scholarly journals In vivo and in vitro 31P-NMR Study of the Phosphate Transport and Polyphosphate Metabolism in Hebeloma cylindrosporum in Response to Plant Roots Signals

BIO-PROTOCOL ◽  
2018 ◽  
Vol 8 (16) ◽  
Author(s):  
Christine Le Guerneve ◽  
Adeline Becquer ◽  
Margarita Torres-Aquino ◽  
Laurie Amenc ◽  
Carlos Trives-Segura ◽  
...  
Keyword(s):  
31P Nmr ◽  
Phosphate Transport ◽  
Plant Roots ◽  
Hebeloma Cylindrosporum ◽  
Nmr Study ◽  
Polyphosphate Metabolism

Oxidative Stress Induces Changes and Shedding of Membrane Phospholipids from Thalassemic RBCs - A NMR Study.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1782-1782
Author(s):  
Eitan Fibach ◽  
Inna Freikman ◽  
Johnny Amer ◽  
Jack S. Cohen ◽  
Israel Ringel
Keyword(s):  
Oxidative Stress ◽  
31P Nmr ◽  
Oxidative Status ◽  
Beta Thalassemia ◽  
Globin Genes ◽  
Membrane Phospholipids ◽  
Flip Flop ◽  
Nmr Study

Abstract Changes in the membrane phospholipid (PL) asymmetry is one of the hallmarks of apoptosis in nucleated cells. Although mature anucleated RBCs, do not undergo the classical pattern of apoptosis, upon trauma or aging they present changes in the membrane asymmetry. These changes include a flip-flop of phosphatidylserine (PS) from the inner leaflet of the membrane to its outer leaflet. This externalization of PS stimulates RBC phagocytosis by macrophages of the reticulo-endothelial system and their removal from the circulation. Oxidative stress is among the causes of PS externalization on RBCs. In beta-thalassemia and sickle cell disease, although the primary defects are mutations in the globin genes, oxidative stress is thought to mediate part of the damage to the RBCs, and particularly to its membrane, including PS externalization. In the present study, we used Nuclear Magnetic Resonance (NMR) spectroscopy to analyze normal and beta-thalassemic RBCs in order to study the relationship between their oxidative status and the content and shedding of their PL. Using 1H-NMR, we demonstrated a higher lactate/pyruvate ratio in thalassemic RBCs, confirming their state of oxidative stress. Using 31P-NMR, we measured the content of various PLs, and found 30±3% more phosphatidylcholine (PC), and unexpectedly, less PS in thalassemic RBCs than in normal RBCs. The PS was increased in thalassemic RBC, but not in normal RBC, by treatment with anti-oxidants (vitamin C and N-acetyl cysteine) and decreased by oxidants (t-butylhydroxyperoxide and H2O2) in normal and thalassemic RBCs. PC showed the opposite behavior, indicating a correlation between PS and PC contents and the oxidative status. Since RBCs with exposed PS have been reported to be more frequent in thalassemic blood than in normal blood, we hypothesized that the decrease in PS is a result of shedding from the external membrane, either as free PS moieties or as part of membrane vesicles. NMR analysis of blood plasma obtained from normal and thalassemic donors indicated a 2.6-fold and 1.8-fold increase in PS and PC, respectively in the latter plasma. In vitro incubation of RBC produced much higher PS in supernatants derived from thalassemic RBCs compared with those of normal RBCs. Anti-oxidants reduced the PS shedding from thalassemic RBCs into their supernatants while oxidants increased the PS shedding by normal RBCs. RBCs are known to shed membranous particles (termed vesicles or microparticles) in vitro and in vivo during their physiologic and pathological senescence. We studied this point by purifying microparticles from plasma and RBC supernatants of normal and thalassemic donors, and measuring the PLs content in their lipophilic extracts by 31P-NMR. We found that the PS content and its proportion out of the total PLs were higher in microparticles purified from thalassemic plasma (0.25±0.04 mM, 19% of the plasma total PS) or RBC supernatants than in normal plasma microparticles (0.045±0.06 mM, 9.5% of the plasma total PS) or supernatants. The results also show that although microparticles are enriched in PS compared to their intact RBCs, the bulk of the shed PS is not associated with microparticles. These results suggest that oxidative stress in RBCs causes them to shed their PS and that the increase in PC levels maybe be a compensating mechanism. The pathological consequences of these phenomena on the survival of RBCs in thalassemia warrants further study.

31P-NMR study of resting in vitro rat diaphragm exposed to hypercapnia

1988 ◽  
Vol 65 (5) ◽  
pp. 2270-2277 ◽  
Author(s):  
R. S. Fitzgerald ◽  
S. Howell ◽  
W. E. Jacobus
Keyword(s):  
31P Nmr ◽  
Mechanical Performance ◽  
High Energy ◽  
31P Nmr Spectroscopy ◽  
High Energy Phosphates ◽  
Rat Diaphragm ◽  
Nmr Study

We have reported previously that, when exposed to hypercapnia of various intensities, the diaphragm reduces its force of twitch and tetanic contractions in the in vitro rat preparation as well as in the in vivo dog preparation. The experiments reported here with 31P nuclear magnetic resonance (31P-NMR) spectroscopy attempt to examine cellular mechanisms that might be responsible for this deterioration in mechanical performance. Specifically they describe certain characteristics of this preparation and cautions needed to study the resting in vitro rat diaphragm with such techniques. Second, they report the response of intracellular pH (pHi), phosphocreatine (PCr), ATP, and inorganic phosphate (Pi) in the resting in vitro rat diaphragm exposed to long-term normocapnia or to long-term hypercapnia. The results show that 1) to maintain a viable preparation, it was necessary to keep the diaphragm extended to an area approximating that at functional residual capacity, 2) the diaphragm seemed quite capable of maintaining a constant pHi and constant contents of ATP and Pi during normocapnia, but there was a gradual decline in PCr, and 3) during hypercapnia there was a significant decrease in pHi, but the behavior of the phosphate metabolites was exactly as during normocapnia. The results suggest that the decrease in mechanical performance of the diaphragm is probably not due to a decrease in the availability of the high-energy phosphates, although they do not completely exclude this possibility or possibilities related to regional compartmentation.

scholarly journals 31P-NMR study of dog submandibular gland in vivo and in vitro using the Topical Magnetic Resonance.

1985 ◽  
Vol 35 (5) ◽  
pp. 729-740 ◽  
Author(s):  
Takashi NAKAHARI ◽  
Yoshiteru SEO ◽  
Masataka MURAKAMI ◽  
Hirohiko MORI ◽  
Shinichi MIYAMOTO ◽  
...  
Keyword(s):  
Magnetic Resonance ◽  
Submandibular Gland ◽  
31P Nmr ◽  
Nmr Study

scholarly journals Response of Cisplatin Resistant Skov-3 Cells to [Pt(O,O′-Acac)(γ-Acac)(DMS)] Treatment Revealed by a Metabolomic 1H-NMR Study

Molecules ◽  
2018 ◽  
Vol 23 (9) ◽  
pp. 2301 ◽  
Author(s):  
Federica De Castro ◽  
Michele Benedetti ◽  
Giovanna Antonaci ◽  
Laura Del Coco ◽  
Sandra De Pascali ◽  
...  
Keyword(s):  
Ovarian Carcinoma ◽  
1H Nmr ◽  
Mechanism Of Action ◽  
Culture Media ◽  
Epithelial Ovarian Carcinoma ◽  
Multivariate Statistical ◽  
Nmr Study ◽  
First Time

The novel [Pt(O,O′-acac)(γ-acac)(DMS)], Ptac2S, Pt(II) complex has recently gained increasing attention as a potential anticancer agent for its pharmacological activity shown in different tumor cell lines, studied both in vitro and in vivo. The mechanism of action of Ptac2S, operating on non-genomic targets, is known to be very different from that of cis-[PtCl2(NH3)2], cisplatin, targeting nucleic acids. In this work, we evaluated the cytotoxicity of Ptac2S on the cisplatin resistant Epithelial Ovarian Carcinoma (EOC), SKOV-3 cells, by the MTT assay. A 1H-NMR metabolomic approach coupled with multivariate statistical analysis was used for the first time for Ptac2S to figure out the biological mechanisms of action of the complex. The metabolic variations of intracellular metabolites and the composition of the corresponding extracellular culture media were compared to those of cisplatin (cells were treated at the IC50 doses of both drugs). The reported comparative metabolomic analysis revealed a very different metabolic profile between Ptac2S and cisplatin treated samples, thus confirming the different mechanism of action of Ptac2S also in the Epithelial Ovarian Carcinoma (EOC), SKOV-3 cells line. In particular, higher levels of pyruvate were observed in Ptac2S treated, with respect to cisplatin treated, cells (in both aqueous and culture media). In addition, a very different lipid expression resulted after the exposure to the two drugs (Ptac2S and cisplatin). These results suggest a possible explanation for the Ptac2S ability to circumvent cisplatin resistance in SKOV-3 cells.

scholarly journals Multiple isomers of inositol pentakisphosphate in Epstein-Barr-virus- transformed (T5-1) B-lymphocytes. Identification of inositol 1,3,4,5,6-pentakisphosphate, d-inositol 1,2,4,5,6-pentakisphosphate and l-inositol 1,2,4,5,6-pentakisphosphate

1991 ◽  
Vol 280 (2) ◽  
pp. 323-329 ◽  
Author(s):  
F M McConnell ◽  
L R Stephens ◽  
S B Shears
Keyword(s):  
Inositol Polyphosphate ◽  
Permeabilized Cells ◽  
Barr Virus ◽  
Epstein Barr ◽  
Polyphosphate Metabolism ◽  
And Function ◽  
Lymphocyte Cell ◽  
Lymphocyte Cell Line

Substantial amounts of three [3H]InsP5 isomers were detected in [3H]inositol-labelled human lymphoblastoid (T5-1) cells. Their structures were determined by h.p.l.c. [Phillippy & Bland (1988) Anal. Biochem. 175, 162-166], and by utilizing a stereospecific D-inositol 1,2,4,5,6-pentakisphosphate 3-kinase from Dictyostelium discoideum [Stephens & Irvine (1990) Nature (London) 346, 580-583]. The structures were: inositol 1,3,4,5,6-pentakisphosphate, D-inositol 1,2,4,5,6-pentakisphosphate and L-inositol 1,2,4,5,6-pentakisphosphate. The relative proportions of these isomers (approx. 73:14:14 respectively) were unaffected by cross-linking anti-IgD receptors. The T5-1 cells also contained InsP6 and three Ins P4s, which were identified as the 1,3,4,5, 1,3,4,6 and 3,4,5,6 isomers. In incubations with permeabilized T5-1 cells, both 1,3,4,6 and 3,4,5,6 isomers of InsP4 were phosphorylated solely to Ins(1,3,4,5,6)P5. Permeabilized cells also dephosphorylated InsP6, even in the presence of a large excess of glucose 6-phosphate to saturate non-specific phosphatases. In the latter experiments the following isomers of InsP5 accumulated: D- and/or L-Ins(1,2,3,4,5)P5, plus D- and/or L-Ins(1,2,4,5,6)P5. This demonstration that multiple isomers of InsP5 may be formed in vivo and in vitro by a transformed lymphocyte cell line adds a new level of complexity to the study of inositol polyphosphate metabolism and function.

NAD-glycohydrolase in renal brush border membranes

1985 ◽  
Vol 249 (3) ◽  
pp. F366-F373
Author(s):  
S. A. Kempson
Keyword(s):  
Brush Border ◽  
Osmotic Shock ◽  
Phosphate Transport ◽  
Reaction Products ◽  
Brush Border Membranes ◽  
Nad Glycohydrolase ◽  
Renal Brush Border Membranes ◽  
Renal Brush Border

NAD is hydrolyzed during incubation with isolated renal brush border membranes (BBM). The specific enzymatic mechanisms have not been identified apart from the activity of ADP-ribosyltransferase, which accounts for a very small proportion of the total hydrolysis. In the present study, an NAD-glycohydrolase (NGH) was identified in the renal BBM using the cyanide-addition assay to monitor hydrolysis of NAD at the nicotinamide-ribose bond. The production of nicotinamide and ADP-ribose, the expected reaction products, was determined by thin-layer chromatography. The NGH was enriched ninefold in the BBM fraction and accounted for 36% of the total rate of NAD hydrolysis by BBM enzymes at pH 7.4. Assay of NGH in sealed BBM vesicles subjected to osmotic shock indicated that about 23% of the NGH is exposed on the cytoplasmic surface of the BBM. The enzyme was inhibited by nicotinamide in vitro and also when the nicotinamide was administered in vivo, suggesting, indirectly, that the enzyme may play a role in mediating the effects of nicotinamide on BBM phosphate transport.

scholarly journals Fibroblast growth factor-23 increases mouse PGE2 production in vivo and in vitro

2006 ◽  
Vol 290 (2) ◽  
pp. F450-F455 ◽  
Author(s):  
Ashu Syal ◽  
Susan Schiavi ◽  
Sumana Chakravarty ◽  
Vangipuram Dwarakanath ◽  
Raymond Quigley ◽  
...  
Keyword(s):  
Fibroblast Growth Factor 23 ◽  
Phosphate Transport ◽  
Proximal Tubules ◽  
Fibroblast Growth ◽  
Transport Defect ◽  
Map Kinase Pathway ◽  
Fgf 23 ◽  
Kinase Pathway

Fibroblast growth factor-23 (FGF-23) has been implicated in the renal phosphate wasting in X-linked hypophosphatemia, tumor-induced osteomalacia, and autosomal dominant hypophosphatemic rickets. Recently, we demonstrated that Hyp mice have greater urinary PGE2 levels compared with C57/B6 mice and that indomethacin administration in vivo and in vitro ameliorates the phosphate transport defect in Hyp mice. To determine further whether altered prostaglandin metabolism plays a role in the renal phosphate transport defect in Hyp mice, we incubated renal proximal tubules with arachidonic acid. We find that PGE2 production was higher in Hyp mice than in C57/B6 mice. Incubation of C57/B6 mouse renal proximal tubules with FGF-23R176Q, an active mutant form of FGR23, increased tubular PGE2 production, an effect that was inhibited by 50 μM PD-98059 and 10 μM SB-203580, inhibitors of the MAP kinase pathway. C57/B6 mice injected with FGF-23R176Q had a ∼10-fold increase in PGE2 excretion 24 h after intraperitoneal injection of FGF-23R176Q compared with vehicle-treated controls. Finally, we show that PGE2 inhibited both phosphate and volume absorption in mouse proximal convoluted tubules perfused in vitro and reduced brush-border membrane vesicle NaPi-2a protein abundance from renal cortex incubated in vitro with PGE2. In conclusion, FGF-23 increases urinary and renal tubular PGE2 production via the MAP kinase pathway and PGE2 inhibits proximal tubule phosphate transport.

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