Induction of an extracellular cyclic nucleotide phosphodiesterase as an accessory ribonucleolytic activity during phosphate starvation of cultured tomato cells.

During growth under conditions of phosphate limitation, suspension-cultured cells of tomato (Lycopersicon esculentum Mill.) secrete phosphodiesterase activity in a similar fashion to phosphate starvation-inducible ribonuclease (RNase LE), a cyclizing endoribonuclease that generates 2':3'-cyclic nucleoside monophosphates (NMP) as its major monomeric products (T. Nürnberger, S. Abel, W. Jost, K. Glund [1990] Plant Physiol 92: 970-976). Tomato extracellular phosphodiesterase was purified to homogeneity from the spent culture medium of phosphate-starved cells and was characterized as a cyclic nucleotide phosphodiesterase. The purified enzyme has a molecular mass of 70 kD, a pH optimum of 6.2, and an isoelectric point of 8.1. The phosphodiesterase preparation is free of any detectable deoxyribonuclease, ribonuclease, and nucleotidase activity. Tomato extracellular phosphodiesterase is insensitive to EDTA and hydrolyzes with no apparent base specificity 2':3'-cyclic NMP to 3'-NMP and the 3':5'-cyclic isomers to a mixture of 3'-NMP and 5'-NMP. Specific activities of the enzyme are 2-fold higher for 2':3'-cyclic NMP than for 3':5'-cyclic isomers. Analysis of monomeric products of sequential RNA hydrolysis with purified RNase LE, purified extracellular phosphodiesterase, and cleared -Pi culture medium as a source of 3'-nucleotidase activity indicates that cyclic nucleotide phosphodiesterase functions as an accessory ribonucleolytic activity that effectively hydrolyzes primary products of RNase LE to substrates for phosphate-starvation-inducible phosphomonoesterases. Biosynthetical labeling of cyclic nucleotide phopshodiesterase upon phosphate starvation suggests de novo synthesis and secretion of a set of nucleolytic enzymes for scavenging phosphate from extracellular RNA substrates.

Pharmacokinetics of mycophenolate mofetil in renal transplant recipients on peritoneal dialysis.

Little is known about the pharmacokinetics of mycophenolatic acid (MPA) in the early posttransplant period after renal transplantation. We studied the impact of peritoneal dialysis on the pharmacokinetics of MPA in 5 patients following renal transplantation (3-6 weeks after transplantation). Three patients had a glomerular filtration rate (GFR) of less than 10 ml/min, 1 patient had a GFR of 32, and 1 of 58 ml/min. Pharmacokinetics of MPA and its main metabolite mycophenolic acid glucuronide (MPAG) were studied on 2 consecutive days (12-hour intervals: with and without peritoneal dialysis). Dosing of MPA was 2 x 1 g/day. MPA and MPAG concentrations were determined by HPLC methods. After initiation of peritoneal dialysis in patients with severe renal impairment (GFR < 10 ml/min) MPA area under the concentration curve (AUC) decreased substantially (15-59%). The calculated clearance of MPA was higher (14.6 vs 8.1 ml/min/kg) on the day of peritoneal dialysis than during the dwell-free day. MPAG-AUC decreased up to 26% in these patients. In both patients with a GFR > 30 ml/min we observed an increase of MPA-AUC on the day of peritoneal dialysis and a decreased MPA clearance. MPAG-AUCs remained stable. Patients with a reduced GFR had much higher MPAG values than patients with a GFR 30 ml/l, however, we did not observe any differences for the MPA levels. We found a significant inverse correlation between GFR and MPAG-AUC (r = 0.91, p < 0.05). While MPA was found only in traces in the peritoneal ultrafiltrate, the cumulative amount of MPAG removed by peritoneal dialysis reached up to 2 g per 12 hours, representing up to 1.2 g of MPA. This is the first report describing a reduction of MPA- and MPAG-AUC during peritoneal dialysis. Further studies are needed to better understand the pharmacokinetics of mycophenolat mofetil during peritoneal dialysis.

Mycophenolate mofetil pharmacokinetics in renal transplant recipients on peritoneal dialysis.

We prospectively studied the impact of peritoneal dialysis (PD) on the pharmacokinetics of mycophenolic acid (MPA) in five patients following renal transplantation. Three patients had a glomerular filtration rate (GFR) of less than 10 ml/min and two had a GFR of more than 40 ml/min. Pharmacokinetics of MPA and of its main metabolite, mycophenolic acid glucuronide (MPAG), were studied during two consecutive 12-h periods (with and without PD). After initiation of PD in patients with severe renal impairment (GFR < 10 ml/ min), MPA-area-under-the-concentration-curve (AUC) decreased up to 59% and MPAG-AUC decreased up to 26%. We did not observe any substantial changes in the MPA-AUC or MPAG-AUC of either patient with a GFR above 40 ml/min. Patients with a reduced GFR had much higher MPAG values than patients with a GFR above 40 ml/l; yet, we did not observe any differences in the MPA values. We found a significant inverse correlation between GFR and MPA-AUC (r = 0.81, P < 0.05) and between GFR and MPAG-AUC (r = 0.94, P < 0.01). While MPA was found only in traces in the peritoneal ultrafiltrate, the cumulative amount of MPAG removed by PD reached up to 2 g/ 12 h, representing 1.2 g of MPA. This is the first report describing a reduction in MPA-AUC and MPAG-AUC during PD. Further studies are needed to completely understand the pharmacokinetics of mycophenolate mofetil during PD.

Cucumber T-complex protein. Molecular cloning, bacterial expression and characterization within a 22-S cytosolic complex in cotyledons and hypocotyls.

T-complex protein (TCP) found in mammalian cells and yeast has been proposed as cytosolic folding machinery. We report here the cloning and initial characterization of a plant TCP cDNA. CSTCP-1 cDNA prepared from mRNA of cotyledons of germinating cucumber seeds encodes a polypeptide composed of 535 amino acid residues. The 59157-Da protein exhibits only 28% identity to both TCP-1p from yeast or and its homolog in Arabidopsis thaliana. Antibodies raised against the bacterially expressed plant protein were used to analyze the intracellular localization of TCP in two different plant tissues: fat-degrading non-dividing cotyledons and meristematic hypocotyls during seed germination. Cell fractionations included differential centrifugation and sedimentation of large complexes at 23000O x g for 4h. The latter fraction was further fractionated by sedimentation velocity centrifugation. This enrichment was required to detect by Western blotting cytosolic 59-kDa species as constituents of 22-S particles. From hypocotyls, a preparation of T-complex was obtained which consisted almost exclusively of proteins in the molecular range of 57-62 kDa. Likewise, the radioactive Cucumis sativus TCP-1 synthesized from CSTCP-1 mRNA in vitro using reticulocyte lysate was shown to migrate as a 61-kDa species.