Generation of monoclonal antibodies against tumor-associated antigen MX35/sodium-dependent phosphate transporter NaPi2b.

Tumor-associated antigen MX35, which is overexpressed in 70-90% of epithelial ovarian cancers, has been recently identified as phosphate transporter NaPi2b. This finding has raised significant interest in understanding NaPi2b function under physiological conditions and its deregulation in human pathologies, such as cancer. As a member of the sodium-dependent phosphate transporter family, NaPi2b is primarily involved in the maintenance of phosphate homeostasis in the human body.

Immunohistochemical analysis of S6K1 and S6K2 expression in human breast tumors.

Aim: To express recombinant S6K2 in baculovirus expression system; to purify large quantities of recombinant S6K2 for biochemical studies; to generate and characterise specific MABs against recombinant S6K2; to study the patterns S6K1 and S6K2 expression and subcellular localization in normal, benign and malignant breast tissues.

Methods: Recombinant baculovirus, expressing wild type S6K2 was generated using Bac-to-Bac system (Invitrogen); recombinant S6K was purified from infected Sf9 cells using affinity purification approach; monoclonal antibodies against recombinant S6K2 were generated; the specificity of generated MABs towards recombinant and endogenous S6K2 were examined by ELISA, Western blotting, immunoprecipitation and immuhohistochemical staining; immunohistochemical detection of S6K1 and S6K2 in human breast tissues was performed using specific monoclonal antibodies towards S6K1 and S6K2.

Results: Large amounts of enzymatically active S6K2 were purified using baculovirus expression system; highly purified preparations of S6K2 were used to generate and characterize anti-S6K2 MABs; elevated levels of S6K1 and S6K2 were found in breast tumors when compared to normal breast tissues; S6K2 is frequently localized in the nuclei of adenocarcinoma tissues, but rarely in fibroadenoma or "normal" breast tissues.

Molecular cloning of CoA Synthase. The missing link in CoA biosynthesis.

Coenzyme A functions as a carrier of acetyl and acyl groups in living cells and is essential for numerous biosynthetic, energy-yielding, and degradative metabolic pathways. There are five enzymatic steps in CoA biosynthesis. To date, molecular cloning of enzymes involved in the CoA biosynthetic pathway in mammals has been only reported for pantothenate kinase.