On-Surface Synthesis of BN-Substituted Heteroaromatic Networks

We report on the bottom-up fabrication of BN-substituted heteroaromatic networks achieved by surface-assisted polymerization and subsequent cyclodehydrogenation of specifically designed BN-substituted precursor monomers based on a borazine core structural element. To get insight into the cyclodehydrogenation pathway and the influence of molecular flexibility on network quality, two closely related precursor monomers with different degrees of internal cyclodehydrogenation have been employed. Scanning tunneling microscopy shows that, for both monomers, surface-assisted cyclodehydrogenation allows for complete monomer cyclization and the formation of covalently interlinked BN-substituted polyaromatic hydrocarbon networks on the Ag(111) surface. In agreement with experimental observations, density functional theory calculations reveal a significantly lower energy barrier for the cyclodehydrogenation of the conformationally more rigid precursor monomer, which is also reflected in a higher degree of long-range order of the obtained heteroaromatic network. Our proof-of-concept study will allow for the fabrication of atomically precise substitution patterns within BNC heterostructures.

Immunophenotyping of acute leukemia and lymphoproliferative disorders: a consensus proposal of the European LeukemiaNet Work Package 10

The European LeukemiaNet (ELN), workpackage 10 (WP10) was designed to deal with diagnosis matters using morphology and immunophenotyping. This group aimed at establishing a consensus on the required reagents for proper immunophenotyping of acute leukemia and lymphoproliferative disorders. Animated discussions within WP10, together with the application of the Delphi method of proposals circulation, quickly led to post-consensual immunophenotyping panels for disorders on the ELN website. In this report, we established a comprehensive description of these panels, both mandatory and complementary, for both types of clinical conditions. The reason for using each marker, sustained by relevant literature information, is provided in detail. With the constant development of immunophenotyping techniques in flow cytometry and related software, this work aims at providing useful guidelines to perform the most pertinent exploration at diagnosis and for follow-up, with the best cost benefit in diseases, the treatment of which has a strong impact on health systems.

Molecular and biochemical characterisation of a novel mutation in POLG associated with Alpers syndrome

Background DNA polymerase γ (POLG) is the only known mitochondrial DNA (mtDNA) polymerase. It mediates mtDNA replication and base excision repair. Mutations in the POLG gene lead to reduction of functional mtDNA (mtDNA depletion and/or deletions) and are therefore predicted to result in defective oxidative phosphorylation (OXPHOS). Many mutations map to the polymerase and exonuclease domains of the enzyme and produce a broad clinical spectrum. The most frequent mutation p.A467T is localised in the linker region between these domains. In compound heterozygote patients the p.A467T mutation has been described to be associated amongst others with fatal childhood encephalopathy. These patients have a poorer survival rate compared to homozygotes. Methods mtDNA content in various tissues (fibroblasts, muscle and liver) was quantified using quantitative PCR (qPCR). OXPHOS activities in the same tissues were assessed using spectrophotometric methods and catalytic stain of BN-PAGE. Results We characterise a novel splice site mutation in POLG found in trans with the p.A467T mutation in a 3.5 years old boy with valproic acid induced acute liver failure (Alpers-Huttenlocher syndrome). These mutations result in a tissue specific depletion of the mtDNA which correlates with the OXPHOS-activities. Conclusions mtDNA depletion can be expressed in a high tissue-specific manner and confirms the need to analyse primary tissue. Furthermore, POLG analysis optimises clinical management in the early stages of disease and reinforces the need for its evaluation before starting valproic acid treatment.

Dopamine-related deficit in reward learning after catecholamine depletion in unmedicated, remitted subjects with bulimia nervosa

Disturbances in reward processing have been implicated in bulimia nervosa (BN). Abnormalities in processing reward-related stimuli might be linked to dysfunctions of the catecholaminergic neurotransmitter system, but findings have been inconclusive. A powerful way to investigate the relationship between catecholaminergic function and behavior is to examine behavioral changes in response to experimental catecholamine depletion (CD). The purpose of this study was to uncover putative catecholaminergic dysfunction in remitted subjects with BN who performed a reinforcement-learning task after CD. CD was achieved by oral alpha-methyl-para-tyrosine (AMPT) in 19 unmedicated female subjects with remitted BN (rBN) and 28 demographically matched healthy female controls (HC). Sham depletion administered identical capsules containing diphenhydramine. The study design consisted of a randomized, double-blind, placebo-controlled crossover, single-site experimental trial. The main outcome measures were reward learning in a probabilistic reward task analyzed using signal-detection theory. Secondary outcome measures included self-report assessments, including the Eating Disorder Examination-Questionnaire. Relative to healthy controls, rBN subjects were characterized by blunted reward learning in the AMPT-but not in placebo-condition. Highlighting the specificity of these findings, groups did not differ in their ability to perceptually distinguish between stimuli. Increased CD-induced anhedonic (but not eating disorder) symptoms were associated with a reduced response bias toward a more frequently rewarded stimulus. In conclusion, under CD, rBN subjects showed reduced reward learning compared with healthy control subjects. These deficits uncover disturbance of the central reward processing systems in rBN related to altered brain catecholamine levels, which might reflect a trait-like deficit increasing vulnerability to BN.

Evolution of bombesin conjugates for targeted PET imaging of tumors

Bombesin receptors are under intense investigation as molecular targets since they are overexpressed in several prevalent solid tumors. We rationally designed and synthesized a series of modified bombesin (BN) peptide analogs to study the influence of charge and spacers at the N-terminus, as well as amino acid substitutions, on both receptor binding affinity and pharmacokinetics. This enabled development of a novel (64/67)Cu-labeled BN peptide for PET imaging and targeted radiotherapy of BN receptor-positive tumors. Our results show that N-terminally positively charged peptide ligands had significantly higher affinity to human gastrin releasing peptide receptor (GRPr) than negatively charged or uncharged ligands (IC(50): 3.2±0.5 vs 26.3±3.5 vs 41.5±2.5 nM). The replacement of Nle(14) by Met, and deletion of D-Tyr(6), further resulted in 8-fold higher affinity. Contrary to significant changes to human GRPr binding, modifications at the N-terminal and at the 6(th), 11(th), and 14(th) position of BN induced only slight influences on affinity to mouse GRPr. [Cu(II)]-CPTA-[βAla(11)] BN(7-14) ([Cu(II)]-BZH7) showed the highest internalization rate into PC-3 cells with relatively slow efflux because of its subnanomolar affinity to GRPr. Interestingly, [(64/67)Cu]-BZH7 also displayed similar affinities to the other 2 human BN receptor subtypes. In vivo studies showed that [(64/67)Cu]-BZH7 had a high accumulation in PC-3 xenografts and allowed for clear-cut visualization of the tumor in PET imaging. In addition, a CPTA-glycine derivative, forming a hippurane-type spacer, enhanced kidney clearance of the radiotracer. These data indicate that the species variation of BN receptor plays an important role in screening radiolabeled BN. As well, the positive charge from the metallated complex at the N-terminal significantly increases affinity to human GRPr. Application of these observations enabled the novel ligand [(64/67)Cu]-BZH7 to clearly visualize PC-3 tumors in vivo. This study provides a strong starting point for optimizing radiopeptides for targeting carcinomas that express any of the BN receptor subtypes.

Novel 111In-labelled bombesin analogues for molecular imaging of prostate tumours

PURPOSE: It has been shown that some primary human tumours and their metastases, including prostate and breast tumours, overexpress gastrin-releasing peptide (GRP) receptors. Bombesin (BN) is a neuropeptide with a high affinity for these GRP receptors. We demonstrated successful scintigraphic visualisation of BN receptor-positive tumours in preclinical studies using the radiolabelled BN analogue [(111)In-DTPA-Pro(1),Tyr(4)]BN. However, the receptor affinity as well as the serum stability of this analogue leave room for improvement. Therefore new (111)In-labelled BN analogues were synthesised and evaluated in vitro and in vivo. METHODS AND RESULTS: The receptor affinity of the new BN analogues was tested on human GRP receptor-expressing prostate tumour xenografts and rat colon sections. Analogues with high receptor affinity (low nM range) were selected for further evaluation. Incubation in vitro of GRP receptor-expressing rat CA20948 and human PC3 tumour cells with the (111)In-labelled analogues resulted in rapid receptor-mediated uptake and internalisation. The BN analogue with the best receptor affinity and in vitro internalisation characteristics, Cmp 3 ([(111)In-DTPA-ACMpip(5),Tha(6),betaAla(11),Tha(13),Nle(14)]BN(5-14)), was tested in vivo in biodistribution studies using rats bearing GRP receptor-expressing CA20948 tumours, and nude mice bearing human PC3 xenografts. Injection of (111)In-labelled Cmp 3 in these animals showed high, receptor-mediated uptake in receptor-positive organs and tumours which could be visualised using planar gamma camera and microSPECT/CT imaging. CONCLUSION: With their enhanced receptor affinity and their rapid receptor-mediated internalisation in vitro and in vivo, the new BN analogues, and especially Cmp 3, are promising candidates for use in diagnostic molecular imaging and targeted radionuclide therapy of GRP receptor-expressing cancers.

DOTA-PESIN, a DOTA-conjugated bombesin derivative designed for the imaging and targeted radionuclide treatment of bombesin receptor-positive tumours

PURPOSE: We aimed at designing and developing a novel bombesin analogue, DOTA-PEG(4)-BN(7-14) (DOTA-PESIN), with the goal of labelling it with (67/68)Ga and (177)Lu for diagnosis and radionuclide therapy of prostate and other human cancers overexpressing bombesin receptors. METHODS: The 8-amino acid peptide bombesin (7-14) was coupled to the macrocyclic chelator DOTA via the spacer 15-amino-4,7,10,13-tetraoxapentadecanoic acid (PEG(4)). The conjugate was complexed with Ga(III) and Lu(III) salts. The GRP receptor affinity and the bombesin receptor subtype profile were determined in human tumour specimens expressing the three bombesin receptor subtypes. Internalisation and efflux studies were performed with the human GRP receptor cell line PC-3. Xenografted nude mice were used for biodistribution. RESULTS: [Ga(III)/Lu(III)]-DOTA-PESIN showed good affinity to GRP and neuromedin B receptors but no affinity to BB3. [(67)Ga/(177)Lu]-DOTA-PESIN internalised rapidly into PC-3 cells whereas the efflux from PC-3 cells was relatively slow. In vivo experiments showed a high and specific tumour uptake and good retention of [(67)Ga/(177)Lu]-DOTA-PESIN. [(67)Ga/(177)Lu]-DOTA-PESIN highly accumulated in GRP receptor-expressing mouse pancreas. The uptake specificity was demonstrated by blocking tumour uptake and pancreas uptake. Fast clearance was found from blood and all non-target organs except the kidneys. High tumour-to-normal tissue ratios were achieved, which increased with time. PET imaging with [(68)Ga]-DOTA-PESIN was successful in visualising the tumour at 1 h post injection. Planar scintigraphic imaging showed that the (177)Lu-labelled peptide remained in the tumour even 3 days post injection. CONCLUSION: The newly designed ligands have high potential with regard to PET and SPECT imaging with (68/67)Ga and targeted radionuclide therapy with (177)Lu.

Gamma-tocopherol supplementation inhibits protein nitration and ascorbate oxidation in rats with inflammation

Gamma-tocopherol (gammaT) complements alpha-tocopherol (alphaT) by trapping reactive nitrogen oxides to form a stable adduct, 5-nitro-gammaT [Christen et al., PNAS 94:3217-3222; 1997]. This observation led to the current investigation in which we studied the effects of gammaT supplementation on plasma and tissue vitamin C, vitamin E, and protein nitration before and after zymosan-induced acute peritonitis. Male Fischer 344 rats were fed for 4 weeks with either a normal chow diet with basal 32 mg alphaT/kg, or the same diet supplemented with approximately 90 mg d-gammaT/kg. Supplementation resulted in significantly higher levels of gammaT in plasma, liver, and kidney of control animals without affecting alphaT, total alphaT+gammaT or vitamin C. Intraperitoneal injection of zymosan caused a marked increase in 3-nitrotyrosine and a profound decline in vitamin C in all tissues examined. Supplementation with gammaT significantly inhibited protein nitration and ascorbate oxidation in the kidney, as indicated by the 29% and 56% reduction of kidney 3-nitrotyrosine and dehydroascorbate, respectively. Supplementation significantly attenuated inflammation-induced loss of vitamin C in the plasma (38%) and kidney (20%). Zymosan-treated animals had significantly higher plasma and tissue gammaT than nontreated pair-fed controls, and the elevation of gammaT was strongly accentuated by the supplementation. In contrast, alphaT did not significantly change in response to zymosan treatment. In untreated control animals, gammaT supplementation lowered basal levels of 3-nitrotyrosine in the kidney and buffered the starvation-induced changes in vitamin C in all tissues examined. Our study provides the first in vivo evidence that in rats with high basal amounts of alphaT, a moderate gammaT supplementation attenuates inflammation-mediated damage, and spares vitamin C during starvation-induced stress without affecting alphaT.

Analysis of plasma tocopherols alpha, gamma, and 5-nitro-gamma in rats with inflammation by HPLC coulometric detection

Reactive nitrogen oxide species (RNOS) have been implicated as effector molecules in inflammatory diseases. There is emerging evidence that gamma-tocopherol (gammaT), the major form of vitamin E in the North American diet, may play an important role in these diseases. GammaT scavenges RNOS such as peroxynitrite by forming a stable adduct, 5-nitro-gammaT (NGT). Here we describe a convenient HPLC method for the simultaneous determination of NGT, alphaT, and gammaT in blood plasma and other tissues. Coulometric detection of NGT separated on a deactivated reversed-phase column was linear over a wide range of concentrations and highly sensitive (approximately 10 fmol detection limit). NGT extracted from blood plasma of 15-week-old Fischer 344 rats was in the low nM range, representing approximately 4% of gammaT. Twenty-four h after intraperitoneal injection of zymosan, plasma NGT levels were 2-fold higher compared to fasted control animals when adjusted to gammaT or corrected for total neutral lipids, while alpha- and gammaT levels remained unchanged. These results demonstrate that nitration of gammaT is increased under inflammatory conditions and highlight the importance of RNOS reactions in the lipid phase. The present HPLC method should be helpful in clarifying the precise physiological role of gammaT.

Immunodetection of 3-nitrotyrosine in the liver of zymosan-treated rats with a new monoclonal antibody: comparison to analysis by HPLC

Zymosan-induced peritonitis is associated with an increased production of reactive nitrogen oxides that may contribute to the often-observed failure of multiple organ systems in this model of acute inflammation. Quantitative biochemical evidence is provided for a marked 13-fold increase in protein-bound 3-nitrotyrosine (NTyr), a biomarker of reactive nitrogen oxides, in liver tissue of zymosan-treated rats. In order to investigate the localization of NTyr in this affected tissue, a monoclonal antibody, designated 39B6, was raised against 3-(4-hydroxy-3-nitrophenylacetamido) propionic acid-bovine serum albumin conjugate and its performance characterized. 39B6 was judged by competition ELISA to be approximately 2 orders of magnitude more sensitive than a commercial anti-NTyr monoclonal antibody. Binding characteristics of 39B6 were similar, but not identical, to that of a commercial affinity-purified polyclonal antibody in ELISA and immunohistochemical analyses. Western blot experiments revealed high specificity of 39B6 against NTyr and increased immunoreactivity of specific proteins from liver tissue homogenates of zymosan-treated rats. Immunohistochemical analysis of liver sections indicated a marked zymosan-induced increase in immunofluorescent staining, which was particularly intense in or adjacent to nonparenchymal cells, but not in the parenchymal cells of this tissue. Quantitative analysis of fractions enriched in these cell populations corroborated the immunofluorescent data, although the relative amounts detected in response to zymosan treatment was greatly reduced compared to whole liver tissue. These results demonstrate the high specificity of the newly developed antibody and its usefulness in Western blot and immunohistochemical analysis for NTyr, confirm the presence of NTyr by complementary methods, and suggest the possible involvement of reactive nitrogen oxides in hepatic vascular dysfunction.

gamma-tocopherol, the major form of vitamin E in the US diet, deserves more attention

gamma-tocopherol is the major form of vitamin E in many plant seeds and in the US diet, but has drawn little attention compared with alpha-tocopherol, the predominant form of vitamin E in tissues and the primary form in supplements. However, recent studies indicate that gamma-tocopherol may be important to human health and that it possesses unique features that distinguish it from alpha-tocopherol. gamma-Tocopherol appears to be a more effective trap for lipophilic electrophiles than is alpha-tocopherol. gamma-Tocopherol is well absorbed and accumulates to a significant degree in some human tissues; it is metabolized, however, largely to 2,7,8-trimethyl-2-(beta-carboxyethyl)-6-hydroxychroman (gamma-CEHC), which is mainly excreted in the urine. gamma-CEHC, but not the corresponding metabolite derived from alpha-tocopherol, has natriuretic activity that may be of physiologic importance. Both gamma-tocopherol and gamma-CEHC, but not alpha-tocopherol, inhibit cyclooxygenase activity and, thus, possess antiinflammatory properties. Some human and animal studies indicate that plasma concentrations of gamma-tocopherol are inversely associated with the incidence of cardiovascular disease and prostate cancer. These distinguishing features of gamma-tocopherol and its metabolite suggest that gamma-tocopherol may contribute significantly to human health in ways not recognized previously. This possibility should be further evaluated, especially considering that high doses of alpha-tocopherol deplete plasma and tissue gamma-tocopherol, in contrast with supplementation with gamma-tocopherol, which increases both. We review current information on the bioavailability, metabolism, chemistry, and nonantioxidant activities of gamma-tocopherol and epidemiologic data concerning the relation between gamma-tocopherol and cardiovascular disease and cancer.

Ascorbate is depleted by smoking and repleted by moderate supplementation: a study in male smokers and nonsmokers with matched dietary antioxidant intakes

BACKGROUND: Lack of reliable dietary data has hampered the ability to effectively distinguish between effects of smoking and diet on plasma antioxidant status. As confirmed by analyses of comprehensive food-frequency questionnaires, the total dietary intakes of fruit and vegetables and of dietary antioxidants were not significantly different between the study groups in the present study, thereby enabling isolation of the effect of smoking. OBJECTIVE: Our objective was to investigate the effect of smoking on plasma antioxidant status by measuring ascorbic acid, alpha-tocopherol, gamma-tocopherol, beta-carotene, and lycopene, and subsequently, to test the effect of a 3-mo dietary supplementation with a moderate-dose vitamin cocktail. DESIGN: In a double-blind, placebo-controlled design, the effect of a vitamin cocktail containing 272 mg vitamin C, 31 mg all-rac-alpha-tocopheryl acetate, and 400 microg folic acid on plasma antioxidants was determined in a population of smokers (n = 37) and nonsmokers (n = 38). The population was selected for a low intake of fruit and vegetables and recruited from the San Francisco Bay area. RESULTS: Only ascorbic acid was significantly depleted by smoking per se (P < 0.01). After the 3-mo supplementation period, ascorbic acid was efficiently repleted in smokers (P < 0.001). Plasma alpha-tocopherol and the ratio of alpha- to gamma-tocopherol increased significantly in both supplemented groups (P < 0.05). CONCLUSIONS: Our data suggest that previous reports of lower concentrations of plasma vitamin E and carotenoids in smokers than in nonsmokers may primarily have been caused by differences in dietary habits between study groups. Plasma ascorbic acid was depleted by smoking and repleted by moderate supplementation.

gamma-tocopherol traps mutagenic electrophiles such as NO(X) and complements alpha-tocopherol: physiological implications

Peroxynitrite, a powerful mutagenic oxidant and nitrating species, is formed by the near diffusion-limited reaction of .NO and O2.- during activation of phagocytes. Chronic inflammation induced by phagocytes is a major contributor to cancer and other degenerative diseases. We examined how gamma-tocopherol (gammaT), the principal form of vitamin E in the United States diet, and alpha-tocopherol (alphaT), the major form in supplements, protect against peroxynitrite-induced lipid oxidation. Lipid hydroperoxide formation in liposomes (but not isolated low-density lipoprotein) exposed to peroxynitrite or the .NO and O2.- generator SIN-1 (3-morpholinosydnonimine) was inhibited more effectively by gammaT than alphaT. More importantly, nitration of gammaT at the nucleophilic 5-position, which proceeded in both liposomes and human low density lipoprotein at yields of approximately 50% and approximately 75%, respectively, was not affected by the presence of alphaT. These results suggest that despite alphaT's action as an antioxidant gammaT is required to effectively remove the peroxynitrite-derived nitrating species. We postulate that gammaT acts in vivo as a trap for membrane-soluble electrophilic nitrogen oxides and other electrophilic mutagens, forming stable carbon-centered adducts through the nucleophilic 5-position, which is blocked in alphaT. Because large doses of dietary alphaT displace gammaT in plasma and other tissues, the current wisdom of vitamin E supplementation with primarily alphaT should be reconsidered.