A methodology for the selection of a routine purity test for insoluble beta(1-->3)glucan.

Beta(1-->3)glucan isolated from the cell wall of Saccharomyces cerevisiae is a biological response modifier (BRM) stimulating resistance against bacterial, viral, fungal and protozoal diseases. This polysaccharide has a high molecular weight, which makes it very difficult to achieve a purity test. A comparative study of different analytical procedures for beta(1-->3)glucan from S. cerevisiae was conducted in order to establish a reliable routine analytical methodology for quality control of this active ingredient in pharmaceutical products. With this aim, different combinations of the analytical procedure steps were tested, including three alternatives for the acid hydrolysis step, three for neutralization, two for gas-liquid chromatographic derivatization and two internal standards. The glucose yield, precision, time consumption and reagent cost per sample were determined for 10 sample replicates. All gas chromatographic determinations were conducted using packed GLC columns and an FID detector. The selected analytical method showed 83.61 +/- 3.48% glucose yield, the shortest relative time consumption (54.2%) and the lowest cost of reagents (7.4%) and consisted of a combination of 72% sulfuric acid hydrolysis, 25% ammonium hydroxide neutralization and alditol acetate derivatization using xylose as internal standard.

High expression of PKA regulatory subunit 1A protein is related to proliferation of human melanoma cells.

The cAMP-protein kinase A (PKA) pathway is the major signal transduction pathway involved in melanocyte-stimulating hormone receptor-mediated signaling and melanin production, whereas its role in the control of melanocyte proliferation is still controversial. In this study, we evaluated the effects of selective activation of the different PKA regulatory subunits type 1A (R1A) and type 2B (R2B) on melanocyte proliferation. Immunohistochemistry demonstrated that normal melanocytes lacked R1A protein whereas this subunit was highly expressed in all human melanomas studied (N=20) and in six human melanoma cell lines. Pharmacological activation of the R2 subunits by the cAMP analogue 8-Cl-cAMP inhibited proliferation and increased caspase-3 activity by 68.77+/-10.5 and 72+/-9% respectively, in all cell lines with the exception of the only p53-mutated one. Similar effects were obtained by activating R2 subunits with other analogues and by silencing R1A expression. The antiproliferative and proapoptotic effects of 8-Cl-cAMP were comparable to those observed with commonly used antitumoral drugs. Moreover, 8-Cl-cAMP potentiated the effects of these drugs on both cell proliferation and caspase-3 activity. In conclusion, this study first reports that human melanomas are characterized by a high R1/R2 ratio and that pharmacological and genetic manipulations able to revert this unbalanced expression cause significant antiproliferative and proapoptotic effects in melanoma cells.