Differential metabolism of androst-5-ene-3β,17β-diol between rats, canines, monkeys and humans

The potent anti-inflammatory activity of exogenous dehydroepiandrosterone (DHEA) in rodents has not translated to humans. This disparity in pharmacological effects has been attributed to factors such as differences in expression and function of molecular targets and differential metabolism. Hepatocytes from rats, dogs, monkeys, and humans were used to measure species-specific metabolism of a related compound, androst-5-ene-3β,17β-diol (5-AED) using reversed-phase radio-HPLC, to explore the metabolic contribution to this interspecies disparity. We found that rat hepatocytes transformed 5-AED predominantly into an array of highly oxidized metabolites. Canine metabolites overlapped with rat, but contained a greater abundance of less hydrophilic species. Monkey and human metabolites were strikingly less hydrophilic, dominated by 5-AED and DHEA conjugates. From the accumulating evidence indicating that the DHEA anti-inflammatory activity may actually reside in its more highly oxidized metabolites, we advance a hypothesis that the virtual absence of these metabolites in humans is central to the failure of exogenous DHEA to produce a potent pharmacological effect in clinical investigations. Accordingly, emulation of its anti-inflammatory activity in humans will require administration of an active native metabolite or a synthetic pharmaceutical derivative.     

The in vivo preventive and therapeutic properties of curcumin in bile reflux‐related oncogenesis of the hypopharynx

Bile at strongly acidic pH exerts a carcinogenic effect on the hypopharynx, based upon recent pre‐clinical studies that support its role as an independent risk factor. We recently demonstrated in vitro that curcumin can prevent oncogenic profile of bile in human hypopharyngeal cells, by inhibiting NF‐κB. We hypothesize that topically applied curcumin to the hypopharynx can similarly block early oncogenic molecular events of bile, by inhibiting NF‐κB and consequently altering the expression of genes with oncogenic function. Using Mus musculus (C57Bl/6J), we topically applied curcumin (250 μmol/L; three times per day; 10 days) to the hypopharynx, 15 minutes before, 15 minutes after or in combination with bile acids (pH 3.0). Immunohistochemical analysis and qPCR revealed that topically applied curcumin either before, after or in combination with acidic bile exposure significantly suppressed its induced NF‐κB activation in regenerating epithelial cells, and overexpression of Rela, Bcl2, Egfr, Stat3, Wnt5a, Tnf, Il6, Ptgs2. Akt1 was particularly inhibited by curcumin when applied simultaneously with bile. We provide novel evidence into the preventive and therapeutic properties of topically applied curcumin in acidic bile‐induced early oncogenic molecular events in hypopharyngeal mucosa, by inhibiting NF‐κB, and shaping future translational development of effective targeted therapies using topical non‐pharmacologic inhibitors of NF‐κB.     

Multi-Site N-glycan mapping study 1: Capillary electrophoresis – laser induced fluorescence

An international team that included 20 independent laboratories from biopharmaceutical companies, universities, analytical contract laboratories and national authorities in the United States, Europe and Asia was formed to evaluate the reproducibility of sample preparation and analysis of N-glycans using capillary electrophoresis of 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled glycans with laser induced fluorescence (CE-LIF) detection (16 sites) and ultra high-performance liquid chromatography (UHPLC, 12 sites; results to be reported in a subsequent publication). All participants used the same lot of chemicals, samples, reagents, and columns/capillaries to run their assays. Migration time, peak area and peak area percent values were determined for all peaks with >0.1% peak area. Our results demonstrated low variability and high reproducibility, both, within any given site as well across all sites, which indicates that a standard N-glycan analysis platform appropriate for general use (clone selection, process development, lot release, etc.) within the industry can be established.     

Suppression of tumorigenicity by the IncW R plasmid pSa in Agrobacterium tumefaciens

Summary The effect of the IncW R plasmid, pSa, on tumorigenicity and on the expression and maintenance of the Ti plasmid in tumorigenic strains of A. tumefaciens was determined. Plasmid pSa could be transferred into and stably maintained by both octopine-and nopaline-utilizing A. tumefaciens strains. The R plasmid had no effect on Ti plasmid maintenance or on Ti plasmid functions, such as octopine utilization or conjugal bacterial transfer. However, A. tumefaciens strains harboring both the R plasmid and the Ti plasmid in most instances failed to induce tumors on a number of plant species. This effect on tumorigenicity is specific to pSa. When pSa is cured from the A. tumefaciens transconjugants or when their Ti plasmids are genetically transferred to an appropriate recipient, the resultant strains lacking the R plasmid regain tumorigenicity. Restriction endonuclease analysis of plasmid DNA isolated from transconjugants harboring pSa showed no difference in Ti plasmid cleavage patterns when compared to plasmid DNA isolated from the tumorigenic parent strain. These results indicate that pSa does not induce detectable permanent genetic alteration of the Ti plasmid. Rather, it appears that the R plasmid suppresses some Ti plasmid function(s) necessary for tumorigenicity.     

THE INFLUENCE OF THE BACKWARD REACTION IN THE PEPTIC HYDROLYSIS OF ALBUMIN

1. No destruction of pepsin by heat is demonstrable at pH 1.6 until a temperature of 40°C. is exceeded. 2. The influence of the backward reaction in peptic hydrolysis is shown in the diminishing rate at which increasing concentrations of protein are hydrolyzed. 3. The backward reaction causes the optimum for the hydrolysis of higher concentrations of protein to be attained at a lower temperature than with more dilute solutions. 4. The proteose and peptone associated with commercial pepsin retard hydrolysis in the same sense as the products due to the action of the enzyme.