipids in a neuroblastoma x glioma hybrid cell line (NG 108-15). Long term ethanol exposure induced a potentiation of the incorporation of [¹⁴C]serine into phosphatidylserine. Moreover, the phosphorus content of PS was found to be increased after long-term ethanol exposure. No concomitant changes in the phosphorus content of other phospholipids were observed. The results indicate that in NG 108-15 cells, the incorporation of radiolabelled serine into PS is potentiated during chronic ethanol exposure.     

Bacteria,phages and pigs: the effects of in-feed antibiotics on the microbiome at different gut locations

Disturbance of the beneficial gut microbial community is a potential collateral effect of antibiotics, which have many uses in animal agriculture (disease treatment or prevention and feed efficiency improvement). Understanding antibiotic effects on bacterial communities at different intestinal locations is essential to realize the full benefits and consequences of in-feed antibiotics. In this study, we defined the lumenal and mucosal bacterial communities from the small intestine (ileum) and large intestine (cecum and colon) plus feces, and characterized the effects of in-feed antibiotics (chlortetracycline, sulfamethazine and penicillin (ASP250)) on these communities. 16S rRNA gene sequence and metagenomic analyses of bacterial membership and functions revealed dramatic differences between small and large intestinal locations, including enrichment of Firmicutes and phage-encoding genes in the ileum. The large intestinal microbiota encoded numerous genes to degrade plant cell wall components, and these genes were lacking in the ileum. The mucosa-associated ileal microbiota harbored greater bacterial diversity than the lumen but similar membership to the mucosa of the large intestine, suggesting that most gut microbes can associate with the mucosa and might serve as an inoculum for the lumen. The collateral effects on the microbiota of antibiotic-fed animals caused divergence from that of control animals, with notable changes being increases in Escherichia coli populations in the ileum, Lachnobacterium spp. in all gut locations, and resistance genes to antibiotics not administered. Characterizing the differential metabolic capacities and response to perturbation at distinct intestinal locations will inform strategies to improve gut health and food safety.     

CHANGES IN LIPID CONCENTRATIONS AND FATTY ACID COMPOSITIONS IN RAT CEREBRUM DURING MATURATION

Abstract— Rat cerebrum was analysed at 20 different ages from birth to 45 days of age, for its concentration of protein, cholesterol, cerebrosides, phospholipids and gangliosides, and for the concentration of fatty acids of the linoleic and linolenic acid series. The fatty acid patterns of choline phosphoglycerides and ethanolamine phosphoglycerides were determined at the same ages. Phases of rapid accretion were found for protein, phospholipids, gangliosides and cholesterol. The accretion of the fatty acids of the linoleic acid series ceased at 20 days of age, while that of the fatty acids of the linolenic acid series continued. The fatty acid composition of the phosphoglycerides changed during the maturation of rat cerebrum and these changes consisted of chain elongation, increased unsaturation and variation in the pattern of the polyenoic acids. These changes varied irregularly with age and each developmental stage had characteristic fatty acid patterns of choline and ethanolamine phosphoglycerides.     

Kinetics of the antibody response to type III pneumococcal polysaccharide. I. Evidence that suppressor cells function by inhibiting the recruitment and proliferation of antibody-producing cells.

For the first 126 hr after immunization of mice with an optimally immunogenic dose (0.5 mug) of Type III pneumococcal polysaccharide (SSS-III), splenic antibody-forming PFC and serum antibody levels were measured at 2- and 8-hr intervals, respectively. PFC were detected at 28 hr after immunization and then increased through 86 hr after immunization; thereafter, the number of PFC remained nearly constant for the next 20 to 24 hr, and then began to decline. In contrast, serum antibody was first detected 60 hr after immunization. The accumulation of serum antibody continued to lag behind the increase in numbers of PFC by 16 to 20 hr until maximal serum antibody levels were attained; curves fitted to the values obtained for each parameter were nearly parallel.