Modulation of Enzyme-Catalyzed Synthesis of Prostaglandins by Components Contained in Kidney Microsomal Preparations

In the kidney, prostaglandins formed by cyclooxygenase 1 and 2 (COX-1 and COX-2) play an important role in regulating renal blood flow. In the present study, we report our observations regarding a unique modulatory effect of renal microsomal preparation on COX-1/2-mediated formation of major prostaglandin (PG) products in vitro. We found that microsomes prepared from pig and rat kidneys had a dual stimulatory–inhibitory effect on the formation of certain PG products catalyzed by COX-1 and COX-2. At lower concentrations, kidney microsomes stimulated the formation of certain PG products, whereas at higher concentrations, their presence inhibited the formation. Presence of kidney microsomes consistently increased the Km values of the COX-1/2-mediated reactions, while the Vmax might be increased or decreased depending on stimulation or inhibition observed. Experimental evidence was presented to show that a protein component present in the pig kidney microsomes was primarily responsible for the activation of the enzyme-catalyzed arachidonic acid metabolism leading to the formation of certain PG products.

Effect of non-competitive inhibitors of aminopeptidase N on viability of human and murine tumor cells

Cancer is the second leading cause of death worldwide. Peptidases participate in tumor development and growth. Mammalian neutral aminopeptidase (APN, EC 3.4.11.2, M1 family) catalyzes the cleavage of neutral and basic amino acids from the N-terminus of substrates. APN expression is dysregulated in several types of cancer, being a target for the development of new anticancer agents. Recently, we identified three new non-competitive inhibitors of soluble porcine APN (pAPN) by virtual screening (BTB11079, JFD00064, BTB07018, from Maybridge). In the present contribution we assayed their effect on the activity of APN in a microsomal preparation of porcine kidney cortex, a model of the physicochemical environment of the enzyme. These classical inhibitors had an IC50 value of 3–5 µM. Additionally, using a kinetic approach and a specific substrate, we quantified APN activity on the cell surface of human and murine lung, colon, prostate, and skin tumor cells. APN inhibitors reduced tumor cells viability, more efficiently in the higher APN activity tumor cell lines, but not in non-tumoral cells. BTB11079, JFD00064, BTB07018 effects on cell viability were stronger than that of bestatin, a positive control. Thus, these non-competitive APN inhibitors may be useful tools for cancer treatment.

Efficient Reduction of Prions by the Manufacturing Process of a VWF/FVIII Product

Variant Creutzfeldt-Jakob disease (vCJD) is a fatal neurodegenerative disease acquired through infection with prions which cause bovine spongiform encephalopathy (BSE) by consumption of beef products from infected cattle. Presumptive transfusion transmitted cases of vCJD have been reported in the UK increasing the concern that medicinal products manufactured from plasma might also pose a risk of vCJD transmission. Therefore, investigational studies were performed to assess the prion reduction capacity of the manufacturing process of a VWF/FVIII product (Haemate® P / Humate-P®). In these studies hamster brain derived prion material in the form of a microsomal preparation were used to spike plasma product intermediates. As the purification process of a desired plasma protein may impact the physico-chemical form of the spiked prion protein, the addition of reduction factors from single step studies possibly might not reflect the true prion reduction of the full process; in particular where the spike material is heterogeneous, one fraction, i.e. gross aggregates of prion material could be preferentially removed by one step and the same subfraction by a subsequent step. Therefore, we employed a combined step study approach and not a single spike step approach to address that issue of the impact of heterogeneous spike fractions regarding the overall reduction factor. The manufacturing process of the VWF/FVIII product was divided in two parts which were studied independently twice: Pooled plasma donations (4,500 ml) were spiked and processed covering cryoprecipitation, Al(OH)3 adsorption and subsequent precipitations by glycine and NaCl. The second part of the VWF/FVIII manufacturing process was studied starting with spiked dissolved NaCl precipitate (154 ml) and processed further by pasteurization, second NaCl precipitation, dialysis, ultracentrifugation and sterile filtration. The prion spiked starting material and product intermediate were processed according to the manufacturing conditions based on a valid down-scale model. The prion reduction factors were determined as the difference of the prion load in the spiked starting materials and in the respective final samples using a biochemical assay (Conformation-Dependent Immunoassay (CDI)) or a bioassay in hamsters for quantification of PrPSc (dose dependent incubation period measurement). The results of these investigational studies for the two prion quantification methods are shown below PrionReduction Factors [log10] demonstrated by Manufacturing steps biochemical methods (CDI) bioassay (incubation time) Cryo precipitation / Al(OH)3adsorption / Glycine precipitation 3.2 ± 0.1 2.8 ± 0.3 Pasteurization / NaCl precipitation / Dialysis / Ultracentrifugation / Sterile filtration 2.9 ± 0.2 3.1 ± 0.1 Filling / Lyophilization n.d. n.d. Overall Prion Reduction Factor 6.1 ± 0.2 5.9 ± 0.3 These results demonstrate (i) comparable prion reduction factors quantified either by biochemical methods or by a bioassay and (ii) an appropriate overall prion reduction capacity of the manufacturing process of Haemate® P / Humate-P®. Based on complementary safety procedures, i.e., collection of plasma by stringent donor selection due to geographic donor deferral policy and the overall prion reduction factor of approximately 6 log10, which clearly exceeds a potential prion load in the manufacturing pool, a risk assessment results in an extremely remote risk of prion transmission by the VWF / FVIII product Haemate® P / Humate-P®.

Selective increase in acyl hydrolase activity by graminicides in wheat

Seedlings of wheat were grown for 24 h in control nutrient solution and in solutions containing haloxyfop, alloxydim, diquat or paraquat, and thereafter the roots were used for microsomal preparations. Phosphatidylcholine or diacyl-glycerol with various 1-14C-labelled fatty acids (oleic, linoleic, linolenic or ricinoleic acids) in position sn-2 were added to the prepared microsomes. After incubation for 2 h at 30 °C, the lipids were extracted and the distribution of radioactivity among lipid classes was determined. In the microsomal preparations of plants treated with diquat and paraquat, the amounts of fatty acids released were similar to the control, whereas they were 1.4–2 times higher in the microsomal preparation of plants treated with haloxyfop and alloxydim. Thus, the data indicate that graminicides could increase lipid catabolism in sensitive plants and that this is not a general phenomenon connected with inhibition of growth.