Manganese- and Platinum-Driven Oxidative and Nitrosative Stress in Oxaliplatin-Associated CIPN with Special Reference to Ca4Mn(DPDP)5, MnDPDP and DPDP.

Platinum-containing chemotherapeutic drugs are efficacious in many forms of cancer but are dose-restricted by serious side effects, of which peripheral neuropathy induced by oxidative-nitrosative-stress-mediated chain reactions is most disturbing. Recently, hope has been raised regarding the catalytic antioxidants mangafodipir (MnDPDP) and calmangafodipir [Ca4Mn(DPDP)5; PledOx®], which by mimicking mitochondrial manganese superoxide dismutase (MnSOD) may be expected to overcome oxaliplatin-associated chemotherapy-induced peripheral neuropathy (CIPN). Unfortunately, two recent phase III studies (POLAR A and M trials) applying Ca4Mn(DPDP)5 in colorectal cancer (CRC) patients receiving multiple cycles of FOLFOX6 (5-FU + oxaliplatin) failed to demonstrate efficacy. Instead of an anticipated 50% reduction in the incidence of CIPN in patients co-treated with Ca4Mn(DPDP)5, a statistically significant increase of about 50% was seen. The current article deals with confusing differences between early and positive findings with MnDPDP in comparison to the recent findings with Ca4Mn(DPDP)5. The POLAR failure may also reveal important mechanisms behind oxaliplatin-associated CIPN itself. Thus, exacerbated neurotoxicity in patients receiving Ca4Mn(DPDP)5 may be explained by redox interactions between Pt2+ and Mn2+ and subtle oxidative-nitrosative chain reactions. In peripheral sensory nerves, Pt2+ presumably leads to oxidation of the Mn2+ from Ca4Mn(DPDP)5 as well as from Mn2+ in MnSOD and other endogenous sources. Thereafter, Mn3+ may be oxidized by peroxynitrite (ONOO-) into Mn4+, which drives site-specific nitration of tyrosine (Tyr) 34 in the MnSOD enzyme. Conformational changes of MnSOD then lead to the closure of the superoxide (O2•-) access channel. A similar metal-driven nitration of Tyr74 in cytochrome c will cause an irreversible disruption of electron transport. Altogether, these events may uncover important steps in the mechanism behind Pt2+-associated CIPN. There is little doubt that the efficacy of MnDPDP and its therapeutic improved counterpart Ca4Mn(DPDP)5 mainly depends on their MnSOD-mimetic activity when it comes to their potential use as rescue medicines during, e.g., acute myocardial infarction. However, pharmacokinetic considerations suggest that the efficacy of MnDPDP on Pt2+-associated neurotoxicity depends on another action of this drug. Electron paramagnetic resonance (EPR) studies have demonstrated that Pt2+ outcompetes Mn2+ and endogenous Zn2+ in binding to fodipir (DPDP), hence suggesting that the previously reported protective efficacy of MnDPDP against CIPN is a result of chelation and elimination of Pt2+ by DPDP, which in turn suggests that Mn2+ is unnecessary for efficacy when it comes to oxaliplatin-associated CIPN.

A Comparative Study on the Effects of the Lysine Reagent Pyridoxal 5-Phosphate and Some Thiol Reagents in Opening the Tl+-Induced Mitochondrial Permeability Transition Pore.

Lysine residues are essential in regulating enzymatic activity and the spatial structure maintenance of mitochondrial proteins and functional complexes. The most important parts of the mitochondrial permeability transition pore are F1F0 ATPase, the adenine nucleotide translocase (ANT), and the inorganic phosphate cotransporter. The ANT conformation play a significant role in the Tl+-induced MPTP opening in the inner membrane of calcium-loaded rat liver mitochondria. The present study tests the effects of a lysine reagent, pyridoxal 5-phosphate (PLP), and thiol reagents (phenylarsine oxide, tert-butylhydroperoxide, eosin-5-maleimide, and mersalyl) to induce the MPTP opening that was accompanied by increased swelling, membrane potential decline, and decreased respiration in 3 and 3UDNP (2,4-dinitrophenol uncoupled) states. This pore opening was more noticeable in increasing the concentration of PLP and thiol reagents. However, more significant concentrations of PLP were required to induce the above effects comparable to those of these thiol reagents. This study suggests that the Tl+-induced MPTP opening can be associated not only with the state of functionally active cysteines of the pore parts, but may be due to a change in the state of the corresponding lysines forming the pore structure.

Identification of biomarkers and candidate small-molecule drugs in lipopolysaccharide (LPS)-induced acute lung injury by bioinformatics analysis.

BACKGROUND/OBJECTIVE: Acute lung injury (ALI) is a critical clinical syndrome with high rates of incidence and mortality. However, its molecular mechanism remains unclear. The current work aimed to explore the molecular mechanisms of ALI by identifying different expression genes (DEGs) and candidate drugs using a combination of chip analysis and experimental validation. METHODS: Three microarray datasets were downloaded from Gene Expression Omnibus (GEO) database to obtain DEGs. We conducted a Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway-enrichment analyses of overlapping DEGs among three databases. The expression level of key gene was verified by Western blotting analysis in LPS-treated ALI cell models. Finally, we predicted the candidate drugs targeting the key gene that might be effective for ALI treatment, and the role of candidate drug in treating ALI was verified by investigation. RESULTS: A total 29 overlapping DEGs were up-regulated in LPS-induced ALI groups. They were enriched in inflammation and inflammation-related pathways. Serpin family A member 3 (SERPINA3) was defined as a key gene because it was associated with inflammation pathway and up-regulated in microarray datasets in LPS-induced ALI. In LPS-induced human bronchial epithelial cells transformed with Ad12-SV40-2B (BEAS-2B) cells, SERPINA3 was enhanced. Pyridoxal phosphate as an upstream drug of SERPINA3 could improve cell viability and reduce expression inflammatory factors in LPS-treated BEAS-2B cells. CONCLUSION: Our study suggested that pyridoxal phosphate could be a candidate drug targeting SERPINA3 gene in LPS-induced ALI. It has protective and anti-inflammatory effects in BEAS-2B cells, and may become a potential novel treatment for ALI.

Milk metabolome reveals pyrimidine and its degradation products as the discriminant markers of different corn silage-based nutritional strategies.

The purpose of this study was to evaluate the effect of 6 different feeding systems (based on corn silage as the main ingredient) on the chemical composition of milk and to highlight the potential of untargeted metabolomics to find discriminant marker compounds of different nutritional strategies. Interestingly, the multivariate statistical analysis discriminated milk samples mainly according to the high-moisture ear corn (HMC) included in the diet formulation. Overall, the most discriminant compounds, identified as a function of the HMC, belonged to AA (10 compounds), peptides (71 compounds), pyrimidines (38 compounds), purines (15 compounds), and pyridines (14 compounds). The discriminant milk metabolites were found to significantly explain the metabolic pathways of pyrimidines and vitamin B6. Interestingly, pathway analyses revealed that the inclusion of HMC in the diet formulation strongly affected the pyrimidine metabolism in milk, determining a significant up-accumulation of pyrimidine degradation products, such as 3-ureidopropionic acid, 3-ureidoisobutyric acid, and 3-aminoisobutyric acid. Also, some pyrimidine intermediates (such as l-aspartic acid, N-carbamoyl-l-aspartic acid, and orotic acid) were found to possess a high discrimination degree. Additionally, our findings suggested that the inclusion of alfalfa silage in the diet formulation was potentially correlated with the vitamin B6 metabolism in milk, being 4-pyridoxic acid (a pyridoxal phosphate degradation product) the most significant and up-accumulated compound. Taken together, the accumulation trends of different marker compounds revealed that both pyrimidine intermediates and degradation products are potential marker compounds of HMC-based diets, likely involving a complex metabolism of microbial nitrogen based on total splanchnic fluxes from the rumen to mammary gland in dairy cows. Also, our findings highlight the potential of untargeted metabolomics in both foodomics and foodomics-based studies involving dairy products.

Vitamin B6 rescues insulin resistance and glucose-induced DNA damage caused by reduced activity of Drosophila PI3K.

The insulin signaling pathway controls cell growth and metabolism, thus its deregulation is associated with both cancer and diabetes. Phosphatidylinositol 3-kinase (PI3K) contributes to the cascade of phosphorylation events occurring in the insulin pathway by activating the protein kinase B (PKB/AKT), which phosphorylates several substrates, including those involved in glucose uptake and storage. PI3K inactivating mutations are associated with insulin resistance while activating mutations are identified in human cancers. Here we show that RNAi-induced depletion of the Drosophila PI3K catalytic subunit (Dp110) results in diabetic phenotypes such as hyperglycemia, body size reduction, and decreased glycogen content. Interestingly, we found that hyperglycemia produces chromosome aberrations (CABs) triggered by the accumulation of advanced glycation end-products and reactive oxygen species. Rearing PI3KRNAi flies in a medium supplemented with pyridoxal 5'-phosphate (PLP; the catalytically active form of vitamin B6) rescues DNA damage while, in contrast, treating PI3KRNAi larvae with the PLP inhibitor 4-deoxypyridoxine strongly enhances CAB frequency. Interestingly, PLP supplementation rescues also diabetic phenotypes. Taken together, our results provide a strong link between impaired PI3K activity and genomic instability, a crucial relationship that needs to be monitored not only in diabetes due to impaired insulin signaling but also in cancer therapies based on PI3K inhibitors. In addition, our findings confirm the notion that vitamin B6 is a good natural remedy to counteract insulin resistance and its complications.

Thiamine-dependent regulation of mammalian brain pyridoxal kinase in vitro and in vivo.

Vitamins B1 (thiamine) and B6 (pyridox (al/ine/amine)) are crucial for central nervous system (CNS) function and neurogenesis due to the coenzyme action of their phosphorylated derivatives in the brain metabolism of glucose and neurotransmitters. Here, the non-coenzyme action of thiamine on the major mammalian producers of pyridoxal-5'-phosphate (PLP), such as pyridoxal kinase (PdxK) and pyridoxine 5'-phosphate oxidase (PNPO), is characterized. Among the natural thiamine compounds, thiamine triphosphate (ThTP) is the best effector of recombinant human PdxK (hPdxK) in vitro, inhibiting hPdxK in the presence of Mg2+ but activating the Zn2+ -dependent reaction. Inhibition of hPdxK by thiamine antagonists decreases from amprolium to pyrithiamine to oxythiamine, highlighting possible dysregulation of both the B1 - and B6 -dependent metabolism in the chemical models of thiamine deficiency. Compared with the canonical hPdxK, the D87H and V128I variants show a twofold increase in Kapp of thiamine inhibition, and the V128I and H246Q variants show a fourfold and a twofold decreased Kapp of thiamine diphosphate (ThDP), respectively. Thiamine administration changes diurnal regulation of PdxK activity and phosphorylation at Ser213 and Ser285, expression of the PdxK-related circadian kinases/phosphatases in the rat brain, and electrocardiography (ECG). In contrast to PdxK, PNPO is not affected by thiamine or its derivatives, either in vitro or in vivo. Dephosphorylation of the PdxK Ser285, potentially affecting mobility of the ATP-binding loop, inversely correlates with the enzyme activity. Dephosphorylation of the PdxK Ser213, which is far away from the active site, does not correlate with the activity. The correlations analysis suggests the PdxK Ser213 to be a target of kinase MAP2K1 and phosphatase Ppp1ca. Diurnal effects of thiamine administration on the metabolically linked ThDP- and PLP-dependent enzymes may support the brain homeostatic mechanisms and physiological fitness.

Pyridoxal-5-phosphate induced cardioprotection in aging associated with up-expression of cystathionine-γ-lyase, 3-mercaptopyruvate sulfurtransferase, and ATP-sensitive potassium channels.

BACKGROUND: In the present work, we investigated the cardioprotective potential of pyridoxal-5-phosphate (PLP) in old rats as a cofactor of enzymes that synthesize hydrogen sulphide (H2 S). MATERIALS AND METHODS: PLP was administered per os in a dose of 0.7 mg per kg daily for 2 weeks. Rats were divided into three groups (adult, old and old +PLP) of 20 animals. The cardiac mRNA levels of genes encoding H2 S-synthesizing enzymes cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST), uncoupling proteins (UCP3), subunits of ATP-sensitive potassium (KATP ) channels were determined using real-time polymerase chain reaction analysis. We also studied the effect of PLP-administration on the content of H2 S, oxidative stress, the activities of inducible and constitutive NO-synthase (iNOS, cNOS), arginase and nitrate reductase in the heart homogenates as well as cardiac resistance to ischemia-reperfusion in Langendorff-isolated heart model. RESULTS: It was shown that PLP restored mRNA levels of CSE, 3-MST and UCP3 genes, and H2 S content and also significantly increased the expression of SUR2 and Kir6.1 (2.2 and 3.3 times, respectively) in the heart of old rats. PLP significantly reduced the formation of superoxide, malondialdehyde, diene conjugates as well as the activity of iNOS and arginase. PLP significantly increased constitutive synthesis of NO and prevented reperfusion disturbances of the heart function after ischemia. CONCLUSIONS: Thus, PLP-administration in old rats was associated with up-expression of CSE, 3-MST, UCP3 and SUR2 and Kir6.1 subunits of KATP channels, and also increased cNOS activity and reduced oxidative stress and prevented reperfusion dysfunction of the heart in ischemia-reperfusion.

Assessment of stunned and viable myocardium using manganese-enhanced MRI.

OBJECTIVE: In a proof-of-concept study, to quantify myocardial viability in patients with acute myocardial infarction using manganese-enhanced MRI (MEMRI), a measure of intracellular calcium handling. METHODS: Healthy volunteers (n=20) and patients with ST-elevation myocardial infarction (n=20) underwent late gadolinium enhancement (LGE) using gadobutrol and MEMRI using manganese dipyridoxyl diphosphate. Patients were scanned ≤7 days after reperfusion and rescanned after 3 months. Differential manganese uptake was described using a two-compartment model. RESULTS: After manganese administration, healthy control and remote non-infarcted myocardium showed a sustained 25% reduction in T1 values (mean reductions, 288±34 and 281±12 ms). Infarcted myocardium demonstrated less T1 shortening than healthy control or remote myocardium (1157±74 vs 859±36 and 835±28 ms; both p<0.0001) with intermediate T1 values (1007±31 ms) in peri-infarct regions. Compared with LGE, MEMRI was more sensitive in detecting dysfunctional myocardium (dysfunctional fraction 40.5±11.9 vs 34.9%±13.9%; p=0.02) and tracked more closely with abnormal wall motion (r2=0.72 vs 0.55; p<0.0001). Kinetic modelling showed reduced myocardial manganese influx between remote, peri-infarct and infarct regions, enabling absolute discrimination of infarcted myocardium. After 3 months, manganese uptake increased in peri-infarct regions (16.5±3.5 vs 22.8±3.5 mL/100 g/min, p<0.0001), but not the remote (23.3±2.8 vs 23.0±3.2 mL/100 g/min, p=0.8) or infarcted (11.5±3.7 vs 14.0±1.2 mL/100 g/min, p>0.1) myocardium. CONCLUSIONS: Through visualisation of intracellular calcium handling, MEMRI accurately differentiates infarcted, stunned and viable myocardium, and correlates with myocardial dysfunction better than LGE. MEMRI holds major promise in directly assessing myocardial viability, function and calcium handling across a range of cardiac diseases. TRIAL REGISTRATION NUMBERS: NCT03607669; EudraCT number 2016-003782-25.

Expression of purinergic receptors on microglia in the animal model of choroidal neovascularisation.

To investigate the effect of P2 receptor on microglia and its inhibitor PPADS on choroidal neovascularization. Forty CX3CR1GFP/+ mice were randomly divided into 8 groups. In addition to the normal group, the rest of groups were receiving laser treatment. The retina and choroid from the second, third, fourth and fifth group of mice were taken in the 1, 4, 7, 14 days after laser treatment. The mice in the sixth and seventh group received intravitreal injection of 2 µl PPADS or PBS respectively immediately after laser treatment. The mice in the eighth group received topical application of PPADS once per day of three days. The mice in sixth, seventh and eighth group received AF and FFA examination on the fourth day after laser treatment. Immunofluorescence histochemical staining and real-time quantitative PCR were used to evaluate P2 expression and its effect on choroidal neovascularization. After laser treatment, activated microglia can express P2 receptors (P2X4, P2X7, P2Y2 and P2Y12). The expression of P2 increased on the first day after laser damage, peaked on the fourth day (tP2X4 = 6.05, tP2X7 = 2.95, tP2Y2 = 3.67, tP2Y12 = 5.98, all P < 0.01), and then decreased. After PPADS inhibition, compared with the PBS injection group, the mRNA of P2X4, P2X7, P2Y2 and P2Y12 were decreased significantly in the PPADS injection group (tP2X4 = 5.54, tP2X7 = 9.82, tP2Y2 = 3.86, tP2Y12 = 7.91, all P < 0.01) and the PPADS topical application group (tP2X4 = 3.24, tP2X7 = 5.89, tP2Y2 = 6.75, tP2Y12 = 4.97, all P < 0.01). Compared with the PBS injection group, not only the activity of microglia cells but also the leakage of CNV decreased significantly (P < 0.01) in the PPADS injection group and the PPADS topical application group. But between two PPADS groups, the leakage of CNV had no difference (P = 0.864). After laser induced CNV, activated microglia can express P2 receptors. The P2 receptor inhibitor, PPADS, can significantly affect the function of microglia and inhibit the formation of choroidal neovascularization.

Contribution of ATP-Mediated Positive Feedback to Sympathetic Nerve-Induced Positive Inotropy in Guinea Pig Ventricular Myocardium.

The functional role of ATP released from sympathetic nerve terminals was examined in isolated guinea pig ventricular papillary muscles. The contractile force of papillary muscles was increased by field electrical stimulation of sympathetic nerve endings. This increase was attenuated by pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) or suramin, blockers of the P2X receptor, and was abolished by propranolol and prazosin. PPADS, suramin, and ATP affected neither the basal contractile force nor the positive inotropic effect of noradrenaline. These results provide functional evidence that ATP released from sympathetic nerve terminals enhances noradrenaline release and contributes to sympathetic nerve-induced inotropy.

P2X7 receptor antagonism increases regulatory T cells and reduces clinical and histological graft-versus-host disease in a humanised mouse model.

Graft-versus-host disease (GVHD) is a severe inflammatory response arising from allogeneic haematopoietic stem cell transplantation. Previous studies revealed that antagonism of the P2X7 receptor with Brilliant Blue G (BBG) reduced liver GVHD but did not alter clinical GVHD in a humanised mouse model. Therefore, the present study aimed to trial a modified injection regime using more frequent dosing of BBG to improve outcomes in this model of GVHD. NOD-scid IL2Rγnull (NSG) mice were injected intraperitoneally (i.p.) with 10 × 106 human peripheral blood mononuclear cells (hPBMCs) (day 0), then daily with BBG (50 mg/kg) or saline (days 0-10). BBG significantly reduced clinical score, mortality and histological GVHD compared with saline treatment (endpoint). BBG significantly increased proportions of human regulatory T cells (Tregs) and human B cells and reduced serum human interferon-γ compared with saline treatment prior to development of clinical GVHD (day 21). To confirm the therapeutic benefit of P2X7 antagonism, NSG mice were injected i.p. with 10 × 106 hPBMCs (day 0), then daily with pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (300 mg/kg) or saline (days 0-10). PPADS increased human Treg proportions compared with saline treatment (day 21), but potential clinical benefits were confounded by increased weight loss with this antagonist. To investigate the role of P2X7 antagonism on Treg survival, hPBMCs were cultured in reduced serum conditions to promote cell death. BBG increased proportions of Tregs (and B cells) compared with saline under these conditions. In conclusion, P2X7 antagonism reduces clinical and histological GVHD in a humanised mouse model corresponding to an increase in human Tregs.

Bladder urothelium converts bacterial lipopolysaccharide information into neural signaling via an ATP-mediated pathway to enhance the micturition reflex for rapid defense.

When bacteria enter the bladder lumen, a first-stage active defensive mechanism flushes them out. Although urinary frequency induced by bacterial cystitis is a well-known defensive response against bacteria, the underlying mechanism remains unclear. In this study, using a mouse model of acute bacterial cystitis, we demonstrate that the bladder urothelium senses luminal extracellular bacterial lipopolysaccharide (LPS) through Toll-like receptor 4 and releases the transmitter ATP. Moreover, analysis of purinergic P2X2 and P2X3 receptor-deficient mice indicated that ATP signaling plays a pivotal role in the LPS-induced activation of L6-S1 spinal neurons through the bladder afferent pathway, resulting in rapid onset of the enhanced micturition reflex. Thus, we revealed a novel defensive mechanism against bacterial infection via an epithelial-neural interaction that induces urinary frequency prior to bacterial clearance by neutrophils of the innate immune system. Our results indicate an important defense role for the bladder urothelium as a chemical-neural transducer, converting bacterial LPS information into neural signaling via an ATP-mediated pathway, with bladder urothelial cells acting as sensory receptor cells.

Pyridoxal 5'-phosphate enhances the growth and morpho-physiological characteristics of rice cultivars by mitigating the ethylene accumulation under salinity stress.

Salinity-induced ethylene accumulation caused by high production of 1-aminocyclopropane-1-carboxylic acid (ACC) hinders rice plant growth and development. Nevertheless, ACC deaminase may alleviate salt stress and high ethylene production in rice cultivars under salinity stress. Pyridoxal 5'-phosphate (PLP), an ACC deaminase co-factor, could be a useful ACC inhibitor in plants; however, it has not been studied before. In the present study, the effects of PLP on the growth and morphophysiological characteristics of rice cultivars (Jinyuan 85 (JY85) and Nipponbare (NPBA) were investigated under salinity stress (control (CK), low salinity (LS), and high salinity (HS) in hydroponic conditions. The experiment was laid out in a completely randomized design (CRD) under factorial arrangement of treatments. The results showed that, compared with no PLP, exogenous application of PLP significantly inhibited ACC and ethylene production in the roots, leaves and panicles of both cultivars under salinity, and PLP was more effective at improving the physiological characteristics of both cultivars under salinity stress. Further, root morphophysiological traits and pollen viability were triggered in the PLP treatment compared to the no-PLP treatment under various salinity levels. ACC production inhibited by PLP was useful for improving the 1000-grain weight, grain yield per plant, and total plant biomass under the CK, LS and HS treatments in both rice cultivars. These results revealed that PLP, as an ACC deaminase cofactor, is a key tool for mitigating ethylene-induced effects under salinity stress and for enhancing the agronomic and morphophysiological traits of rice under saline conditions.

SAR of non-hydrolysable analogs of pyridoxal 5'-phosphate against low molecular weight protein tyrosine phosphatase isoforms.

Kinases and phosphatases are key enzymes in cell signal transduction pathways. Imbalances in these enzymes have been linked to numerous disease states ranging from cancer to diabetes to autoimmune disorders. The two isoforms (IFA and IFB) of Low Molecular Weight Protein Tyrosine Phosphatase (LMW-PTP) appear to play a role in these diseases. Pyridoxal 5'-phosphate (PLP) has been shown to act as a potent but, impractical micromolar inhibitor for both isoforms. In this study, a series of non-hydrolysable phosphonate analogs of PLP were designed, synthesized and tested against the two isoforms of LMW-PTP. Assay results demonstrated that the best inhibitor for both isoforms was compound 5 with a Kis of 1.84 µM (IFA) and 15.6 µM (IFB). The most selective inhibitor was compound 16, with a selectivity of roughly 370-fold for IFA over IFB.

Identification of Phosphate-Containing Compounds as New Inhibitors of 14-3-3/c-Abl Protein-Protein Interaction.

The 14-3-3/c-Abl protein-protein interaction (PPI) is related to carcinogenesis and in particular to pathogenesis of chronic myeloid leukemia (CML). Previous studies have demonstrated that molecules able to disrupt this interaction improve the nuclear translocation of c-Abl, inducing apoptosis in leukemia cells. Through an X-ray crystallography screening program, we have identified two phosphate-containing compounds, inosine monophosphate (IMP) and pyridoxal phosphate (PLP), as binders of human 14-3-3σ, by targeting the protein amphipathic groove. Interestingly, they also act as weak inhibitors of the 14-3-3/c-Abl PPI, demonstrated by NMR, SPR, and FP data. A 37-compound library of PLP and IMP analogues was investigated using a FP assay, leading to the identification of three further molecules acting as weak inhibitors of the 14-3-3/c-Abl complex formation. The antiproliferative activity of IMP, PLP, and the three derivatives was tested against K-562 cells, showing that the parent compounds had the most pronounced effect on tumor cells. PLP and IMP were also effective in promoting the c-Abl nuclear translocation in c-Abl overexpressing cells. Further, these compounds demonstrated low cytotoxicity on human Hs27 fibroblasts. In conclusion, our data suggest that 14-3-3σ targeting compounds represent promising hits for further development of drugs against c-Abl-dependent cancers.

Stimulation of the endogenous hydrogen sulfide synthesis suppresses oxidative-nitrosative stress and restores endothelial-dependent vasorelaxation in old rats.

Hydrogen sulfide (H2S) is an endogenous gas transmitter with profound effects on the cardiovascular system. We hypothesized that stimulation of H2S synthesis might alleviate age-associated changes in vascular reactivity. Pyridoxal-5-phosphate (PLP), the coenzyme of H2S-synthesizing enzymes, was administrated to old male Wistar rats per os at a dose of 0.7 mg/kg body mass once a day for 2 weeks. H2S content in the aortic tissue, markers of oxidative stress, inducible nitric oxide synthase (iNOS) and constitutive nitric oxide synthase (cNOS), arginase activities, and endothelium-dependent vasorelaxation of the aortic rings were studied. Our results showed that PLP restored endogenous H2S and low molecular weight S-nitrosothiol levels in old rat aorta to the levels detected in adults. PLP significantly reduced diene conjugate content, hydrogen peroxide and peroxynitrite generation rates, and iNOS and arginase activity in the aortic tissue of old rats. PLP also greatly improved acetylcholine-induced relaxation of old rat aorta (47.7% ± 4.8% versus 18.4% ± 4.1% in old rats, P < 0.05) that was abolished by NO inhibition with N-nitro-l-arginine methyl ester hydrochloride (L-NAME) or H2S inhibition with O-carboxymethylhydroxylamine (O-CMH). Thus, PLP might be used for stimulation of endogenous H2S synthesis and correction of oxidative and nitrosative stress and vessel tone dysfunction in aging and age-associated diseases.

High sensitivity and non-background SERS detection of endogenous hydrogen sulfide in living cells using core-shell nanoparticles.

Endogenous hydrogen sulfide (H2S) exists in multiple physiological processes. In order to further understand the action mechanism of H2S in cells and human body, we proposed a smart surface-enhanced Raman scattering (SERS) nanoprobe, Au core-4-mercaptobenzonitrile-Ag shell nanoparticle (Au@4-MBN@Ag), for the detection of endogenous H2S in living cells based on the reaction between Ag shell and sulfide species. 4-MBN was selected as the SERS reporter to avoid interference from cellular molecules. With the sulfide concentration increasing, the Ag2S constantly formed, and consequently the SERS signal intensity of Au@4-MBN@Ag gradually decreased owing to the weaker SERS activity of Ag2S. With the nanoprobes, this method not only offers a high sensitivity for H2S detection at an nM level, but also achieves the goal of non-background analysis. It displays satisfactory anti-interference capability and a good linear relationship with sulfide concentration ranging from 50 nM to 500 µM, and an estimated detection limit is 0.14 nM. The Au@4-MBN@Ag nanoprobes were successfully applied to detect endogenous H2S in living HepG2 cells stimulated by pyridoxal 5-phosphate monohydrate. This work offers a potential analytical method in the related research of H2S physiological function.

Manganese-Enhanced MRI of the Brain in Healthy Volunteers.

BACKGROUND AND PURPOSE: The manganese ion is used as an intracellular MR imaging contrast agent to study neuronal function in animal models, but it remains unclear whether manganese-enhanced MR imaging can be similarly useful in humans. Using mangafodipir (Teslascan, a chelated manganese-based contrast agent that is FDA-approved), we evaluated the dynamics of manganese enhancement of the brain and glandular structures in the rostral head and neck in healthy volunteers. MATERIALS AND METHODS: We administered mangafodipir intravenously at a rate of 1 mL/minute for a total dose of 5 µmol/kg body weight. Nine healthy adult volunteers (6 men/3 women; median age, 43 years) completed baseline history and physical examination, 3T MR imaging, and blood work. MR imaging also followed mangafodipir administration at various time points from immediate to 7 days, with delayed scans at 1-3 months. RESULTS: The choroid plexus and anterior pituitary gland enhanced within 10 minutes of infusion, with enhancement persisting up to 7 and 30 days, respectively. Exocrine (parotid, submandibular, sublingual, and lacrimal) glands also enhanced avidly as early as 1 hour postadministration, generally resolving by 1 month; 3 volunteers had residual exocrine gland enhancement, which resolved by 2 months in 1 and by 3 months in the other 2. Mangafodipir did not affect clinical parameters, laboratory values, or T1-weighted signal in the basal ganglia. CONCLUSIONS: Manganese ions released from mangafodipir successfully enable noninvasive visualization of intra- and extracranial structures that lie outside the blood-brain barrier without adverse clinical effects, setting the stage for future neuroradiologic investigation in disease.

Development of Recombinant Methioninase for Cancer Treatment.

The elevated requirement for methionine (MET) of cancer cells is termed MET dependence. To selectively target the MET dependence of tumors for treatment on a large-scale preclinical and clinical basis, the L-methionine α-deamino-γ-mercaptomethane-lyase (EC 4.4.1.11) (methioninase, [METase]) gene from Pseudomonas putida has been cloned in Escherichia coli using the polymerase chain reaction (PCR). Purification using two DEAE Sepharose FF ion-exchange column and one ActiClean Etox endotoxin-affinity chromatography column has been established. Plasmid pMGLTrc03, which has a trc promoter and a spacing of 12 nucleotides between the Shine-Dalgarno sequence and the ATG translation initiation codon, was selected as the most suitable plasmid. The recombinant bacteria produced rMETase at 43% of the total proteins in soluble fraction by simple batch fermentation using a 500 L fermentor. Crystals were directly obtained from crude enzyme with 87% yield by a crystallization in the presence of 9.0% polyethylene glycol 6000, 3.6% ammonium sulfate, and 0.18 M sodium chloride using a 100 L crystallizer. After recrystallization, the enzyme was purified by anion-exchange column chromatography to remove endotoxins and by gel filtration for polishing. Purified rMETase is stable to lyophilization. In order to prevent immunological reactions which might be produced by multiple dosing of rMETase and to prolong the serum half-life of rMETase, the N-hydroxysuccinimidyl ester of methoxypolyethylene glycol propionic acid (M-SPA-PEG 5000) has been coupled to rMETase. The PEGylated molecules (PEG-rMETase) were purified from unreacted PEG with Amicon 30 K centriprep concentrators or by Sephacryl S-300 HR gel-filtration chromatography. Unreacted rMETase was removed by DEAE Sepharose FF anion-exchange chromatography. The resulting PEG-rMETase subunit, produced from a PEG/rMETase ratio of 30/1 in the synthetic reaction, had a molecular mass of approximately 53 kda determined by matrix-assisted laser desorption/ionization mass spectrometry, indicating the conjugation of two PEG molecules per subunit of rMETase and eight per tetramer. PEG-rMETase molecules obtained from reacting ratios of PEG/rMETase of 30/1 had an enzyme activity of 70% of unmodified rMETase. PEGylation of rMETase increased the serum half-life of the enzyme in rats to approximately 160 min compared to 80 min for unmodified rMETase. PEG-rMETase could deplete serum MET levels to less than 0.1 µM for approximately 8 h compared to 2 h for rMETase in rats. A significant prolongation of in vivo activity and effective MET depletion by the PEG-rMETase were achieved by the simultaneous administration of pyridoxal 5'-phosphate. rMETase was also conjugated with methoxypolyethylene glycol succinimidyl glutarate 5000 (MEGC-PEG). Miniosmotic pumps containing various concentrations of PLP were implanted in BALB-C mice. PLP-infused mice were then injected with a single dose of 4000 or 8000 units/kg PEG-rMETase. Mice infused with 5, 50, 100, 200, and 500 mg/mL PLP-containing miniosmotic pumps increased plasma PLP to 7, 24, 34, 60, and 95 µM, respectively, from the PLP baseline of 0.3 µM. PLP increased the half-life of MEGC-PEG-rMETase holoenzyme in a dose-dependent manner. The extended time of MET depletion by MEGC-PEG-rMETase was due to the maintenance of active MEGC-PEG-rMETase holoenzyme by infused PLP.

Melanopsin for precise optogenetic activation of astrocyte-neuron networks.

Optogenetics has been widely expanded to enhance or suppress neuronal activity and it has been recently applied to glial cells. Here, we have used a new approach based on selective expression of melanopsin, a G-protein-coupled photopigment, in astrocytes to trigger Ca2+ signaling. Using the genetically encoded Ca2+ indicator GCaMP6f and two-photon imaging, we show that melanopsin is both competent to stimulate robust IP3-dependent Ca2+ signals in astrocyte fine processes, and to evoke an ATP/Adenosine-dependent transient boost of hippocampal excitatory synaptic transmission. Additionally, under low-frequency light stimulation conditions, melanopsin-transfected astrocytes can trigger long-term synaptic changes. In vivo, melanopsin-astrocyte activation enhances episodic-like memory, suggesting melanopsin as an optical tool that could recapitulate the wide range of regulatory actions of astrocytes on neuronal networks in behaving animals. These results describe a novel approach using melanopsin as a precise trigger for astrocytes that mimics their endogenous G-protein signaling pathways, and present melanopsin as a valuable optical tool for neuron-glia studies.