sCD30 and neopterin as risk factors of chronic renal transplant rejection: impact of cyclosporine A, tacrolimus, and mycophenolate mofetil.

High pretransplantation sCD30 levels have been shown to be associated with lower 5-year kidney graft survival in mainly Cyclosporine A (CsA)-treated recipients (Collaborative Transplant Study database). To analyze the effect of different immunosupressive regimens (CsA/Azathioprine [Aza], CsA/Mycophenolate Mofetil [MMF], Tacrolimus [Tacr]/Aza) on sCD30, we assessed serum sCD30 and neopterin together with in vitro cytokine responses in a prospective randomized study of 84 renal transplant recipients before, 4 months, and 1 year after transplantation. Panel-reactive antibody (PRA) formation, HLA matching, ATG induction therapy, and acute rejections had no impact on sCD30 levels, whereas cytomegalovirus (CMV) infections induced an up-regulation of sCD30 4 months posttransplantation (P = .003). Whereas MMF showed no effect on sCD30 compared with Aza therapy, we found a significant impact of Tacr versus CsA treatment (1-year sCD30 > or = 60 U/mL: 14/42 (33%), CsA; 1/38 (3%), Tacr; P < .0005). Chronic rejection 2 years posttransplantation was associated with elevated 1-year sCD30 (P = .001) and neopterin levels (P = .006). Our data indicate that the Th2 activation marker sCD30 provides a risk factor for chronic rejection independent of classical immunological risk factors and may be down-regulated using Tacr treatment.

A novel superoxide dismutase with a high isoelectric point in higher plants. expression, regulation, and protein localization.

Several isoforms of superoxide dismutase (SOD) with a high isoelectric point (pI) have been identified by isoelectric focusing chromatography in protein extracts from Scots pine (Pinus sylvestris) needles. One of these isoforms, a CuZn-SOD with a pI of about 10 and thus denoted hipI-SOD, has been isolated and purified to apparent homogeneity. A cDNA encoding the hipI-SOD protein was cloned and sequenced. Northern hybridization of mRNA isolated from different organs and tissues showed that hipI-SOD has a markedly different pattern of expression compared with chloroplastic and cytosolic SOD. Furthermore, the transcript levels of hipI-SOD and cytosolic SOD were found to respond differently to mechanical wounding, treatment with oxidized glutathione, paraquat, and ozone. Immunogold electron microscopy localized the hipI-SOD in the plasma membrane of sieve cells and the Golgi apparatus of albuminous cells. Moreover, high protein density was also detected in extracellular spaces such as secondary cell wall thickenings of the xylem and sclerenchyma and in intercellular spaces of parenchyma cells.

Isolation and purification of mitochondrial Mn-superoxide dismutase from the gymnosperm Pinus sylvestris L.

Manganese superoxide dismutase (Mn-SOD; EC 1.15.1.1) was purified from germinating seeds of Scots pine (Pinus sylvestris L.) 3 days after the start of imbibition. The purification schedule included (NH4)2SO4 fractionation, anion-exchange and hydrophobic-interaction chromatographies and chromatofocusing. Purified Mn-SOD had an apparent specific activity of 4,130 McCord-Fridovich units (mg protein)-1. The molecular mass of the holoenzyme was estimated to be 91 kDa by size-exclusion chromatography, and a molecular mass of 23 kDa was determined by SDS-PAGE. However, isoelectric focusing demonstrated that the purified enzyme consisted of three similarly migrating isoforms, with isoelectric points of approximately 6.5. NH2-terminal amino acid sequencing of purified Mn-SOD revealed no differences among the three isoforms. The comparison of the first 32 NH2-terminal amino acids with sequences of NH2-terminal amino acids of Mn-SODs from angiosperms reflected the phylogenetic distances between Scots pine, which is a gymnosperm, and angiospermic species. Cell fractionation suggested the mitochondrial localization of Mn-SODs and no evidence for glyoxysomal localization was found. Mn-SOD activity was absent from dry seeds. It was detectable at a considerable level after imbibition for 24 h, and it was again absent from 3-week-old seedlings.

Pinus sylvestris L. needles contain extracellular CuZn superoxide dismutase.

Four new isoforms of superoxide dismutase (SOD; superoxide: superoxide oxidoreductase, EC 1.15.1.1.) were identified in extracellular washing fluid from Scots pine (Pinus sylvestris L.) needles. The isoforms had an apparent molecular mass of 33 kDa. No neutral carbohydrates were present in the enzymes. The enzymatic activities were inhibited by 3 mM NaCN. One of the putative extracellular SOD isoforms was purified and NH2-terminal-sequenced. The sequence contained the domain KAVAVL. The domains VEG and V(K/S)G, present in chloroplastic and cytosolic CuZn SODs of plants, respectively, were not detected. The enzyme was composed of two subunits of 17.8 kDa each. The isoelectric point was determined to be 6.5. The results suggest the existence of an extracellular SOD in Scots pine.