Pi Treatment Research Articles

The WRKY6 Transcription Factor ModulatesPHOSPHATE1Expression in Response to Low Pi Stress inArabidopsis

Arabidopsis thaliana WRKY family comprises 74 members and some of them are involved in plant responses to biotic and abiotic stresses. This study demonstrated that WRKY6 is involved in Arabidopsis responses to low-Pi stress through regulating PHOSPHATE1 (PHO1) expression. WRKY6 overexpression lines, similar to the pho1 mutant, were more sensitive to low Pi stress and had lower Pi contents in shoots compared with wild-type seedlings and the wrky6-1 mutant. Immunoprecipitation assays demonstrated that WRKY6 can bind to two W-boxes of the PHO1 promoter. RNA gel blot and beta-glucuronidase activity assays showed that PHO1 expression was repressed in WRKY6-overexpressing lines and enhanced in the wrky6-1 mutant. Low Pi treatment reduced WRKY6 binding to the PHO1 promoter, which indicates that PHO1 regulation by WRKY6 is Pi dependent and that low Pi treatment may release inhibition of PHO1 expression. Protein gel blot analysis showed that the decrease in WRKY6 protein induced by low Pi treatment was inhibited by a 26S proteosome inhibitor, MG132, suggesting that low Pi-induced release of PHO1 repression may result from 26S proteosome-mediated proteolysis. In addition, WRKY42 also showed binding to W-boxes of the PHO1 promoter and repressed PHO1 expression. Our results demonstrate that WRKY6 and WRKY42 are involved in Arabidopsis responses to low Pi stress by regulation of PHO1 expression.

Chromosomal Abnormalities in Transformed Ph-Negative Myeloproliferative Neoplasm Are Independent of the JAK2 and the TET2 Statuses.

Abstract 2900Poster Board II-876Ph-negative myeloproliferative neoplasms: polycythemia vera (PV), essential thrombocythemia (ET) and primitive myelofibrosis (PMF) carry an acquired somatic mutation JAK2V617F in 95% (PV), and in 50 to 60% (ET or PMF) of the patients. Mutations of the TET2 gene have been observed with roughly similar frequencies in the three MPN, irrespective of the presence of JAK2V617F. Evolution to myelofibrosis or acute leukemia may occur with time in MPN patients. Although its molecular bases are poorly understood, the evolution is likely due to the acquisition of additional mutations.To investigate whether cytogenetic abnormalities are distributed differently according to type of transformation and to the JAK2 and TET2 statuses, the Groupe Francophone de Cytogénétique Hématologique has collected 82 patients with transformation of MPN.There were 66 (80%) acute myeloid leukemia or myelodysplastic syndromes (AML/MDS) and 16 (20%) myelofibroses (MF). Of note pipobroman (Pi) treatment seems to be associated with MF, and hydroxyuera (Hu) with AML/MDS evolution in our series. Statistical analyses of clinical, cytogenetic and molecular data are shown Table 1.On the cytogenetical point of view, several points are noteworthy.Some abnormalities were unevenly distributed: there were significantly more -7/del7q and -5/del5q in AML/MDS and tri1q and tri9 in MF. MF and PMF cytogenetic profile looked similar, suggesting a potential link between cytogenetic markers and the phenotype.Although the derivative chromosome der(1;7), observed in 9 patients, is responsible for a loss of 7q, it seemed different from patients with -7/del7q [excluding der(1;7)]. In the -7/del7q group, AML/MDS patients were more numerous than MF patients and the overall survival was shorter compared with the der(1;7) group (22/22 (100%) vs 6/9 (67%) AML/MDS, p=0.02; median: 4 vs 41 months, p=0.0007 respectively).Some specific associations could be observed, such as 17p deletions with 5q deletion (12/30, 40% vs 4/48, 8%, p=0.0007) and 20q deletion with der(1;7) (4/9 (44%) vs11/69 (16%), p=0.03).We detected 24/40 (60%) JAK2V617F and 8/25 (32%) TET2 mutations in transformed MPN, with all possible combinations between the wildtype and mutated forms of both genes. For one post-ET AML patient, JAK2V617F had been observed in a fraction of the granulocytes at the chronic phase. Analysis of blood cDNA obtained at chronic phase showed the same TET2 mutation as observed at acute phase. Because the blast cells were JAK2wt-TET2mut and carried a t(10;16)(q22;q23) affecting the CBFB gene, it is likely that the resulting non-MYH11 CBFB fusion gene transformed a JAK2wt-TET2 mutated progenitor that predominated in the chronic phase.In conclusion, no specific chromosomal abnormality was associated with TET2 or JAK2 mutations. Chromosomal abnormalities were associated with a type of transformation (AML/MDS or MF), suggesting a specific role in the process. In addition, association between some chromosomal abnormalities suggest a specific oncogenic cooperation.Table 1n=82AML/MDS n=66MF n=16p univariatep multivariateSex F39 (59%)5 (31%)nsnsPV/ET/PMF30/26/1013/3/0nsnsAge at diagnosis of MPN54 [20-82]55.5 [31-69]nsnsChronic Phase (duration, years)12 [2-34]14.5 [3-28]nsnsPrior treatments (n=73*)57*16..No treatment (n=6)60nsnsOne treatment (n=40)33 (58%)7 (44%)nsnsTreatments with Hu (n=57)48 (73%)9 (56%)0.03Treatments with Pi (n=41)26 (46%)15 (93%)0.00060.05Age at transformation66.5[37-92]68 [45-80]nsnsAbnormal karyotype62 (94%)16 (100%)nsnsComplex karyotype45 (68%)7 (44%)nsns-7/del7q28 (42%)3 (18%)0.07ns-7/del7q[without der(1;7)]22 (33%)00.0040.04-5/del5q28 (42%)2 (12%)0.03ns-13/del13q5 (8%)3 (19%)nsns-20/del20q11 (17%)4 (25%)nsns-17/del17p15 (23%)1(6%)nsns+1q14 (22%)9 (56%)0.01ns+95 (8%)4 (25%)0.04ns+811 (17%)3 (19%)nsnsdic17 (26%)3 (19%)nsnsder(1;7)6 (9%)3 (19%)nsnsAmplification MLL0 (0%)nsnsJAK2mut17/31 (55%)7/9 (78%)nsnsTET2mut6/19 (32%)2/6 (33%)nsnsMedian overall survival (months)448<0.00010.001*treatment unknown for 3 patients Disclosures:No relevant conflicts of interest to declare.

Clinical Evidence of Interaction Between Itraconazole and Nonnucleoside Reverse Transcriptase Inhibitors in HIV-Infected Patients with Disseminated Histoplasmosis

Itraconazole is the preferred drug for chronic maintenance therapy in HIV-infected patients with disseminated histoplasmosis. Unfortunately, few clinical data exist confirming a presumed interaction between itraconazole and nonnucleoside reverse transcriptase inhibitors (NNRTIs). To determine whether serum itraconazole concentrations are affected by the type of antiretroviral therapy (NNRTI or protease inhibitor [PI]) being taken concomitantly. This retrospective cohort identified patients on antiretroviral therapy and itraconazole for disseminated histoplasmosis between January 2003 and December 2006 at a large HIV clinic in Houston, TX. Available laboratory values were abstracted from medical records. Thirteen itraconazole concentrations from 10 patients were available for analysis: 7 patients were on concomitant PIs, 4 on concomitant NNRTIs, and 2 on antiretroviral regimens containing both PIs and NNRTIs. Six of the itraconazole concentrations during concomitant PI treatment were therapeutic (>1.0 microg/mL), in contrast with none in patients taking an NNRTI. All patients taking concomitant NNRTIs had undetectable serum itraconazole concentrations (<0.05 microg/mL). Two patients switched from NNRTI-based to PI-based antiretroviral regimens and subsequently reached therapeutic itraconazole concentrations. Although limited by small sample size, this study provides the largest clinical data among HIV-infected patients demonstrating that coadministration of an NNRTI and itraconazole results in significant decreases in itraconazole blood concentrations, likely by inducing the CYP3A4 enzyme system. Itraconazole concentrations should be monitored in patients taking concomitant NNRTIs. PI-based highly active antiretroviral therapy (HAART) may be preferred over NNRTI-based HAART when itraconazole is used to treat HIV-infected patients with disseminated histoplasmosis.

Improving phosphorus acquisition of white clover (Trifolium repens L.) by transgenic expression of plant-derived phytase and acid phosphatase genes

Phosphate is one of the least available macronutrients restricting crop production in many ecosystems. A phytase gene (MtPHY1) and a purple acid phosphatase gene (MtPAP1), both isolated from the model legume Medicago truncatula, were introduced into white clover (Trifolium repens L.) by Agrobacterium-mediated transformation. The transgenes were driven by the constitutive CaMV35S promoter or the root-specific MtPT1 promoter. Transcripts were detected in roots of the transgenic plants. Phytase or acid phosphatase (APase) activities in root apoplasts of the transgenic plants were increased up to three-fold compared to the wild type control. After the plants were grown 80 days in sand pots supplied with organic phosphorus (Po) as the sole P source, dry weights of shoot tissues of the best performing transgenic plants almost doubled that of the control and were comparable to the counterparts supplied with inorganic phosphorus (Pi). Relative biomass production of the transgenics under Po treatment was over 90% and 80% of that from the Pi treatment when the plants were grown in hydroponics (40 days) and sand pots (80 days), respectively. In contrast, biomass of the wild type controls under Po treatment was only about 50% of the Pi treatment in either hydroponic cultures or sand pots. In addition, shoot P concentrations of the transgenic plants were significantly increased compared to the control. Transgenic plants accumulated much higher amounts of total P (up to 2.6-fold after 80 days of growth) than the control in Po supplied sand pots. The results showed that transgenic expression of MtPHY1 or MtPAP1 in white clover plants increased their abilities of utilizing organic phosphorus in response to P deficiency.

Reduction of root flavonoid level and its potential involvement in lateral root emergence in Arabidopsis thaliana grown under low phosphate supply.

Although it is well known that phosphate (Pi) deficiency affects flavonoid accumulation in higher plants, knowledge on the regulation and potential function of flavonoids in the plants grown with low Pi supply is lacking. In this work, we found that low Pi treatment caused significant reduction of root flavonoid (e.g. quercetin, kaempferol and their derivatives) levels in both Columbia (Col-0) and Landsberg erecta (Ler) ecotypes of Arabidopsis thaliana (L.) Heynh. Further investigations revealed that the dysfunction of PHR1, PHO1, PHO2 and NPC4 did not affect the decrease of root flavonoid level by low Pi treatment. In contrast, pldζ2, a knockout mutant of the Arabidopsis phospholipase Dζ2, exhibited defects in the reduction of root flavonoid level and lateral root (LR) emergence under low Pi conditions. When grown under low Pi supply, the transport of auxin from the shoot apex into the root, expression of the auxin responsive DR5::GUS marker and induction of the auxin responsive genes were all significantly less efficient in pldζ2 than in wild-type (WT) control. This is the first report on the reduction of root flavonoid level and its likely contribution to increased LR emergence in Arabidopsis under Pi deficiency conditions, which may facilitate the adaptation of plants to the growth environments with poor Pi availability.

Long-Term Outcome in Polycythemia Vera (PV): Final Analysis of a Randomized Trial Comparing Hydroxyurea (HU) to Pipobroman (Pi).

Background: Only 4 randomized clinical trials in PV have directly compared cytoreductive therapies (32P, busulfan, chlorambucil, HU, and Pi), all published after a median follow-up of 10 years or less. Thrombosis was the main cause of mortality and morbidity in all trials. Cumulative incidence of evolution to myelofibrosis (MF), and acute myeloid leukemia or myelodysplastic syndromes (AML/MDS) was about 8%, and 6%, respectively. In 1 of those trials, conducted between 1980 and 1996 by the French Polycythemia Study Group (FPSG) in 292 PV patients (pts) younger than 65 yrs, no difference between HU and Pi was found in terms of survival, risk of thrombosis, and AML/MDS/MF evolution at the time of first analysis with a median follow up of 7 years (Najean, Blood , 1997, 90:3370).Methods: We updated the results of the FPSG trial at the reference date of April 15, 2008, with a median follow-up of 16.3 years. After randomization in the trial (136 pts in the HU arm, 149 in the Pi arm), 94 (33%) pts had received only HU, 130 (46%) only Pi, and 61 (21%) both drugs (including 42 who had switched from HU to Pi, and 19 from Pi to HU) during the whole follow-up period (7 patients were excluded from the final analysis because of incomplete follow up data). To take into account treatment crossovers, statistical analysis was performed both in “intention to treat” (ITT) and according to the main treatment received (i.e. treatment effectively received).Results: Median survival was 20.3 years (95%CI: 16.4 – 25) in HU arm, and 15.4 years (95%CI: 13.4 – 17) in Pi arm (p=0.008). 95 pts have died, the 3 main causes of death being evolution to AML/MDS in 51 pts (54%), vascular events in 19 pts (20%), and solid tumor in 11 (12%) pts. The 51 cases of evolution to AML/MDS included 10 MDS, and 41 AML (including 5 preceded by an MDS phase). Cumulative incidence (CI) of AML/MDS at 10, 15, and 20 years in ITT analysis was 6.6%, 16.5%, and 24% in the HU arm, and 13%, 34%, and 52% in the Pi arm, respectively (p= 0.004). As the median duration of HU treatment (12 years) was significantly longer than that of Pi treatment (9.5 years, p=0.0002), we also performed the analyses according to the main treatment actually received by pts, which showed cumulative incidence of AML/MDS of 7%, 14%, and 22% with HU, and 12%, 37%, and 56% with Pi at 10, 15, and 20 years, respectively (p=0.008), confirming the results obtained in ITT analyses. Regarding MF, the CI at 10, 15 and 20 years was comparable in both arms in ITT analysis: 12.6%, 19%, and 27% in the HU arm, and 7.8%, 16%, and 27% in the Pi arm, respectively (p=0.7). However, a significantly higher incidence of MF was found in pts who had received HU as main treatment: 15%, 24%, and 32% at 10, 15, and 20 years, compared to 5%, 10%, and 21%, respectively, in patients who received mainly Pi (p=0.02).Conclusion: Although medium-term analysis of FPSG trial did not show differences between HU and Pi, current update after a median follow-up of 16.3 years finally showed that median survival was shorter, and incidence of AML/MDS higher in Pi treated pts. By contrast, risk of MF was higher in pts treated predominantly with HU. Of note, was the clearly higher than previously reported cumulative incidence of AML/MDS with HU. Evolution to AML/MDS represented by far the first cause of death in the long-term (54% versus 20% for vascular events). Those results suggest that Pi should not be used as first line therapy in PV patients, and that very long term may be required to draw conclusions regarding leukemic evolution in PV trials.

Influence of Phosphate on the Arsenic Uptake by Wheat (Triticum durum L.) Irrigated with Arsenic Solutions at Three Different Concentrations

In this study we have investigated the uptake and distribution of arsenic (As) and phosphate (Pi) in roots, shoots, and grain of wheat grown in an uncontaminated soil irrigated with solutions containing As at three different concentrations (0.5, 1 and 2 mg l−1) and in the presence or in the absence of P fertilization. Arsenic in irrigation water reduced plants growth and decreased grain yield. When Pi was not added (P−), plants were more greatly impacted compared to the plus Pi (P+) treatments. The differences in mean biomass between P− and P+ treatments at the higher As concentrations demonstrated the role of Pi in preventing As toxicity and growth inhibition. Arsenic concentrations in root, shoot and grain increased with increasing As concentration in irrigation water. It appears that P fertilization minimizes the translocation of As to the shoots and grain whilst enhancing P status of plant. The observation that P fertilization minimises the translocation of arsenic to the shoots and grain is interesting and may be useful for certain regions of the world that has high levels of As in groundwater or soils.

INFLUENCE OF CASSAVA (MANIHOT ESCULENTA) INTERCROP ON GROWTH AND FRUIT YIELDS OF PEPPER (CAPSICUM SPP.) IN SOUTH-WESTERN NIGERIA

In tropical Africa, pepper (Capsicum spp.) is grown as a rainfed crop, and its production is limited by the long, hot growing season. Field experiments were conducted in Nigeria to evaluate the effects of cassava (Manihot esculenta) on the growth and yields of three pepper cultivars and gross returns in 2001–2003. In Experiment 1, pepper (cv. Sombo) was planted between rows of cassava cvs Idileru (PI), Odongbo (PO) and TMS 30572 (PT). In Experiment 2, pepper cvs Sombo, Tatase and Atarodo, were mixed with TMS 30572 (MS, MT or MA). The growth environment for the intercropped pepper differed from sole crops of pepper. Radiant energy reaching the soil surface, maximum diurnal soil and canopy temperatures, and weed growth were lower with intercropping, with the lowest values being observed in the PI and PT intercrops. Similarly, soil moisture content and the number of earthworm casts were greater with intercropping, with the highest values also occurring in the PI and PT intercrops. In both experiments, fresh fruit yields of pepper depended on the duration of harvest, the number of fruits per plant and the weight of fruits. In Experiment 1, although the number of fruits and fruit yield of cv. Sombo were greater in the sole crop (SP) than the PO intercrop, the fruit yields in the PI and PT intercrops were similar to those of the SP plot. In Experiment 2, the number of fruits and yield of intercropped pepper cvs Tatase, Sombo and Atarodo were 25–28 % higher, on average, than in pure stands. Cassava tuber yield was not affected by intercropped pepper in either experiment. Total gross returns were greater than growing either pepper or cassava in monoculture. Increased total gross returns in the intercrops were obtained in the PI and PT treatments and in the MS and MA treatments without a significant reduction in pepper fruit yield. By promoting early fruit set and harvest, and bearing in mind the cumulative gross returns, mixing pepper and cassava enhanced the value of the vegetable, as early fresh pepper fruits command a premium price. It is concluded that pepper can be grown between cassava rows to provide a suitable environment for growth, but that this depends on the cassava cultivar. Using the less tall early cassava cultivar, with a relatively moderate leaf area index in a mixture with pepper is therefore recommended.

Inorganic phosphate inhibits growth of human osteosarcoma U2OS cells via adenylate cyclase/cAMP pathway

In order to elucidate how phosphate regulates cellular functions, we investigated the effects of inorganic phosphate (Pi) on adenylate cyclase (AC)/cyclic AMP (cAMP) axis. Here we describe that Pi treatment of human osteosarcoma U2OS cells results in a decrease of both intracellular cAMP levels and AC activity, and in a cell growth inhibition. The phosphate-triggered effects observed in U2OS cells are not a widespread phenomenon regarding all cell lines, since other cell lines screened respond differently to parallel Pi treatments. In U2OS cell line, the AC activity/cAMP downregulation is accompanied by significant variations in the levels of some membrane proteins belonging to the AC system. Remarkably, the above effects are blunted by pharmacological inhibition of sodium-dependent phosphate transport. Moreover, 8-Br-cAMP and other cAMP-elevating agents, such as IBMX and forskolin, interestingly, prevent the cell growth inhibition in response to phosphate. Our results enforce the increasing evidences of phosphate as a signaling molecule, identifying in U2OS cell line the AC/cAMP axis, as a novel-signaling pathway modulated by phosphate to ultimately affect cell growth.

Pioglitazone prevents cardiac remodeling in high-fat, high-calorie-induced Type 2 diabetes mellitus

The agonists of peroxisome proliferator-activated receptor-gamma (PPARgamma) ameliorate cardiovascular complications associated with diabetes mellitus. We tested the hypothesis that recovery from ailing to failing myocardium in diabetes by PPARgamma agonist is in part due to decreased matrix metalloproteinase-9 (MMP-9) activation and left ventricular (LV) tissue levels of homocysteine (Hcy). C57BL/6J mice were made diabetic (D) by feeding them a high-fat calorie diet. PPARgamma was activated by adding pioglitazone (Pi) to the diet. After 6 wk, mice were grouped into: normal calorie diet (N), D, N + Pi and D + Pi (n = 6 in each group). LV variables were measured by echocardiography, endothelial-myocyte (E-M) coupling was measured in cardiac rings, and MMP-9 activation was measured by zymography. Blood glucose levels were twofold higher in D mice compared with N mice. Pi decreased the levels of glucose in D mice to the levels in N mice. LV Hcy levels were 3.5 +/- 0.5 microM in N groups compared with 12.4 +/- 0.6 microM in D groups. Treatment with Pi normalized the LV levels of Hcy but had no effect on plasma levels of Hcy. In the D group, LV contraction was reduced compared with that of the N group and was ameliorated by treatment with Pi. LV wall thickness was reduced to 0.25 +/- 0.02 mm in the D group compared with 0.42 +/- 0.01 mm in the N group. LV diastolic diameter was 3.05 +/- 0.01 mm in the D group compared with 2.20 +/- 0.02 mm in the N group. LV systolic diameter was 1.19 +/- 0.02 mm in the D group and 0.59 +/- 0.01 mm in the N group. Pi normalized the LV variables in D mice. The responses to ACh and nitroprusside were attenuated in diabetic hearts, suggesting that there was E-M uncoupling in the D group compared with the N group, which was ameliorated by Pi. Plasma and LV levels of MMP-2 and -9 activities were higher in the D group than in the N group but normalized after Pi treatment. These results suggest that E-M uncoupling in the myocardium, in part, is due to increased MMP activities secondary to suppressing PPARgamma activity in high-fat, calorie-induced Type 2 diabetes mellitus.

Effect of ischemia reperfusion on sodium-dependent phosphate transport in renal brush border membranes

The effect of ischemia induced acute renal failure (ARF) on the transport of phosphate (Pi) after early (15–30 min) and prolonged (60 min) ischemia in the brush border membrane vesicles (BBMV) from rat renal cortex was studied. Sodium-dependent transport of Pi declined significantly and progressively due to ischemia. Western blot analysis of BBM from ischemic rats showed decreased expression of NaPi-2. A compensatory increase was observed in Pi uptake in BBMV from contralateral kidneys. There was no significant difference in NaPi-2 expression between BBMV from sham and contralateral kidneys. Early blood reperfusion for 15 min after 30 min ischemia caused further decline in Pi uptake. Prolonged reperfusion for 120 min caused partial reversal of transport activities in 30-min ischemic rats. However, no improvement in the transport of Pi was observed in 60-min ischemic rats after 120 min of blood reperfusion. Kinetic studies showed that the effect of ischemia and blood reperfusion was dependent on the V max of the Na-Pi transporter. Western blot analysis showed increased expression of NaPi-2 in the BBMs from ischemia–reperfusion animals. Further, a shift in the association of Na ions to transport one molecule of Pi was observed under different extracellular Na concentrations [Na] o. Feeding rats with low Pi diet and/or treatment with thyroid hormone (T3) prior to ischemia resulted in increased basal Pi transport. Ischemia caused similar decline in Pi transport in BBM from LPD and/or T3 animals. However, recovery in these animals was faster than the normal Pi diet fed (NPD) animals. The study suggests a change in the intrinsic properties of the Na-Pi transporter in rat kidneys due to ischemia. The study also indicates that treatment with T3 and feeding LPD prior to ischemia caused faster recovery of phosphate uptake due to ischemia–reperfusion injury.

Interactive effects of inorganic phosphate nutrition and carbon dioxide enrichment on assimilate partitioning in barley roots

The combined effects of inorganic phosphate (Pi) insufficiency and CO2 enrichment on metabolite levels and carbon partitioning were studied using roots of 9‐, 13‐ and 17‐day‐old barley seedlings (Hordeum vulgare L. cv. Brant). Plants were grown from seed in controlled environment chambers providing 36 ± 1 Pa (ambient) or 100 ± 2 Pa (elevated) CO2 and either 1.0 mM (Pi sufficient) or 0.05 mM (Pi insufficient) Pi. When values were combined for both Pi treatments, plants grown under enhanced CO2 showed increased root dry matter, adenylates (ATP + ADP), glutamine and non‐ structural carbohydrates other than starch. In contrast with shoots, enhanced CO2 partially reversed the inhibition of root dry matter formation imposed by Pi insufficiency. The Pi‐insufficient treatment also increased sucrose, glucose and fructose levels in barley roots. The Pi and CO2 treatments were additive, so that the highest soluble carbohydrate levels were observed in roots of Pi‐insufficient plants from the elevated CO2 treatment. Pi limitation decreased dry matter formation, acid‐extractable Pi, nitrate, hexose‐phosphates, glutamate, glutamine and acid invertase activity of barley roots in plants grown in both ambient and elevated CO2. Adenylate levels in roots were unaffected by the moderate Pi insufficiency described here. Thus, the reduced hexose‐phosphate levels of Pi‐insufficient roots were not likely to be the result of low adenylate concentrations. The above results suggest that the capacity of barley roots to utilize carbohydrates from the shoot is inadequate under both Pi‐insufficient and CO2‐enriched treatments. In addition, the Pi and CO2 treatments used here alter the nitrogen metabolism of barley roots. These findings further emphasize the importance of avoiding nutrient stress during CO2 enrichment experiments.

Role of Ligands in Accumulation and Fractionation of Rare Earth Elements in Plants: Examples of Phosphate and Citrate

Few studies have been carried out on the effects of ligands on rare earth element (REE) bioaccumulation processes. In this study, the effects of phosphate (Pi, an inorganic ligand) and citrate (an organic ligand) on accumulation and fractionation of REEs in wheat were investigated using aqueous culture with extraneous mixed REEs (MRE). The results show that initial Pi solution culture at various levels followed by exposure to a fixed-MRE solution did not significantly change the total concentrations of REEs (SigmaREE) in roots, whereas the SigmaREE in leaves dramatically decreased with increasing levels of Pi applied. Simultaneous culture of wheat with mixture of MRE and citrate solutions caused obvious decreases of the SigmaREE in both roots and leaves. Compared with MRE, significant fractionations of REEs were found in wheat organs when no ligand was applied. Notable middle REE (MREE) enrichment and M-type tetrad effect were observed in the roots, and heavy REE (HREE) enrichment and W-type tetrad effect existed in the leaves. Pi treatments did not significantly affect the fractionations of REEs in the roots, but enrichment of HREEs in the leaves slightly increased at the highest level of Pi applied. Fractionations of REEs in both roots and leaves decreased with increasing levels of citrate applied; at higher levels of citrate (> or =150 microM), no above fractionation features were observed in wheat, but light REE (LREE) enrichment existed in the roots and leaves. The results indicate that ligands might play important roles in accumulation and fractionation of REEs during bioaccumulation processes.

Organic phosphorus utilization by wheat plants under sterile conditions

An experiment was set up to make a critical assessment of the role of organic P (Po) in soil solution in the nutrition of wheat plants under sterile conditions. Three concentration gradients of Po (17.08, 27.31 and 37.61 μM l-1) were created in a sterilized Oxisol by fertilization with a soil solution prepared by a dry freezing technique and containing antibiotics to minimize microbial growth. Due to the high P fixing capacity of the Oxisol and high buffering capacity of inorganic P (Pi), as compared to Po, a negligible change in Pi concentration occurred, due to fertilization after equilibrium in soil solution. Phosphorus supply had a positive effect on dry matter and P concentration of the plants. Acid phosphatase secretion by plant roots was 5–11 times higher in Po treatments than in the respective Pi treatments. No alkaline phosphatase activity was detected, confirming the absence of microbial activity in our system. Net P inflow into plants was significantly higher in the Pi + Po treatments at all three Po concentrations than in the respective Pi (control) treatment, providing evidence for the role of Po in the P nutrition of plants. It was hypothesized that plants secrete phosphatases in response to the presence of Po in soil solution and Po might be responsible for the increase in P influx to wheat plants.

High pressure induces superoxide production in isolated arteries via protein kinase C-dependent activation of NAD(P)H oxidase.

Oxidative stress seems to be present in all forms of hypertension. Thus, we tested the hypothesis that high intraluminal pressure (Pi) itself, by activating vascular oxidases, elicits increased superoxide (O2*-) production interfering with flow-induced dilation. Isolated, cannulated rat femoral arterial branches were exposed in vitro (for 30 minutes) to normal Pi (80 mm Hg) or high Pi (160 mm Hg). High Pi significantly increased vascular O2*- production (as measured by lucigenin chemiluminescence and ethidium bromide fluorescence) and impaired endothelium-dependent dilations to flow; these effects could be reversed by superoxide dismutase. Administration of the NAD(P)H oxidase inhibitor diphenyleneiodonium, apocynin, the protein kinase C (PKC) inhibitor chelerythrine or staurosporin or the removal of extracellular Ca2+ during high Pi treatment prevented the increases in O2*- production, whereas administration of losartan or captopril had no effect. High Pi resulted in significant increases in intracellular Ca2+ ([Ca2+]i) in the vascular wall (fura 2 fluorescence) and phosphorylation of PKCalpha (Western blotting). The PKC activator phorbol myristate acetate significantly increased vascular O2*- production, which was inhibited by superoxide dismutase, diphenyleneiodonium, chelerythrine, or removal of extracellular Ca2+. Both high Pi and phorbol myristate acetate increased the phosphorylation of the NAD(P)H oxidase subunit p47phox. High Pi itself elicits arterial O2.- production, most likely by PKC-dependent activation of NAD(P)H oxidase, thus providing a potential explanation for the presence of oxidative stress and endothelial dysfunction in various forms of hypertension and the vasculoprotective effect of antihypertensive agents of different mechanisms of action.

Prolonged high-dose phosphate treatment: a risk factor for tertiary hyperparathyroidism in X-linked hypophosphatemic rickets.

X-linked hypophosphatemic rickets is characterized by renal phosphate wasting, hypophosphatemia and defective bone mineralization. Treatment with oral phosphate (Pi) and calcitriol improves skeletal changes but associates with secondary hyperparathyroidism and nephrocalcinosis. Tertiary hyperparathyroidism is a rare complication of the treatment. The aim of the present study was to identify treatment-related factors that might be associated with the transition of secondary hyperparathyroidism to tertiary hyperparathyroidism in patients with X-linked hypophosphatemic rickets. Thirteen patients with X-linked hypophosphatemic rickets and secondary or tertiary hyperparathyroidism were included in the study. Their hospital records were reviewed and compared for onset, duration and dosage of treatment, and for age of diagnosis and degree of secondary hyperparathyroidism. Two patients developed tertiary hyperparathyroidism and 11 patients secondary hyperparathyroidism during the treatment. Patients with tertiary hyperparathyroidism had, on average, earlier onset and longer duration of treatment, higher dose of Pi and longer duration of treatment with very high Pi doses (> 100 mg/kg/day) compared to the 11 patients with secondary hyperparathyroidism. However, variation of all parameters was great with considerable overlap. Very high S-PTH levels > or = 42 pmol/l were observed in those who later developed tertiary hyperparathyroidism. Prolonged very high dose oral Pi treatment is a major risk factor for the development of tertiary hyperparathyroidism in X-linked hypophosphatemic rickets.

Modifications in lipoprotein surface charge alter cyclosporine A association with low-density lipoproteins.

The purpose of this study was to examine the influence of lipoprotein surface charge on the plasma distribution of cyclosporine A (CSA). Phosphatidylinositol (PI; 40 micromol) was administered intravenously to rabbits. Blood was removed 10 min after injection and plasma was retrieved. Radiolabeled CSA ([3H] CSA) at a concentration of 1000 ng/mL was incubated for 60 min at 37 degrees C in control and PI-treated rabbit plasma. After incubation, plasma was separated into its lipoprotein and lipoprotein-deficient plasma (LPDP) fractions by density gradient ultracentrifugation, and the percentage of [3H]CSA recovered in each fraction was determined by radioactivity. To determine lipoprotein surface charge within control and PI-treated plasma, the zeta potential of each lipoprotein fraction was measured. The effect of PI on lipoprotein surface charge was further confirmed by gel electrophoresis. PI treatment caused low-density lipoprotein (LDL) fraction to migrate further on the agarose gel, indicative of an increased negative surface charge. Zeta potential analysis further showed that LDL particles had a surface potential of -11.4 +/- 1.9 mV and -17.4 +/- 3 mV in control and PI-treated groups, respectively. A greater percentage of [3H]CSA was recovered within the LDL (16.4 +/-1.1% vs. 7.7 +/- 2.1%; n = 3; p < 0.05) fraction after incubation in PI treated than in control plasma, respectively. These findings suggest that modifications in lipoprotein surface charge alter CSA distribution within the LDL plasma fraction.

Sexual dysfunction in 156 ambulatory HIV-infected men receiving highly active antiretroviral therapy combinations with and without protease inhibitors.

We conducted a cross-sectional study of 156 ambulatory HIV-infected homosexual or bisexual men to assess and compare the prevalence and characteristics of sexual dysfunction according to treatment combinations (group A, protease inhibitor [PI] treatment; group B, no PI treatment; and C, PI treatment interrupted >1 month previously). The study was based on a self-administered 163-item questionnaire that included a French translation of the International Index of Erectile Function, five sections of the Derogatis Sexual Functioning Inventory, and open questions. Data analysis was performed using Mann-Whitney and Kruskal-Wallis H nonparametric tests (quantitative values) and chi2 tests (qualitative values) using SPSS software (SPSS, Chicago, IL, U.S.A.). One hundred fifty-six patients completed the study. The median age +/- SD of the patients was 40.5 +/- 7.7 years, and the median CD4+ cell count +/- SD was 415 +/- 236/mm3. One hundred eleven (71%) of 156 patients reported some degree of sexual dysfunction since the beginning of their treatment (65 [71%] of 91 group A patients; 15 [65%] of 23 group B patients; and 31 [74%] of 42 group C patients), with no significant difference among the groups. Of the 111 patients, 99 (89%) reported decrease or loss of libido, 76 (68%) reported orgasmic perturbation, 96 (86%) reported erectile dysfunction, and 65 (59%) reported ejaculation perturbation, with no significant difference among the three groups. There were no significant differences among the three groups regarding the International Index of Erectile Function and Derogatis Sexual Functioning Inventory scores. These data suggest that PI-based therapy does not seem to increase sexual dysfunction in this patient population.

Vascular calcification and inorganic phosphate

Vascular calcification is highly correlated with elevated serum phosphate levels in uremic patients. To shed light on this process, we examined the ability of extracellular inorganic phosphate (Pi) levels to regulate human aortic smooth muscle cell (HSMC) culture mineralization in vitro. When cultured in media containing normal physiological levels of Pi (1.4 mmol/L Pi), HSMC grew in monolayers and did not mineralize. In contrast, HSMC cultured in media containing Pi levels comparable to those seen in hyperphosphatemic individuals (> 1.4 mmol/L), showed dose-dependent increases in cell culture calcium deposition. Mechanistic studies showed that elevated Pi treatment of HSMC also enhanced the expression of the osteogenic markers, osteocalcin and Cbfa-1. The effects of elevated Pi on HSMC were mediated by a sodium-dependent phosphate cotransporter (NPC), as indicated by the ability of the specific NPC inhibitor, phosphonoformic acid (PFA), to dose-dependently inhibit Pi-induced calcium deposition as well as osteocalcin and Cbfa-1 gene expression. Using polymerase chain reaction and Northern blot analyses, the NPC in HSMC was identified as Pit-1 (Glvr-1), a member of the type III NPCs. Interestingly, platelet-derived growth factor-BB (PDGF-BB), a potent atherogenic stimulus, increased the maximum velocity (Vmax) but not the affinity (Km) of phosphate uptake, enhanced the expression of Pit-1 mRNA, and induced HSMC culture calcification in a time- and dose-dependent manner. Importantly, in the presence of PDGF, HSMC culture calcification occurred under normophosphatemic conditions. These data suggest that elevated Pi may directly stimulate HSMC to undergo phenotypic changes that predispose to calcification and may help explain both the phenomena of human metastatic calcification under hyperphosphatemic conditions as well as increased calcification in PDGF-rich atherosclerotic lesions. © 2001 by the National Kidney Foundation, Inc.

Longitudinal Follow-up of Patients With α1-Protease Inhibitor Deficiency Before and During Therapy With IV α1-Protease Inhibitor

The efficacy of IV augmentation therapy with human alpha(1)-protease inhibitor (alpha(1)-Pi) in patients with severe alpha(1)-Pi deficiency is still under debate. To evaluate the progression of emphysema in patients with alpha(1)-Pi deficiency before and during a period in which they received treatment with alpha(1)-Pi. Multicenter, retrospective cohort study. Outpatient clinics of 26 university clinics and pulmonary hospitals. Ninety-six patients with severe alpha(1)-Pi deficiency receiving weekly augmentation therapy with human alpha(1)-Pi, 60 mg/kg of body weight, had a minimum of two lung function measurements before and two lung function measurements after augmentation therapy was started. Lung function data were followed up for a minimum of 12 months both before and during treatment (mean, 47.5 months and 50.2 months, respectively). Patients were grouped according to the severity of their lung function impairment. The change in FEV(1) was compared during nontreatment and treatment periods. In the whole group, the decline in FEV(1) was significantly lower during the treatment period (49.2 mL/yr vs 34.2 mL/yr, p = 0.019). In patients with FEV(1) > 65%, IV alpha(1)-Pi treatment reduced the decline in FEV(1) by 73.6 mL/yr (p = 0.045). Seven individuals had a rapid decline of FEV(1) before treatment, and the loss in FEV(1) could be reduced from 256 mL/yr to 53 mL/yr (p = 0.001). Some patients with severe alpha(1)-Pi deficiency and well-preserved lung function show a rapid decline in FEV(1). These patients profit from weekly IV therapy with human alpha(1)-Pi and have less rapid decline if treated. Early detection of patients at risk and early start of augmentation therapy may prevent accelerated loss of lung tissue.