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Abstract

2-hydroxyoleic acid (OHOA, Minerval®) is an example of a substance used for membrane lipid therapy, where the cellular membranes rather than specific proteins constitute the therapeutical target. OHOA is thought to mediate its anti-tumor effect by affecting the biophysical properties of membranes, which leads to altered recruitment and activation of amphitropic proteins, altered cellular signaling, and eventual cell death. Little is known about the initial signaling events upon treatment with OHOA, and whether the altered membrane properties would have any impact on the dynamic intracellular transport system. In the present study we demonstrate that treatment with OHOA led to a rapid release of intracellular calcium and activation of multiple signaling pathways in HeLa cells, including the PI3K-AKT1-MTOR pathway and several MAP kinases, in a process independent of the EGFR. By lipidomics we confirmed that OHOA was incorporated into several lipid classes. Concomitantly, OHOA potently increased retrograde transport of the plant toxin ricin from endosomes to the Golgi and further to the endoplasmic reticulum. The OHOA-stimulated ricin transport seemed to require several amphitropic proteins, including Src, phospholipase C, protein kinase C, and also Ca2+/calmodulin. Interestingly, OHOA induced a slight increase in endosomal localization of the retromer component VPS35. Thus, our data show that addition of a lipid known to alter membrane properties not only affects signaling, but also intracellular transport.

OHOA stimulates transport of ricin to the Golgi and the ER

To verify that sorting into the retrograde pathway was stimulated by OHOA, we specifically measured the arrival of ricin to the TGN by the sulfation assay. This assay takes advantage of the trans-Golgi-specific sulfation process, in which tyrosyl protein sulfotransferases couple sulfate groups to proteins displaying a sulfation site []. By addition of recombinant ricin molecules containing a sulfation site, the arrival of toxin molecules to the Golgi can be detected. Indeed, treatment with increasing concentrations of OHOA potentiated the ricin sulfation (Figure ). The total amount of ricin associated with the cells was unchanged. To address the specificity of the stimulatory effect of OHOA, unmodified OA was used in parallel. In agreement with the toxicity data, OA had a very limited effect on ricin sulfation (Figure ).

OHOA stimulates transport of ricin to the Golgi and the ER

Along the same lines, and as previously shown for cholera toxin [], we established a SNAP-tag assay for measuring the arrival of cargo molecules to the ER. The SNAP-tag enzyme was maintained selectively in the ER lumen by fusion to an ER-signal peptide and the KDEL-retrieval sequence (). In line with the data for Golgi-transport, ricin transport to the ER was stimulated by OHOA in a concentration-dependent manner (Figure ).

Abstract

Background. Inflammation and fibrosis are the important pathophysiologic processes in diabetic nephropathy (DN). Maresin 1 is a potential anti-inflammatory lipid mediator, which has displayed powerful proresolving activities. Aim. We determine whether maresin 1 has protective effect on mouse glomerular mesangial cells (GMCs) induced by high glucose. Methods. We cultured GMCs stimulated by high glucose and categorized as follows: normal glucose group (5.6 mmol/L), high glucose group (30 mmol/L), mannitol group, maresin 1 intervention group (1, 10, and 100 nmol/L), maresin 1 and normal glucose group, and the N-acetylcysteine (NAC) intervention group (10 μmol/L NAC). After 24 h, the expression of ROS, NLRP3, caspase-1, procaspase-1, IL-1β, and pro-IL-1β was detected by western-blot, RT-PCR, and immunofluorescence. After 48 h, the expression of TGF-β1 and FN was detected by RT-PCR and ELISA. Results. Compared with normal glucose group, the expression of ROS, NLRP3, caspase-1, IL-1β, TGF-β1, and FN increased in high glucose group (P < 0.05), but it decreased after the treatment of maresin 1 in different concentrations. On the contrary, the expression of procaspase-1 and pro-IL-1β protein was restrained by high glucose and enhanced by maresin 1 in a dose-dependent manner (P < 0.05). Conclusion. Maresin 1 can inhibit NLRP3 inflammasome, TGF-β1, and FN in GMCs; it may have protective effect on DN by mitigating the inflammation and early fibrosis.

2.1. Cell Culture and Stimulation

Mouse glomerular mesangial cells (GMCs) were purchased from Chinese Academy of Sciences and cultured in low glucose Dulbecco's Modified Eagle Medium (DMEM, Hyclone, USA) with 10% fetal bovine serum (FBS, BOVOGEN, Australia) at 37°C and 5% CO2. GMCs were used between the 2nd and 5th passages for all experiments. To observe the effect of maresin 1, we divided the cells into 8 groups: ① normal control group (NC group): 5.6 mmol/L glucose, ② osmotic pressure control group (OP group): 5.6 mmol/L glucose + 24.4 mmol/L mannitol, ③ high glucose group (HG group): 30 mmol/L glucose, ④ different concentrations of maresin 1 + high glucose group (M + HG group): the GMCs being treated with different concentration of maresin 1 (1, 10, and 100 nmol/L, Cayman Chemical, USA) for 30 min before being exposed to high glucose (30 mmol/L) [, ], 1 nmol/L maresin 1 + 30 mmol/L glucose (M1 + HG group), 10 nmol/L maresin 1 + 30 mmol/L glucose (M2 + HG group), 100 nmol/L maresin 1 + 30 mmol/L glucose (M3 + HG group), ⑤ maresin 1 + normal control group (M + NC group): the most obvious effect of maresin 1 concentration + 5.6 mmol/L glucose, ⑥ N-acetylcysteine (NAC) intervention group as positive control (10 μmol/L NAC + HG group) [, ]. Each test was independently repeated more than 3 times.

3.1.1. The Expression of NLRP3, Caspase-1, Procaspase-1, IL-1β, and Pro-IL-1β in GMCs Induced by 30 mmol/L Glucose for Each Group

Compared with NC group, the mRNA expression of NLRP3, caspase-1, and IL-1β increased significantly after exposing to 30 mmol/L glucose for 6 h, 12 h, 24 h, and 48 h (P < 0.05), and the expression was positively correlated with time changes (Figures and ). Moreover, in M + HG group for 24 h, the protein and mRNA expression of NLRP3, caspase-1, and IL-1β decreased, but procaspase-1 and pro-IL-1β were enhanced obviously as a concentration-dependent manner of maresin 1 compared with HG group (P < 0.05) (Figures , , , and ). And we used immunofluorescence to detect the level of NLRP3, caspase-1, and IL-1β in each group, and we observed the same change (). There were no apparent differences among NC group, OP group, and M + NC group (P > 0.05) (Figures , , , and ).

3.1.2. The ROS Level of GMCs Which Were Treated with NAC and Different Doses of Maresin 1 for 30 min and Were Exposed to High Glucose (30 mmol/L) for 24 h by Using Spectrophotometer and Fluorescence Microscope

As ROS plays an important role in high glucose-induced damage, we explored ROS level in GMCs using DCFH-DA. In spectrophotometer analysis, we observed that high glucose caused a significant ROS generation in GMCs (P < 0.05) compared with NC group. Notably, ROS generation was significantly inhibited by NAC and maresin 1 (1, 10, and 100 nmol/L) (P < 0.05), and the ROS level decreased in a concentration-dependent manner of maresin 1 (). Meanwhile, we got a similar result by using fluorescence microscopy ().

3.2. Antifibrosis Effect of Maresin 1 on GMCs Induced by High Glucose

Compared with NC group, the expression of TGF-β1 and FN upregulated in HG group for different time (P < 0.05), especially for 48 h (Figures , , and ). However, it downregulated significantly in M + HG group by a negative correlation with concentration of maresin 1 (P < 0.05) (Figures , , and ). It seemed that 10 nmol/L maresin 1 was effective enough for counteracting the expression of TGF-β1 and FN enhanced by high glucose. Therefore, we chose 10 nmol/L as a protective concentration in the following experiments. And then, we detected that the mRNA level of TGF-β1 and the expression of FN decreased significantly in M2 + HG group compared with HG group (P < 0.05), and there was no significant difference between NC group and other groups (Figures , , and ).

4. Discussion

TGF-β1 is a key regulator of EMT in pathological processes of glomerular sclerosis in DN, which participate in the renal fibrosis of DN commonly [, ]. Extracellular high glucose can be used as profibrotic factors in GMCs to promote the synthesis and secretion of TGF-β1 and increase the expression of FN that would prompt deposition of extracellular matrix and destruct the complete basement membrane. Finally, EMT and stable mesenchymal phenotype occurred []. This study in vitro found that high glucose could upregulate the expression of TGF-β1 and FN in GMCs in time dependence, suggesting that the high glucose may promote glomerular sclerosis and renal fibrosis in DN. What is more, maresin 1 could restrain the expression of TGF-β1 and FN induced by high glucose in GMCs in a dose-dependent manner, indicating that maresin 1 may inhibit EMT in GMCs and reduce DN glomerular fibrosis. Therefore, we hypothesized that maresin 1 potentially offers a therapeutic option for better treatment of DN through its anti-inflammatory effect and inhibition of renal fibrosis. However, this experiment is not enough; since the limit experimental time, we failed to investigate the specific mechanism of action, which needs the further basis experiments to prove.

2.3. Human Leukocyte Antigen (HLA) Class II Binding Assay and Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) Detection of Ximelagatran Bound to HLA-DR Molecules

To reconfirm the inhibitory effect of ximelagatran on the binding of the ligand peptide to HLA-DRB1*07:01 [], a DELFIA-based binding assay was conducted. As previously reported [], ximelagatran inhibited the binding of the ligand peptide to HLA-DRB1*07:01, whereas no inhibitory effect was observed for HLA-DRB1*01:01 and DRB1*15:01 (). LC-MS/MS analysis of the same incubation samples revealed that the presence of HLA-DRB1*01:01 significantly increased the concentration of ximelagatran extracted from HLA class II binding assay samples (). Although the differences were not significant for HLA-DRB1*07:01 and DRB1*15:01, they appeared to be marginally significant (p = 0.087 and p = 0.084, respectively). Taken together, ximelagatran bound to all three HLA-DR molecules was detected in this setting. In contrast, the comparison between ximelagatran concentrations in (1) the presence of HLA-DR molecules and the ligand peptides; and (2) the presence of HLA-DR molecules and absence of the ligand peptides showed that the extent of the decrease of ximelagatran bound to the HLA-DR molecules by the addition of the ligand peptides was different between the three HLA-DR alleles (). The difference was significant (p < 0.05) for HLA-DRB1*01:01 and was marginally significant (p = 0.076) for DRB1*15:01, but just the overall trend for DRB1*07:01.

3. Discussion

In order to evaluate the possible direct interaction of ximelagatran with the peptide binding groove of HLA-DRB1*07:01, a series of in silico simulations and in vitro measurement of ximelagatran bound to HLA-DR molecules were performed.

3. Discussion

Taken together, these results indicate that ximelagatran directly interacts with the peptide binding groove of HLA-DRB1*07:01 and competes with ligand peptides for this binding site. In the cases of abacavir for HLA-B*57:01 [,], and lapatinib for HLA-DRB1*07:01 [], in both of which drugs increase the loading of ligand peptides, it is easy to imagine the immune stimulation by the enhanced antigen presentation and/or the presentation of neo-antigens. Unlike these cases, it may be somewhat difficult to associate the inhibited antigen presentation by ximelagatran with the immune stimulation. However, considering the simulated binding mode of ximelagatran at the peptide binding groove of HLA-DRB1*07:01, there is a possibility that there are some kinds of peptides that are loaded onto HLA-DRB1*07:01 de novo and/or in a different manner in the presence of ximelagatran. Although further investigations are needed to reveal the conclusive mechanism, the present study strongly supports the interaction between ximelagatran and the peptide binding groove of HLA-DRB1*07:01, which could alter the immune response in some way and lead to the idiosyncratic ximelagatran-induced hepatotoxicity. In vitro studies using hepatocyte co-cultured with macrophages or lymphocytes carrying various HLA alleles will support our hypothesis and give us additional information to understand the detailed mechanism of ximelagatran-induced hepatotoxicity.

2.1. Docking Simulations

The interaction modes of ximelagatran or melagatran with HLA-DRB1*07:01 and control alleles DRB1*01:01 and DRB1*15:01, both of which are associated with IDTs caused by other drugs, were predicted by in silico docking stimulations. The binding affinities were judged by a scoring function of generalized-Born volume integral/weighted surface area (GBVI/WSA_dG), which is considered to express protein-ligand binding free energy []. The lowest GBVI/WSA_dG values (kcal/mol) of the complexes between ximelagatran or melagatran and the three HLA-DR molecules are shown in . These values are much lower than those of the complexes between HLA-B*14:02 and nevirapine-related compounds [], and ones between HLA-B*58:01 and allopurinol-related compounds [] simulated by the same software system, indicating ximelagatran and melagatran have high potential to interact with these HLA molecules. The binding modes of ximelagatran and melagatran at the peptide binding grooves of HLA-DRB1*01:01, DRB1*07:01, and DRB1*15:01 with the lowest GBVI/WSA_dG values in each complex are shown in and , respectively. In the complex with HLA-DRB1*07:01, the oxime form of ximelagatran showed a higher binding affinity than the hydroxylamine form. Ximelagatran showed higher simulated binding affinities than melagatran for all three HLA-DR molecules.

1. Introduction

Ximelagatran (Exanta®/Exarta®) is an orally-available anticoagulant agent that directly inhibits thrombin and was developed for the prevention and treatment of thromboembolism. Although ximelagatran was generally well tolerated in short-term use (<12 days), an elevated serum alanine aminotransferase (ALT) level of >3× upper limit of normal was found to develop during long-term treatment (>35 days) in 7.9% of patients []. Due to the concerns for its potential liver toxicity, the development of ximelagatran was terminated and it was withdrawn from the world market in 2006. A retrospective case-control pharmacogenetic study of ALT elevation during long-term treatment of ximelagatran revealed a strong genetic association between elevated ALT and the HLA alleles DRB1*07 and DQA1*02, suggesting a possible immune-mediated pathogenesis []. Furthermore, a competitive binding assay revealed that ximelagatran was able to inhibit the binding of the ligand peptide to HLA-DRB1*07:01, supporting the specific involvement of HLA-DRB1*07:01 in the idiosyncratic ximelagatran-induced hepatotoxicity [].

Methods

VSMCs were harvested from human saphenous veins. VSMCs were analyzed for intracellular cAMP levels and PKA activity after exposure to AT-RvD1. Platelet-derived growth factor (PDGF)-induced migration and cytoskeletal changes in VSMCs were observed through scratch, Transwell, and cell shape assays in the presence or absence of a PKA inhibitor (Rp-8-Br-cAMP). Further investigation of the pathways involved in AT-RvD1 signaling was performed by measuring Rac1 activity, vasodilator stimulated phosphoprotein (VASP) phosphorylation and paxillin translocation. Finally, we examined the role of RvD1 receptors (GPR32 and ALX/FPR2) in AT-RvD1 induced effects on VSMC migration and PKA activity.

Results

Treatment with AT-RvD1 induced a significant increase in cAMP levels and PKA activity in VSMCs at 5 minutes and 30 minutes, respectively. AT-RvD1 attenuated PDGF-induced VSMC migration and cytoskeletal rearrangements. These effects were attenuated by the PKA inhibitor Rp-8-Br-cAMP, suggesting cAMP/PKA involvement. Treatment of VSMC with AT-RvD1 inhibited PDGF-stimulated Rac1 activity, increased VASP phosphorylation, and attenuated paxillin localization to focal adhesions; these effects were negated by the addition of Rp-8-Br-cAMP. The effects of AT-RvD1 on VSMC migration and PKA activity were attenuated by blocking ALX/FPR2, suggesting an important role of this G-protein coupled receptor.

Conclusions

Our results suggest that AT-RvD1 attenuates PDGF-induced VSMC migration via ALX/FPR2 and cAMP/PKA. Interference with Rac1, VASP and paxillin function appear to mediate the downstream effects of AT-RvD1 on VSMC migration.

Cell culture and reagents

Primary VSMCs were isolated from human great saphenous veins discarded at the time of bypass operation under a University of California San Francisco Institutional Review Board-approved protocol (UCSF Committee on Human Research- Number: 10–03395; the committee waived the need for informed consent) as described previously []. VSMCs were cultured at 37°C and 5% CO2 in low glucose Dulbecco’s modified Eagle’s medium (DMEM; HyClone Laboratories, Logan, UT, USA) containing 10% FBS (Life Technologies, Grand Island, NY). VSMCs between passages 2 and 5 were utilized for all experiments. AT-RvD1 (7S,8R,17R-trihydroxy-4Z,9E,11E,13Z,15E19Z-docosahexaenoic acid), 17S-RvD1 (7S,8R,17S-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid) were obtained from Cayman Chemical (Ann Arbor, MI, USA). The anti-GPR32 antibody was purchased from GeneTex (Irvine, CA, USA) and the anti-ALX antibody from Abcam (Cambridge, United Kingdom). PDGF-BB was purchased from Sigma-Aldrich (St. Louis, MO, USA) and Rp-8-Br-cAMP from Enzo Life Sciences (Farmingdale, NY, USA). Phorbol 12,13-dibutyrate (PDBu) was purchased from Biolog (Hayward, CA, USA) and chelerythrine chloride from Sigma-Aldrich (St. Louis, MO, USA).

cAMP measurement

Confluent VSMCs grown in 12-well plates were serum-starved overnight in DMEM containing 0.1% FBS. Cells were treated with AT-RvD1 (10nM) and harvested at different times using a 0.1N HCl, 1% Triton X-100 lysis buffer. After spinning the lysates at 12,000g for 10 minutes and collecting the supernatants, cAMP and total protein contents were measured via ELISA (Enzo Life Sciences, Farmingdale, NY, USA) and BCA assay (Thermo Fisher Scientific, Waltham, MA, USA), respectively, following the manufacturers’ protocols. cAMP concentrations in the lysates were calculated from the absorbance readouts using a four-parameter logistic fit and then normalized by total protein content.

Abstract

18F-fluciclovine (trans-1-amino-3-18F-fluorocyclobutanecarboxylic acid) is an amino acid positron emission tomography (PET) tracer used for cancer staging (e.g., prostate and breast). Patients scheduled to undergo amino acid-PET are usually required to fast before PET tracer administration. However, there have been no reports addressing whether fasting improves fluciclovine-PET imaging. In this study, the authors investigated the influence of fasting on fluciclovine-PET using triple-tracer autoradiography with 14C-fluciclovine, [5,6-3H]-2-fluoro-2-deoxy-d-glucose (3H-FDG), and 99mTc-hydroxymethylene diphosphonate (99mTc-HMDP) in a rat breast cancer model of mixed osteolytic/osteoblastic bone metastases in which the animals fasted overnight. Lesion accumulation of each tracer was evaluated using the target-to-background (muscle) ratio. The mean ratios of 14C-fluciclovine in osteolytic lesions were 4.6 ± 0.8 and 2.8 ± 0.6, respectively, with and without fasting, while those for 3H-FDG were 6.9 ± 2.5 and 5.1 ± 2.0, respectively. In the peri-tumor bone formation regions (osteoblastic), where 99mTc-HMDP accumulated, the ratios of 14C-fluciclovine were 4.3 ± 1.4 and 2.4 ± 0.7, respectively, and those of 3H-FDG were 6.2 ± 3.8 and 3.3 ± 2.2, respectively, with and without fasting. These results suggest that fasting before 18F-fluciclovine-PET improves the contrast between osteolytic and osteoblastic bone metastatic lesions and background, as well as 18F-FDG-PET.

5. Conclusions

Our findings suggest that fasting influences the uptake of 18F-fluciclovine in osteolytic and osteoblastic bone metastasis lesions, and can facilitate clearer visualization of lesions in fluciclovine-PET imaging. However, because AA metabolism is a complex process, further animal and clinical studies are required to confirm our findings.

Abstract

The amino acid 4-fluoro-l-phenylalanine (4F-Phe) was introduced at the positions of Phe6 and Phe22 in the 29-residue polypeptide hormone glucagon by expressing glucagon in E. coli in the presence of an excess of 4F-Phe. Glucagon regulates blood glucose homeostasis by interaction with the glucagon receptor (GCGR), a class B GPCR. By referencing to the 4F-Phe chemical shifts at varying D2O concentrations, the solvent exposure of the two Phe sites along the glucagon sequence was determined, showing that 4F-Phe6 was fully solvent exposed and 4F-Phe22 was only partially exposed. The incorporation of fluorine atoms in polypeptide hormones paves the way for novel studies of their interactions with membrane-spanning receptors, specifically by differentiating between effects on the solvent accessibility, the line shapes, and the chemical shifts from interactions with lipids, detergents and proteins. Studies of interactions of GCGR with ligands in solution is at this point of keen interest, given that recent crystallographic studies revealed that an apparent small molecule antagonist actually binds as an allosteric effector at a distance of ~20 Å from the orthosteric ligand binding site (Jazayeri et al., in Nature 533:274–277, 2016).

19F-NMR study of glucagon solvent accessibility at discrete sequence locations

The two phenylalanine residues in positions 6 and 22 of the glucagon amino acid sequence were substituted by 4F-Phe (Fig. a) in the recombinant variant polypeptide (Fig. b) by incubating E. coli cells with an excess of 4F-Phe, which was then incorporated via the naturally occurring tRNAPhe. 19F-NMR spectra were obtained for 4F-Phe and for 4F-Phe-labeled glucagon in H2O/D2O solutions with variable D2O concentrations (Fig. a, c). Samples of 4 mM 4F-Phe and 0.1 mM 4F-Phe-labeled glucagon in acidic water (pH* 2.5) were prepared with addition of D2O at 10% increments for 19F-NMR spectra collection. Within the NMR tube, a capillary containing 5 mM trifluoroacetic acid (TFA) was used as an external standard, and 19F chemical shift values were referenced to TFA at −76.55 ppm. Except for the spectra shown in Fig. b (see the figure caption for details), the 19F-NMR experiments were performed at 298 K on a Bruker AVANCE-500 spectrometer equipped with a room temperature BBO probehead (Bruker Biospin), using the pulse program zgfhigqn30.2 with 1H-decoupling during 19F-detection. The 19F frequency was about 470 MHz, and the number of scans was 128 for 4F-Phe, and 2048 for 4F-Phe-labeled glucagon. Prior to Fourier transformation, the time-domain NMR data were multiplied with an exponential function with line broadening factors of 1 and 5 Hz for 4F-Phe and 4F-Phe-labeled glucagon, respectively.

19F-NMR study of glucagon solvent accessibility at discrete sequence locations

Just as with 4F-Phe, the 19F chemical shift values of both labels in glucagon decreased linearly with increasing D2O concentration (Fig. d). The maximum 19F chemical shift differences were 0.14 ppm for 4F-Phe6 and 0.08 ppm for 4F-Phe22. These differences suggest that position 6 has full solvent exposure, whereas position 22 is only partially exposed to solvent. This result is readily rationalized by structural data from previous 1H-NMR studies of glucagon in acidic aqueous solutions, which showed that the region around residue six is flexibly disordered and Phe22 is part of a hydrophobic cluster formed by the residues –Phe22–Val23–Gln24–Trp25– (Boesch et al. ).

Introduction

Here, glucagon was labeled by substituting Phe in positions 6 and 22 with the non-proteinogenic amino acid 4-fluoro-l-phenylalanine (4F-Phe) (Bann and Frieden ; Li and Frieden ). The solvent isotope effect on the 19F chemical shift at variable D2O concentrations (Hansen et al. ; Kitevski-LeBlanc et al. ; Shi et al. ) was used to investigate site-specific solvent accessibility of glucagon (Fig. ). As a control, 1D 1H-NMR and cAMP assays were used to show that 4F-Phe incorporation caused at most minimal perturbations of glucagon conformation and activity (Fig. ). Our work lays a foundation for studying glucagon conformation and dynamics when interacting with GCGR with and without the presence of intramolecular partner proteins. Further investigation of glucagon–GCGR interactions in solution is particularly important given recent crystallographic evidence that an apparent small-molecule antagonist actually binds as an allosteric effector to the intracellular side of GCGR at the distance of ~20 Å from the orthosteric ligand binding site (Jazayeri et al. ).

Glucagon conformation and biological activity are preserved after 4F-Phe incorporation

Glucagon activates adenylate cyclase by binding to GCGR and activating G proteins (Bataille and Dalle ). We applied cAMP assays (Norskov-Lauritsen et al. ) to test [4F-Phe6,22]-glucagon for activity. The binding curves demonstrated that [4F-Phe6,22]-glucagon had similar activity as glucagon (Fig. g), indicating that this 19F-labeling does not affect glucagon signaling activity.

Abstract

Vitamin B1 biosynthesis de novo is regulated by duplicated genes and a conserved riboswitch in cassava. Diversity of vitamin B1 levels in cassava leaves negatively correlates with expression of selected vitamin B1 biosynthesis genes.

Abstract

Vitamin B1, which consists of the vitamers thiamin and its phosphorylated derivatives, is an essential micronutrient for all living organisms because it is required as a metabolic cofactor in several enzymatic reactions. Genetic diversity of vitamin B1 biosynthesis and accumulation has not been investigated in major crop species other than rice and potato. We analyzed cassava germplasm for accumulation of B1 vitamers. Vitamin B1 content in leaves and roots of 41 cassava accessions showed significant variation between accessions. HPLC analyses of B1 vitamers revealed distinct profiles in cassava leaves and storage roots, with nearly equal relative levels of thiamin pyrophosphate and thiamin monophosphate in leaves, but mostly thiamin pyrophosphate in storage roots. Unusually, the cassava genome has two genes encoding the 4-amino-2-methyl-5-hydroxymethylpyrimidine phosphate synthase, THIC (MeTHIC1 and MeTHIC2), both of which carry a riboswitch in the 3ʹ-UTR, as well as the adenylated thiazole synthase, THI1 (MeTHI1a and MeTHI1b). The THIC and THI1 genes are expressed at very low levels in storage roots compared with the accumulation of vitamin B1, indicating only limited biosynthesis de novo therein. In leaves, vitamin B1 content is negatively correlated with THIC and THI1 expression levels, suggesting post-transcriptional regulation of THIC by the riboswitch present in the 3ʹ-UTR of the THIC mRNA and regulation of THI1 by promoter activity or alternative post-transcriptional mechanisms.

Abstract

Antioxidants have a number of potential health benefits. The present investigation was designed to determine the relationship between serum alpha- and gamma-tocopherol levels (powerful antioxidants), and leukocyte telomere length (a biomarker of biological aging). A cross-sectional design was employed to study 5768 adults from the National Health and Nutrition Examination Survey (NHANES). DNA was obtained via blood samples. Telomere length was assessed using the quantitative polymerase chain reaction method. Serum concentrations of alpha- and gamma-tocopherol were measured using high performance liquid chromatography (HPLC). Results showed that for each one-year increase in age, telomeres were 15.6 base pairs shorter (F = 410.4, p < 0.0001). After adjusting for differences in the demographic covariates, for each µg/dL higher level of gamma-tocopherol, telomeres were 0.33 base pairs shorter (F = 7.1, p = 0.0126). Telomeres were approximately 1 year shorter (15.6 base pairs) for each increment of 47.3 to 55.7 µg/dL of gamma-tocopherol in the blood, depending on the variables controlled. Adults at the 75th percentile of gamma-tocopherol had 2.8–3.4 years greater cellular aging than those at the 25th percentile, depending on the covariates in the model. However, alpha-tocopherol was not related to telomere length. Evidently, gamma-tocopherol levels, but not alpha-tocopherol, account for meaningful increases in biological aging.

Abstract

Docosahexaenoic acid, enriched in the brain and retina, generates docosanoids in response to disruptions of cellular homeostasis. Docosanoids include neuroprotectin D1 (NPD1), which is decreased in the CA1 hippocampal area of patients with early-stage Alzheimer's disease (AD). We summarize here how NPD1 elicits neuroprotection by up-regulating c-REL, a nuclear factor (NF)-κB subtype that, in turn, enhances expression of BIRC3 (baculoviral inhibitor of apoptosis repeat-containing protein 3) in the retina and in experimental stroke, leading to neuroprotection. Elucidating the mechanisms of action of docosanoids will contribute to managing diseases, including stroke, AD, age-related macular degeneration, traumatic brain injury, Parkinson's disease, and other neurodegenerations.

DHA arrival to the brain and docosanoids

Pre-clinical events in AD include neuroinflammation, dendritic spine damage, and synaptic dysfunction exacerbated by UOS, which leads to dementia. These events coincide with the decreased DHA content in the brain. A study of brains from early-stage AD patients (with neuronal loss) shows that the amount of DHA and its enzymatic derivative, neuroprotectin D1 (NPD1; 10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid), is decreased in the CA1 region of the hippocampus (). NPD1 and other DHA-derived lipid mediators, such as resolvins D1–D5 and maresins, are known as docosanoids and foster overall inflammation resolution (, ).

Significance of bioactive lipids in ischemic stroke

Activation of N-methyl d-aspartate (NMDA) glutamate receptors occurs after stroke and in epilepsy and other neurological diseases (). This activation is accompanied by an increase in membrane phospholipid hydrolysis with concomitant accumulation of free fatty acids (mainly DHA and arachidonic acid) and stearoyl-arachidonoyl-sn-glycerol () due to the rapid activation of phospholipases A2 and C. DHA administration (i.v.) improves behavioral deficit and reduces infarct volume and edema after experimental focal cerebral ischemia (, ). Enzymatic lipoxygenation of DHA in experimental ischemic stroke results in NPD1 formation, which then counteracts pro-inflammatory bioactivity and contributes to sustained neuroprotection (, ). The onset of ischemia causes damage to neurons, and one could argue that in rodent experimental models even after 8 h of reperfusion, accumulation of endogenous neuroprotective mediators (including NPD1) is not sufficient to cope with the resulting damage. Between 24 and 72 h after reperfusion, polymorphonuclear leukocytes migrate to the brain parenchyma, initially removing debris. However, because of their large numbers, these leukocytes induce inflammation and further promote injury, thus increasing stroke size. Continuous injection of NPD1 into the fourth ventricle (400 ng/48 h) after ischemic experimental stroke reduced stroke size in rats by 50% (, ), attenuated polymorphonuclear leukocyte infiltration, and inhibited ischemia induction of cyclooxygenase-2 (COX-2) and nuclear factor κB (NF-κB) (, ). The cerebral ischemic core is surrounded by an area called the penumbra, and due to decreased blood circulation, cell viability is sustained only for 2–4 h post-infarction; therefore, it is critical to protect the penumbra. There is a high correlation between reducing the size of the penumbra and neurological recovery ().