A previous high-performance liquid chromatographic determination of diamine oxidase activity suitable for tissue homogenates was modified in order to adapt it to plasma samples. Simple additional ...steps were introduced after the enzyme reaction and before the chromatographic separation, both according to the previous method. In this way the sensitivity and the reproducibility of the overall procedure was suitable for routine plasma diamine oxidase estimations.
"Transmissible Spongiform Encephalopathies" (TSE) are a group of degenerative progressive fatal disorders of the CNS, affecting both humans and animals. The main pathogenic event is the conversion of ...cellular prion protein from the normal, enzyme-sensitive (PrPsen), to the insoluble proteinase K-resistant isoform (PrPres). Since the new juvenile variant of Creutzfeldt-Jakob disease (vCJD) is probably due to the transmission of Bovine Spongiform Encephalopathy (BSE) prion protein to man, therapeutic and preventive compounds for animals and humans are urgently needed. Congo Red (benzidine-diazo-bis-1-naphthylamine-4-sulfonic acid sodium salt, CAS 573-58-0, CR), an azoic dye that inhibits amyloid deposition, and some newly synthesized derivatives, more lipophilic and less toxic, were tested for their anti-prionic activity, in different experimental models. Cell-free experiments using the synthetic peptide PrP 106-126, homologous to amino acid residues 106-126 of the human PrP, were run to determine the anti-amyloidogenic properties of some of the molecules. Peptide solutions containing each compound were incubated at 37 degrees C, for increasing times, to analyse the kinetics of aggregation of PrP 106-126 peptide. After incubation, the amount of non-aggregated peptide was measured by RP-HPLC. While CR enhanced the amyloidogenicity of PrP 106-126, derivatives "1a" and "1b" both showed the opposite behaviour, reducing aggregation by 15-20%. In other experiments using electron microscopy PrP 106-126 was assayed with the same molecules to assess the number and size of fibrils formed. CR showed its typical interaction, producing amyloid aggregates, "1a" did not interfere with fibril formation, while "1b" seemed to partially affect the structure of PrP 106-126 fibrils. Using a different cell-free model, it was investigated whether CR derivatives could reverse the protease-resistant PrPres, extracted from Syrian hamster infected brain, into the normal protease sensitive PrPsen. Samples containing fixed amounts of PrPres were incubated at 37 degrees C for 1 h with all the newly synthesized molecules, at concentrations ranging from 50 micrograms/mL to 750 micrograms/mL. After treatment with proteinase K, half of each sample was incubated with 3 mol/L guanidine thiocyanate in order to exclude over-stabilisation of the PrPres aggregates already observed with CR. The remaining amount of PrPres was assessed by Enhanced Chemoluminescence (ECL) Western blotting analysis. None of the compounds induced the reversion of PrPres to PrPsen; nevertheless, 6 of the 8 molecules interacted with PrPres molecules, over-stabilising the PrPres aggregates, from this aspect being similar to CR in activity. Finally, the inhibition of the generation of PrPres in the S12 clone of a mouse neuroblastoma cell line (N2a S12), persistently infected by the mouse adapted Chandler strain of scrapple, was evaluated. Increasing amounts of CR, "1a" and "1b" were added to the culture medium at each cell passage. After various days of treatment, the cells were collected, lysed, and the amount of PrPres was assayed by ECL Western blotting after PK treatment. As expected, there was a decrease in pathological PrP expression starting from the 4th day of treatment, with 5 and 10 micrograms/mL CR; PrPres completely disappeared after respectively 10 and 14 days of treatment. "1a" was strongly effective after 3 days of treatment at 5 and 10 micrograms/mL, but it was also highly toxic; at the concentration of 1 microgram/mL, it had a mild inhibitory effect after 8 days. The reduction of PrPres was also evaluated by intracytoplasmic flow-cytometry immunofluorescence on CR- and "1a"-treated N2a S12 cells. CR induced a dose-related decrease of PrP expression from day 3 to 13 of treatment. At the concentrations of 2 and 1.5 micrograms/mL "1a" also strongly affected the expression of PrP starting from the 3rd day of treatment until the end of the experiment (day 13). These results confirm the importance of using an integrated system, based on different experimental models, to obtain useful information on the mechanism of action of anti-prionic compounds.
Adipose tissue (AT) has for a long time been regarded as a simple storage tissue for energy in the form of triglycerides. There is currently accumulating scientific evidence that adipose tissue is a ...highly active organ, that actively takes part in regulating several metabolic processes, including reproduction, inflammatory response, and production and secretion of signalling molecules that have important biological roles, also known as adipokines. These most importantly include tumor necrosis factor alpha (TNF-alpha), interleukins, leptin and adiponectin.
White adipose tissue (AT) has been considered for a long time as the store house of energy and this tissue is now recognized as an active endocrine organ that communicates with the brain and ...peripheral tissues by secreting a wide range of hormones and cytokines collectively termed adipokines; the adipokine family includes also several acute phase proteins (APP) (1). APP are part of the innate immunity and interplay in the restoration of homeostasis and the restraint of microbial growth before acquired immunity is active. Measurements of APP responses from physiological to pathological changes also help in monitoring and diagnosis of diseases in animals (2,3). Liver is considered as the principal organ of APP production, but extra hepatic production of alpha1-acid glycoprotein (AGP), serum amyloid A (SAA), haptoglobin (Hp), and lipopolysaccharide binding protein (LBP) has also been reported (4–9). APP production from AT has been reported for humans and rodents. Recently, SAA mRNA expression (13) from bovine adipose tissue has been confirmed by our group. However, information of APP production from different fat depots is not available at the protein level according to our knowledge. Investigation of APP in fat depots in physiological condition will improve our understanding about the association of AT and APP.
