Influence of GST polymorphisms on busulfan pharmacokinetics in Japanese children.

BACKGROUND: Fatal adverse effects or relapse can occur with excessive or insufficient busulfan exposure in hematopoietic stem cell transplantation. Given that busulfan is mainly metabolized by glutathione S-transferase (GST), we investigated the influence of GST polymorphisms on busulfan pharmacokinetics in Japanese pediatric patients. METHODS: Blood samples were taken from patients receiving high-dose i.v. busulfan as the first dose. Plasma busulfan concentration was measured using high-performance liquid chromatography. The area under the plasma busulfan concentration-time curve (AUC) was calculated. The genotype of GSTA1 was determined on polymerase chain reaction (PCR)-restriction fragment length polymorphism. Multiplex PCR was used to detect the presence or absence of GSTM1 and GSTT1 in the genomic DNA samples. RESULTS: Twenty patients were consecutively enrolled. Phenotype prediction was defined as follows: poor metabolizer (n = 4), one or more GSTA1*B haplotype or GSTM1/GSTT1 double-null genotypes; and extensive metabolizer (n = 16), other genotypes. GSTA1, M1, and T1 independently had no significant differences in AUC0-∞ , clearance or elimination rate constant. For the infant with unexpectedly high AUC0-∞ (2,591 µmol/L min), the GSTA1, M1, and T1 polymorphisms were wild type. On further analysis, the poor metabolizer group had lower clearance and higher AUC0-∞, except for the aforementioned patient, compared with the extensive metabolizer group (1,531 vs 1,010 µmol/L min; P < 0.01). CONCLUSIONS: GST polymorphisms may have affected busulfan pharmacokinetics, but these effects were obscured by other factors, such as underlying disease, systemic conditions, treatment history, and race.

The Neurospora crassa PCL-1 cyclin is a PHO85-1 (PGOV) kinase partner that directs the complex to glycogen metabolism and is involved in calcium metabolism regulation.

Cyclins are a family of proteins characterized by possessing a cyclin box domain that mediates binding to cyclin dependent kinases (CDKs) partners. In this study, the search for a partner cyclin of the PHO85-1 CDK retrieved PCL-1 an ortholog of yeast Pcls (for Pho85 cyclins) that performs functions common to Pcls belonging to different cyclin families. We show here that PCL-1, as a typical cyclin, is involved in cell cycle control and cell progression. In addition, PCL-1 regulates glycogen metabolism; Δpcl-1 cells accumulate higher glycogen levels than wild-type cells and the glycogen synthase (GSN) enzyme is less phosphorylated and, therefore, more active in the mutant cells. Together with PHO85-1, PCL-1 phosphorylates in vitro GSN at the Ser636 amino acid residue. Modeling studies identified PHO85-1 and PCL-1 as a CDK/cyclin complex, with a conserved intermolecular region stabilized by hydrophobic and polar interactions. PCL-1 is also involved in calcium and NaCl stress response. Δpcl-1 cells are sensitive to high NaCl concentration; on the contrary, they grow better and overexpress calcium responsive genes under high calcium chloride concentration compared to the wild-type strain. The expression of the calcium-responsive CRZ-1 transcription factor is modulated by PCL-1, and this transcription factor seems to be less phosphorylated in Δpcl-1 cells since exhibits nuclear location in these cells in the absence of calcium. Our results show that PCL-1 locates at different cell regions suggesting that it may determine its activity by controlling its intracellular location and reveal an interesting functional divergence between yeast and filamentous fungus cyclins.

cDNA cloning and expression of Contractin A, a phospholipase A2-like protein from the globiferous pedicellariae of the venomous sea urchin Toxopneustes pileolus.

Venomous sea urchins contain various biologically active proteins that are toxic to predators. Contractin A is one such protein contained within the globiferous pedicellariae of the venomous sea urchin Toxopneustes pileolus. This protein exhibits several biological activities, such as smooth muscle contraction and mitogenic activity. N-terminal amino acid residues of Contractin A have been determined up to 37 residues from the purified protein. In this study, we cloned cDNA for Contractin A by reverse transcription-PCR using degenerate primers designed on the basis of its N-terminal amino acid sequence. Analysis of the cDNA sequence indicated that Contractin A is composed of 166 amino acid residues including 31 residues of a putative signal sequence, and has homology to the sequence of phospholipase A2 from various organisms. In this study, recombinant Contractin A was expressed in Escherichia coli cells, and the protein was subjected to an assay to determine lipid-degrading activity using carboxyfluorescein-containing liposomes. As a result, Contractin A was found to exhibit Ca(2+)-dependent release of carboxyfluorescein from the liposomes, suggesting that Contractin A has phospholipase A2 activity, which may be closely associated with its biological activities.