A carbon-13 nuclear magnetic resonance investigation of the metabolic fluxes associated withy glucose metabolism in human erythrocytes

We have used [2-¹³C]d-glucose and carbon-13 nuclear magnetic resonance (NMR) spectroscopy to investigate metabolic fluxes through the major pathways of glucose metabolism in intact human erythrocytes and to determine the interactions among these pathways under conditions that perturb metabolism. Using the method described, we have been able to measure fluxes through the pentose phosphate pathway, phosphofructokinase, the 2,3-diphosphoglycerate bypass, and phosphoglycerate kinase, as well as glucose uptake, concurrently and in a single experiment. We have measured these fluxes in normal human erythrocytes under the following conditions: (1) fully oxygenated; (2) treated with methylene blue; and (3) deoxygenated. This method makes it possible to monitor various metabolic effects of stresses in normal and pathological states. Not only has ¹³C-NMR spectroscopy proved to be a useful method for measuring in vivo flux through the pentose phosphate pathway, but it has also provided additional information about the cycling of metabolites through the non-oxidative portion of the pentose phosphate pathway. Our evidence from experiments with [1-¹³C]-, [2-¹³C]-, and [3-¹³C]d-glucoses indicates that there is an observable reverse flux of fructose 6-phosphate through the reactions catalyzed by transketolase and transaldolase, even in the presence of a net flux through the pentose phosphate pathway.     

Decreased reticuloendothelial system clearance and increased blood half-life and immune cell labeling for nano- and micron-sized superparamagnetic iron-oxide particles upon pre-treatment with Intralipid

Background

Superparamagnetic iron-oxide nanoparticles are useful as contrast agents for anatomical, functional and cellular MRI, drug delivery agents, and diagnostic biosensors. Nanoparticles are generally cleared by the reticuloendothelial system (RES), in particular taken up by Kupffer cells in the liver, limiting particle bioavailability and in-vivo applications. Strategies that decrease the RES clearance and prolong the circulation residence time of particles can improve the in-vivo targeting efficiency.

Methods

Intralipid 20.0%, an FDA approved nutritional supplement, was intravenously administered in rats at the clinical dose (2 g/kg) 1 h before intravenous injection of ultra-small superparamagnetic iron-oxide (USPIO) or micron-sized paramagnetic iron-oxide (MPIO) particles. Blood half-life, monocyte labeling efficiency, and particle biodistribution were assessed by magnetic resonance relaxometry, flow cytometry, inductively-coupled plasma MS, and histology.

Results

Pre-treatment with Intralipid resulted in a 3.1-fold increase in USPIO blood half-life and a 2-fold increase in USPIO-labeled monocytes. A 2.5-fold increase in MPIO blood half-life and a 5-fold increase in MPIO-labeled monocytes were observed following Intralipid pre-treatment, with a 3.2-fold increase in mean iron content up to 2.60 pg Fe/monocyte. With Intralipid, there was a 49.2% and 45.1% reduction in liver uptake vs. untreated controls at 48 h for USPIO and MPIO, respectively.

Conclusions

Intralipid pre-treatment significantly decreases initial RES uptake and increases in-vivo circulation and blood monocyte labeling efficiency for nano- and micron-sized superparamagnetic iron-oxide particles.

General significance

Our findings can have broad applications for imaging and drug delivery applications, increasing the bioavailability of nano- and micron-sized particles for target sites other than the liver.     

High-resolution nuclear magnetic resonance spectroscopy studies of polysaccharides crosslinked by sodium trimetaphosphate: a proposal for the reaction mechanism

An NMR spectroscopy study ((31)P, (1)H, (13)C) of the postulated crosslinking mechanism of sodium trimetaphosphate (STMP) on polysaccharides is reported using methyl alpha-D-glucopyranoside as a model. In a first step, reaction of STMP with Glc-OMe gives grafted sodium tripolyphosphate (STPP(g)). On the one hand, STTP(g) can react with a second alcohol functionality to give a crosslinked monophosphate. On the other hand, a monophosphate (grafted phosphate) could be obtained by alkaline degradation of STPP(g). NMR spectroscopy allows to detect the various species formed and to obtain the crosslinking density of STMP-polysaccharides hydrogels.     

Mapping QTLs and candidate genes for iron and zinc concentrations in unpolished rice of Madhukar×Swarna RILs

Identifying QTLs/genes for iron and zinc in rice grains can help in biofortification programs. 168 F(7) RILs derived from Madhukar×Swarna were used to map QTLs for iron and zinc concentrations in unpolished rice grains. Iron ranged from 0.2 to 224ppm and zinc ranged from 0.4 to 104ppm. Genome wide mapping using 101 SSRs and 9 gene specific markers showed 5 QTLs on chromosomes 1, 3, 5, 7 and 12 significantly linked to iron, zinc or both. In all, 14 QTLs were identified for these two traits. QTLs for iron were co-located with QTLs for zinc on chromosomes 7 and 12. In all, ten candidate genes known for iron and zinc homeostasis underlie 12 of the 14 QTLs. Another 6 candidate genes were close to QTLs on chromosomes 3, 5 and 7. Thus the high priority candidate genes for high Fe and Zn in seeds are OsYSL1 and OsMTP1 for iron, OsARD2, OsIRT1, OsNAS1, OsNAS2 for zinc and OsNAS3, OsNRAMP1, Heavy metal ion transport and APRT for both iron and zinc together based on our genetic mapping studies as these genes strictly underlie QTLs. Several elite lines with high Fe, high Zn and both were identified.     

Oxidative changes of lipids monitored by MALDI MS

Oxidation processes of lipids are of paramount interest from many viewpoints. For instance, oxidation processes are highly important under in vivo conditions because molecules with regulatory functions are generated by oxidation of lipids or free fatty acids. Additionally, many inflammatory diseases are accompanied by lipid oxidation and, therefore, oxidation products are also useful disease (bio)markers. Thus, there is also considerable interest in methods of (oxidized) lipid analysis.Nowadays, soft ionization mass spectrometric (MS) methods are regularly used to study oxidative lipid modifications due to their high sensitivities and the extreme mass resolution. Although electrospray ionization (ESI) MS is so far most popular, applications of matrix-assisted laser desorption and ionization (MALDI) MS are increasing. This review aims to summarize the so far available data on MALDI analyses of oxidized lipids. In addition to model systems, special attention will be paid to the monitoring of oxidized lipids under in vivo conditions, particularly the oxidation of (human) lipoproteins. It is not the aim of this review to praise MALDI as the “best” method but to provide a critical survey of the advantages and drawbacks of this method.     

The effect of Ginkgo biloba extract on 3-nitropropionic acid-induced neurotoxicity in rats

3-Nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase enzyme (SDH), induces neurodegeneration similar to that observed in Huntington’s disease (HD). Reduction of prepulse inhibition (PPI) of acoustic startle response, locomotor hypoactivity, bilateral striatal lesions as well as brain oxidative stress are major features of HD. The present study was designed to investigate neuroprotective effect of Ginkgo biloba extract (EGb 761) on 3-NP induced neurobehavioral changes and striatal lesions.Rats administered 3-NP (20 mg/kg, s.c.) for five consecutive days exhibited PPI deficits and locomotor hypoactivity whereas, pretreatment of animals with EGb 761 (100 mg/kg, i.p. for 15 days) ahead of and during the induction of HD by 3-NP (20 mg/kg for 5 days starting at day 8) ameliorated 3-NP-induced neurobehavioral deficits. Administration of 3-NP increased the level of striatal malondialdehyde (MDA). This effect was prevented in animals pre-treated with EGb 761. Changes in the level of apoptotic regulatory gene expressions, following 3-NP treatment, were demonstrated as both an up-regulation and a down-regulation of the expression levels of striatal Bax and Bcl-xl genes, respectively. In addition, an up-regulation of the expression level of striatal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also observed. Pre-treatment with EGb 761 caused a down-regulation in striatal GAPDH and Bax together with an up-regulation of striatal Bcl-xl expression level as compared to the 3-NP treated group. Histochemical examination of striatal tissue showed that EGb 761 significantly prevented 3-NP induced inhibition of SDH activity. Histopathological examination further affirmed the neuroprotective effect of EGb 761 against 3-NP toxicity.Taken together, these results suggest that EGb 761 has a neuroprotective role in the current HD paradigm, which may be related to improvement of energy metabolism, antioxidant properties and antiapoptotic effects.     

Discovery and evaluation of nNav1.5 sodium channel blockers with potent cell invasion inhibitory activity in breast cancer cells

Voltage-gated sodium channels (VGSC) are a well-established drug target for anti-epileptic, anti-arrhythmic and pain medications due to their presence and the important roles that they play in excitable cells. Recently, their presence has been recognized in non-excitable cells such as cancer cells and their overexpression has been shown to be associated with metastatic behavior in a variety of human cancers. The neonatal isoform of the VGSC subtype, Na_v1.5 (nNa_v1.5) is overexpressed in the highly aggressive human breast cancer cell line, MDA-MB-231. The activity of nNa_v1.5 is known to promote the breast cancer cell invasion in vitro and metastasis in vivo, and its expression in primary mammary tumors has been associated with metastasis and patient death. Metastasis development is responsible for the high mortality of breast cancer and currently there is no treatment available to specifically prevent or inhibit breast cancer metastasis. In the present study, a 3D-QSAR model is used to assist the development of low micromolar small molecule VGSC blockers. Using this model, we have designed, synthesized and evaluated five small molecule compounds as blockers of nNa_v1.5-dependent inward currents in whole-cell patch-clamp experiments in MDA-MB-231 cells. The most active compound identified from these studies blocked sodium currents by 34.9?±?6.6% at 1?μM. This compound also inhibited the invasion of MDA-MB-231 cells by 30.3?±?4.5% at 1?μM concentration without affecting the cell viability. The potent small molecule compounds presented here have the potential to be developed as drugs for breast cancer metastasis treatment.     

Ultrastructural changes induced by low concentrations of DDT in the livers of the zebrafish and the guppy

Following exposure to 0.3–3.0 ppb DDT, the livers of zebrafish and guppies were examined by electron microscopy. After 24 h in 1.0 ppb or 72 h in 0.3 ppb, zebrafish had a complete loss of glycogen, decrease in cell size and a cytoplasmic compartment filled with rough endoplasmic reticulum (ER). Guppies exposed for periods of up to 28 days showed only a gradual accumulation of smooth ER and no change in glycogen, lipid, rough ER or cell size. The relationship of these changes to the zebrafish's sensitivity and the guppy's tolerance to DDT is dicussed.