Directed evolution of the fluorescent protein CGP with in situ biosynthesized noncanonical amino acids.

The incorporation of noncanonical amino acids (ncAAs) into proteins can enhance their function beyond the abilities of canonical amino acids and even generate new functions. However, the ncAAs used for such research are usually chemically synthesized, which is expensive and hinders their application on large industrial scales. We believe that the biosynthesis of ncAAs using metabolic engineering and their employment in situ in target protein engineering with genetic code expansion could overcome these limitations. As a proof of principle, we biosynthesized four ncAAs, O-L-methyltyrosine, 3,4-dihydroxy-L-phenylalanine, 5-hydroxytryptophan, and 5-chloro-L-tryptophan using metabolic engineering and directly evolved the fluorescent consensus green protein (CGP) by combination with nine other exogenous ncAAs in Escherichia coli. After screening a TAG scanning library expressing 13 ncAAs, several variants with enhanced fluorescence and stability were identified. The variants CGPV3pMeoF/K190pMeoF and CGPG20pMeoF/K190pMeoF expressed with biosynthetic O-L-methyltyrosine showed an approximately 1.4-fold improvement in fluorescence compared to the original level, and a 2.5-fold improvement in residual fluorescence after heat treatment. Our results demonstrated the feasibility of integrating metabolic engineering, genetic code expansion, and directed evolution in engineered cells to employ biosynthetic ncAAs in protein engineering. These results could further promote the application of ncAAs in protein engineering and enzyme evolution. IMPORTANCE: Noncanonical amino acids (ncAAs) have shown great potential in protein engineering and enzyme evolution through genetic code expansion. However, in most cases, ncAAs must be provided exogenously during protein expression, which hinders their application, especially when they are expensive or have poor cell membrane penetration. Engineering cells with artificial metabolic pathways to biosynthesize ncAAs and employing them in situ for protein engineering and enzyme evolution could facilitate their application and reduce costs. Here, we attempted to evolve the fluorescent consensus green protein (CGP) with biosynthesized ncAAs. Our results demonstrated the feasibility of using biosynthesized ncAAs in protein engineering, which could further stimulate the application of ncAAs in bioengineering and biomedicine.

Development of early diagnosis of Parkinson's disease on animal models based on the intranasal administration of α-methyl-p-tyrosine methyl ester in a gel system.

The fight against neurodegenerative diseases, including Parkinson's disease (PD), is a global challenge of this century. The effectiveness of current PD therapy is limited, since it is diagnosed many years after the onset, following the death of most nigrostriatal dopaminergic neurons regulating motor function. PD treatment could be greatly improved if it was started at an early (preclinical) stage. For this purpose, it is necessary to develop an early diagnosis of PD, which is the goal of our study. We have developed an early diagnosis of PD on animal models using a provocative test by intranasal administration of α-methyl-p-tyrosine methyl ester (αMPTME), a reversible inhibitor of dopamine synthesis. First, we produced the provocative agent, αMPTME in gel, and showed its safety and penetration into the brain bypassing the blood-brain barrier. Then, the optimal dose of αMPTME and time after administration were selected, at which the level of dopamine in the striatum of intact animals decreases, but does not reach the 30% threshold for the appearance of motor disorders in PD patients. Finally, we proved on animal models that intranasal administration of αMPTME can serve as a diagnostic test for preclinical PD. Indeed, intranasal administration of αMPTME to mice in a model of PD at the preclinical stage reversibly reduced the dopamine level in the striatum to the 30% threshold causing short-term motor disorders. Thus, using animal models of PD, we have developed a provocative test for the preclinical diagnosis of PD, a fundamentally new technology in neurology.

Interaction of Halogenated Tyrosine/Phenylalanine Derivatives with Organic Anion Transporter 1 in the Renal Handling of Tumor Imaging Probes.

Halogenated tyrosine/phenylalanine derivatives have been developed for use in tumor imaging and targeted alpha therapy. 3-Fluoro-α-methyl-l-tyrosine (FAMT), targeting amino acid transporter LAT1 (SLC7A5), is a cancer-specific positron emission tomography probe that exhibits high renal accumulation, which is supposed to be mediated by organic anion transporter OAT1 (SLC22A6). In the present study, we investigated the structural requirements of FAMT essential for interaction with OAT1. OAT1 transported FAMT with a K m of 171.9 µM. In structure-activity relationship analyses, removal of either the 3-halogen or 4-hydroxyl group from FAMT or its structural analog 3-iodo-α-methyl-l-tyrosine greatly decreased the interaction with OAT1, reducing the [14C]p-aminohippurate uptake inhibition and the efflux induction. By contrast, the α-methyl group, which is essential for LAT1 specificity, contributed to a lesser degree. In fluorinated tyrosine derivatives, fluorine at any position was accepted by OAT1 when there was a hydroxyl group at the ortho-position, whereas ortho-fluorine was less interactive when a hydroxyl group was at meta- or para-positions. The replacement of the ortho-fluorine with a bulky iodine atom greatly increased the interaction. In in vivo studies, probenecid decreased the renal accumulation (P < 0.001) and urinary excretion (P = 0.0012) of FAMT, whereas the plasma concentration was increased, suggesting the involvement of OAT1-mediated transepithelial organic anion excretion. LAT1-specific 2-fluoro-α-methyltyrosine, which had lower affinity for OAT1, exhibited lower renal accumulation (P = 0.0142) and higher tumor uptake (P = 0.0192) compared with FAMT. These results would provide a basis to design tumor-specific compounds that can avoid renal accumulation for tumor imaging and targeted alpha therapy. SIGNIFICANCE STATEMENT: We revealed the structural characteristics of halogenated tyrosine derivatives essential for interaction with the organic anion transporter responsible for their renal accumulation. We have confirmed that such interactions are important for renal handling and tumor uptake. The critical contribution of hydroxyl and halogen groups and their positions as well as the role of α-methyl group found in the present study may facilitate the development of tumor-specific compounds while avoiding renal accumulation for use in tumor imaging and targeted alpha therapy.

Methylated derivatives of l-tyrosine in reaction catalyzed by l-amino acid oxidase: isotope and inhibitory effects.

l-Amino acid oxidase (LAAO) is widely distributed in nature and shows important biological activity. It induces cell apoptosis and has antibacterial properties. This study was designed to investigate the effect of methyl substituent on its activity as methylated derivatives of l-tyrosine, labelled with short-lived B+ emitters, have been used in oncological diagnostics. To study isotope effects in the oxidative deamination of O-methyl-l-tyrosine, the deuterated isotopomer, i.e. O-methyl-[2-2H]-l-tyrosine, was synthesized by isotope exchange, catalyzed enzymatically by tryptophanase. Isotope effects were determined using the spectrophotometric non-competitive method. The values of isotope effects indicate that the α-C-H bond cleavage occurs in the rate determining step of the investigated reaction and α-hydrogen plays a role in the substrate binding process at the enzyme active site. The inhibitory effect on LAAO activity was studied with α-methyl-l-tyrosine and N-methyl-l-tyrosine. The mode of inhibition was determined based on Lineweavear-Burk plots intersections. α-Methyl-l-tyrosine has been found a mixed type inhibitor of the investigated enzyme, whereas N-methyl-l-tyrosine is a non-competitive inhibitor of LAAO.

Synthesis, experimental and in silico studies of N-fluorenylmethoxycarbonyl-O-tert-butyl-N-methyltyrosine, coupled with CSD data: a survey of interactions in the crystal structures of Fmoc-amino acids.

Recently, fluorenylmethoxycarbonyl (Fmoc) amino acids (e.g. Fmoc-tyrosine or Fmoc-phenylalanine) have attracted growing interest in biomedical research and industry, with special emphasis directed towards the design and development of novel effective hydrogelators, biomaterials or therapeutics. With this in mind, a systematic knowledge of the structural and supramolecular features in recognition of those properties is essential. This work is the first comprehensive summary of noncovalent interactions combined with a library of supramolecular synthon patterns in all crystal structures of amino acids with the Fmoc moiety reported so far. Moreover, a new Fmoc-protected amino acid, namely, 2-{[(9H-fluoren-9-ylmethoxy)carbonyl](methyl)amino}-3-{4-[(2-hydroxypropan-2-yl)oxy]phenyl}propanoic acid or N-fluorenylmethoxycarbonyl-O-tert-butyl-N-methyltyrosine, Fmoc-N-Me-Tyr(t-Bu)-OH, C29H31NO5, was successfully synthesized and the structure of its unsolvated form was determined by single-crystal X-ray diffraction. The structural, conformational and energy landscape was investigated in detail by combined experimental and in silico approaches, and further compared to N-Fmoc-phenylalanine [Draper et al. (2015). CrystEngComm, 42, 8047-8057]. Geometries were optimized by the density functional theory (DFT) method either in vacuo or in solutio. The polarizable conductor calculation model was exploited for the evaluation of the hydration effect. Hirshfeld surface analysis revealed that H...H, C...H/H...C and O...H/H...O interactions constitute the major contributions to the total Hirshfeld surface area in all the investigated systems. The molecular electrostatic potentials mapped over the surfaces identified the electrostatic complementarities in the crystal packing. The prediction of weak hydrogen-bonded patterns via Full Interaction Maps was computed. Supramolecular motifs formed via C-H...O, C-H...π, (fluorenyl)C-H...Cl(I), C-Br...π(fluorenyl) and C-I...π(fluorenyl) interactions are observed. Basic synthons, in combination with the Long-Range Synthon Aufbau Modules, further supported by energy-framework calculations, are discussed. Furthermore, the relevance of Fmoc-based supramolecular hydrogen-bonding patterns in biocomplexes are emphasized, for the first time.

Effect of L1-79 on Core Symptoms of Autism Spectrum Disorder: A Case Series.

PURPOSE: This study examines the effects of the tyrosine hydroxylase inhibitor L1-79, a racemic formulation of α-methylparatyrosine, in patients with autism spectrum disorder (ASD) in a prospective case series. The l-isomer formulation of α-methylparatyrosine, metyrosine, is approved for the management of patients with pheochromocytoma. METHODS: Six male and 2 female patients aged 2.75 to 24 years with ASD were treated for 8 weeks at L1-79 doses ranging from 90 to 400 mg thrice daily. Assessments at weekly intervals included the Aberrant Behavior Checklist-Community (ABC-C), Connor's Parent Rating Scale (CPRS), and Clinical Global Impressions (CGI) scale. The Autism Diagnostic Observation Schedule (ADOS) was administered at baseline and week 10. FINDINGS: The ABC-C and CPRS scores improved between baseline and end of study for 7 of 8 participants; most participants' assessment scores decreased. At week 8, the CGI efficacy index was 05 for 6 of 8 participants, indicating modest improvement with at least partial resolution of symptoms and no medication adverse effects, and 09 for 2 participants, indicating minimal improvement and no change in status or care needs, without adverse effects. The mean ADOS scores improved by ≥31% for 4 of the 6 participants tested, with 1 patient experiencing a 47% improvement. Seven of the 8 participants previously taking psychotropic medications were stable without their legacy medications while receiving L1-79, and 1 patient resumed a single legacy medication at a lower dose. Three adverse events were reported; symptoms were mild and resolved without change in therapy. IMPLICATIONS: These results suggest L1-79 may be a tolerable and effective treatment for the core symptoms of ASD, which must be confirmed with double-blind studies.

Evidence for Naturally Produced Beauvericins Containing N-Methyl-Tyrosine in Hypocreales Fungi.

Beauvericin is a depsipeptide mycotoxin. The production of several beauvericin analogues has previously been shown among various genera among Hypocreales fungi. This includes so-called beauvenniatins, in which one or more N-methyl-phenylalanine residues is exchanged with other amino acids. In addition, a range of "unnatural" beauvericins has been prepared by a precursor addition to growth medium. Our aim was to get insight into the natural production of beauvericin analogues among different Hypocreales fungi, such as Fusarium and Isaria spp. In addition to beauvericin, we tentatively identified six earlier described analogues in the extracts; these were beauvericin A and/or its structural isomer beauvericin F, beauvericin C, beauvericin J, beauvericin D, and beauvenniatin A. Other analogues contained at least one additional oxygen atom. We show that the additional oxygen atom(s) were due to the presence of one to three N-methyl-tyrosine moieties in the depsipeptide molecules by using different liquid chromatography⁻mass spectrometry-based approaches. In addition, we also tentatively identified a beauvenniatin that contained N-methyl-leucine, which we named beauvenniatin L. This compound has not been reported before. Our data show that N-methyl-tyrosine containing beauvericins may be among the major naturally produced analogues in certain fungal strains.

Isotope effects in the tyrosinase catalysed hydroxylation of l-tyrosine methyl derivatives.

To investigate isotope effects in the hydroxylation of [3',5'-2H2]-α-methyl- and [3',5'-2H2]-N-methyl-l-tyrosine, they were synthesised using acid catalysed isotope exchange at high temperature. The kinetic and solvent deuterium isotope effects on Vmax and Vmax/Km parameters of tyrosinase in its action on methylated derivatives of l-tyrosine were determined using the non-competitive spectrophotometric method. Lineweaver-Burk plots were used to consider the inhibition type of O-methyl-l-tyrosine, revealing that it is an uncompetitive inhibitor of tyrosinase.

Usefulness of 18F-α-Methyltyrosine PET for Therapeutic Monitoring of Patients with Advanced Lung Cancer.

BACKGROUND/AIM: L-[3-18F]-α-methyl tyrosine (18F-FAMT) positron emission tomography (PET) has a high specificity for detecting malignant lesions. However, the usefulness of therapeutic monitoring of 18F-FAMT PET against advanced human neoplasms remains unclear. Here, we evaluated 18F-FAMT PET clinical significance regarding therapy response and outcome after systemic chemotherapy in patients with advanced lung cancer, compared to 18F-FDG PET. PATIENTS AND METHODS: All patients with untreated advanced lung cancer received 18F-FAMT PET and 18F-FDG PET before and 4 weeks after one cycle of chemotherapy. Metabolic response (MR) was defined according to the PERCIST guideline. RESULTS: Ninety-five patients were eligible for analysis on both PET scans. The histological type included 87 non-small cell lung cancers and 8 small-cell lung cancers. Post-treatment maximal standardized uptake values (SUVmax) and MR on 18F-FAMT PET were correlated with tumor response. In all patients, post-treatment SUVmax of 18F-FDG and 18F-FAMT PET and MR of 18F-FAMT PET were statistically significant prognostic markers for predicting poor outcome by univariate analysis. Multivariate analysis confirmed that MR on 18F-FAMT PET was a significant independent prognostic factor. CONCLUSION: MR on 18F-FAMT PET may be a potential parameter to predict the prognosis after first-line chemotherapy in patients with advanced lung cancer.

Effect of subacute agomelatine treatment on painful diabetic neuropathy: involvement of catecholaminergic mechanisms.

In this study, we investigated the effects of subacute agomelatine (40 and 80 mg/kg) administration on chronic hyperglycemia, metabolic parameters, and pain perception in streptozotocin-induced diabetic rats. Fasting blood glucose measurements and oral glucose tolerance tests were performed to evaluate the effect of agomelatine on glycemia, while metabolic parameters were monitored using metabolic cages. Potential effect of agomelatine on diabetes-induced mechanical and thermal allodynia was evaluated using dynamic plantar aesthesiometer and warm plate (38 °C) tests, respectively. Additionally, influence of agomelatine on hyperalgesia occurring in connection with diabetic neuropathy was examined using the Randall-Selitto (mechanical nociceptive stimulus), Hargreaves (thermal nociceptive stimulus), and cold plate (4 °C, thermal nociceptive stimulus) tests. Obtained data indicated that, in diabetic rats, agomelatine significantly improved hyperalgesia and allodynia responses, without no effect on hyperglycemia or the associated polydipsia, polyuria, and hyperphagia. Therapeutic potential of agomelatine on neuropathic pain was suppressed with α-methyl-para-tyrosine methyl ester (an inhibitor of catecholamine synthesis), phentolamine (a nonselective α-adrenoceptor antagonist), and propranolol (a nonselective ß-adrenoceptor antagonist) administrations. However, p-chlorophenylalanine methyl ester (an inhibitor of serotonin synthesis) pretreatment could not be achieved to reverse these antihyperalgesic and antiallodynic effects. These results suggest that the curative effect of agomelatine on neuropathic pain is mediated through rising synaptic catecholamine levels as well as through interactions with both α- and ß-adrenoceptors. To our knowledge, this is the first study to show findings that indicate catecholaminergic system mediated antihyperalgesic and antiallodynic effects of agomelatine.

Transport of 3-fluoro-L-α-methyl-tyrosine (FAMT) by organic ion transporters explains renal background in [(18)F]FAMT positron emission tomography.

A PET tracer for tumor imaging, 3-(18)F-l-α-methyl-tyrosine ([(18)F]FAMT), has advantages of high cancer-specificity and low physiological background. In clinical studies, FAMT-PET has been proved useful for the detection of malignant tumors and their differentiation from inflammation and benign lesions. The tumor specific uptake of FAMT is due to its high-selectivity to cancer-type amino acid transporter LAT1 among amino acid transporters. In [(18)F]FAMT PET, kidney is the only organ that shows high physiological background. To reveal transporters involved in renal accumulation of FAMT, we have examined [(14)C]FAMT uptake on the organic ion transporters responsible for the uptake into tubular epithelial cells. We have found that OAT1, OAT10 and OCTN2 transport [(14)C]FAMT. The [(14)C]FAMT uptake was inhibited by probenecid, furosemide and ethacrynic acid, consistent with the properties of the transporters. The amino acid uptake inhibitor, 2-amino-2-norbornanecarboxylic acid (BCH), also inhibited the [(14)C]FAMT uptake, whereas OCTN2-mediated [(14)C]FAMT uptake was Na(+)-dependent. We propose that FAMT uptake by OAT1, OAT10 and OCTN2 into tubular epithelial cells could contribute to the renal accumulation of FAMT. The results from this study would provide clues to the treatments to reduce renal background and enhance tumor uptake as well as to designing PET tracers with less renal accumulation.

Specific transport of 3-fluoro-l-α-methyl-tyrosine by LAT1 explains its specificity to malignant tumors in imaging.

3-(18)F-l-α-methyl-tyrosine ([18F]FAMT), a PET probe for tumor imaging, has advantages of high cancer-specificity and lower physiologic background. FAMT-PET has been proved useful in clinical studies for the prediction of prognosis, the assessment of therapy response and the differentiation of malignant tumors from inflammation and benign lesions. The tumor uptake of [18F]FAMT in PET is strongly correlated with the expression of L-type amino acid transporter 1 (LAT1), an isoform of system L upregulated in cancers. In this study, to assess the transporter-mediated mechanisms in FAMT uptake by tumors, we examined amino acid transporters for FAMT transport. We synthesized [14C]FAMT and measured its transport by human amino acid transporters expressed in Xenopus oocytes. The transport of FAMT was compared with that of l-methionine, a well-studied amino acid PET probe. The significance of LAT1 in FAMT uptake by tumor cells was confirmed by siRNA knockdown. Among amino acid transporters, [14C]FAMT was specifically transported by LAT1, whereas l-[14C]methionine was taken up by most of the transporters. Km of LAT1-mediated [14C]FAMT transport was 72.7 µM, similar to that for endogenous substrates. Knockdown of LAT1 resulted in the marked reduction of [14C]FAMT transport in HeLa S3 cells, confirming the contribution of LAT1 in FAMT uptake by tumor cells. FAMT is highly specific to cancer-type amino acid transporter LAT1, which explains the cancer-specific accumulation of [18F]FAMT in PET. This, vice versa, further supports the cancer-specific expression of LAT1. This study has established FAMT as a LAT1-specific molecular probe to monitor the expression of a potential tumor biomarker LAT1.

Cancer stratification by molecular imaging.

The lack of specificity of traditional cytotoxic drugs has triggered the development of anticancer agents that selectively address specific molecular targets. An intrinsic property of these specialized drugs is their limited applicability for specific patient subgroups. Consequently, the generation of information about tumor characteristics is the key to exploit the potential of these drugs. Currently, cancer stratification relies on three approaches: Gene expression analysis and cancer proteomics, immunohistochemistry and molecular imaging. In order to enable the precise localization of functionally expressed targets, molecular imaging combines highly selective biomarkers and intense signal sources. Thus, cancer stratification and localization are performed simultaneously. Many cancer types are characterized by altered receptor expression, such as somatostatin receptors, folate receptors or Her2 (human epidermal growth factor receptor 2). Similar correlations are also known for a multitude of transporters, such as glucose transporters, amino acid transporters or hNIS (human sodium iodide symporter), as well as cell specific proteins, such as the prostate specific membrane antigen, integrins, and CD20. This review provides a comprehensive description of the methods, targets and agents used in molecular imaging, to outline their application for cancer stratification. Emphasis is placed on radiotracers which are used to identify altered expression patterns of cancer associated markers.

Synthesis of 6-[(11)C]methyl-m-tyrosine ([(11)C]6MemTyr) for dopamine synthesis imaging in living brain using PET.

A novel PET probe, 6-[(11)C]methyl-m-tyrosine ([(11)C]6MemTyr), was developed for quantitative imaging of presynaptic dopamine (DA) synthesis in the living brain using positron emission tomography (PET). This probe was evaluated by comparison with conventional 6-[(18)F]fluoro-l-dopa ([(18)F]FDOPA). [(11)C]6MemTyr was labeled using rapid Pd(0)-mediated C-[(11)C]methylation with [(11)C]methyl iodide. The synthesis time was only 35min, and its radiochemical yield was 76%, with radiochemical purity of >99%. PET measurements indicated that [(11)C]6MemTyr could image presynaptic DA synthesis in the striatum of living monkey brain, providing much higher contrast between the striatum and the cerebellum than that with [(18)F]FDOPA.

Effects of intratumoral inflammatory process on 18F-FDG uptake: pathologic and comparative study with 18F-fluoro-α-methyltyrosine PET/CT in oral squamous cell carcinoma.

UNLABELLED: The accurate depiction of both biologic and anatomic profiles of tumors has long been a challenge in PET imaging. An inflammation, which is innate in the carcinogenesis of oral squamous cell carcinoma (OSCC), frequently complicates the image analysis because of the limitations of (18)F-FDG and maximum standardized uptake values (SUV(max)). New PET parameters, metabolic tumor volume (MTV) and total lesion glycolysis (TLG), as well as (18)F-fluoro-α-methyltyrosine ((18)F-FAMT), a malignancy-specific amino acid-based PET radiotracer, are considered more comprehensive in tumor image analysis. Here, we showed the substantial effects of the intratumoral inflammatory process on (18)F-FDG uptake and further study the possibility of MTV and TLG to predict both tumor biology (proliferation activity) and anatomy (pathologic tumor volume). METHODS: (18)F-FDG and (18)F-FAMT PET images from 25 OSCC patients were analyzed. SUV(max) on the tumor site was obtained. PET volume computerized-assisted reporting was used to generate a volume of interest to obtain MTV and TLG for (18)F-FDG and total lesion retention (TLR) for (18)F-FAMT. The whole tumor dissected from surgery was measured and sectioned for pathologic analysis of tumor inflammation grade and Ki-67 labeling index. RESULTS: The high SUV(max) of (18)F-FDG was related to the high inflammation grade. The SUV(max )ratio of (18)F-FDG to (18)F-FAMT was higher in inflammatory tumors (P < 0.05) whereas the corresponding value in tumors with a low inflammation grade was kept low. All (18)F-FAMT parameters were correlated with Ki-67 labeling index (P < 0.01). Pathologic tumor volume predicted from MTV of (18)F-FAMT was more accurate (R = 0.90, bias = 3.4 ± 6.42 cm(3), 95% confidence interval = 0.77-6.09 cm(3)) than that of (18)F-FDG (R = 0.77, bias = 8.1 ± 11.17 cm(3), 95% confidence interval = 3.45-12.67 cm(3)). CONCLUSION: (18)F-FDG uptake was overestimated by additional uptake related to the intratumoral inflammatory process, whereas (18)F-FAMT simply accumulated in tumors according to tumor activity as evaluated by Ki-67 labeling index in OSCC.

L-[3-¹8F]-α-methyltyrosine uptake by lymph node metastasis is a predictor of complete response to CRT in esophageal cancer.

BACKGROUND/AIM: The amino acid positron emission tomography (PET) tracer [(18)F]-3-fluoro-alpha-methyltyrosine ((18)F-FAMT) is known to be highly specific for malignancies. We evaluated the accumulation of (18)F-FDG or (18)F-FAMT in lymph nodes (LN) prior to definitive chemoradiotherapy (CRT) for esophageal cancer. PATIENTS AND METHODS: We retrospectively reviewed 30 patients with esophageal squamous cell carcinoma. All patients received definitive CRT. The relationship between the accumulation of (18)F-FDG PET or (18)F-FAMT PET in LNs prior to CRT and clinical outcomes was assessed. RESULTS: A correlation was observed between LNs in which most of (18)F-FAMT was accumulated and complete response (CR) rate, but was not for (18)F-FDG. Additionally, for (18)F-FAMT, the CR rate was significantly higher in the LN accumulated lesion ≤ 1 group than in the LN accumulated lesion >2 group. DISCUSSION: To predict the outcome of definitive CRT in patients with esophageal cancer, it is important to evaluate the LN status.

Use of pre-treatment 18F-FAMT PET to predict patient survival in squamous cell carcinoma of the esophagus treated by curative surgery.

BACKGROUND: [18F]-3-fluoro-alpha-methyl tyrosine (18F-FAMT) as an amino acid tracer in positron emission tomography (PET) has been widely investigated in several tumor types. Herein we investigated the clinical significance of 18F-FAMT PET uptake as a prognostic marker together in our updated data of patients with esophageal cancer. PATIENTS AND METHODS: We retrospectively assessed the treatment outcomes of 42 patients with histologically-confirmed esophageal cancer. The survival rate was analyzed using the median peak standardized uptake value (SUV) with 2.2 as the cut-off value. RESULTS: FAMT uptakes were significantly correlated with factors reflecting tumor progression. Moreover, a significant correlation was observed between FAMT uptake and disease-free survival (p=0.023). Moreover, on evaluation of individual lymph node groups, the specificity and positive predictive value were significantly higher for 18F-FAMT-PET than for 18F-FDG-PET and computed tomography (CT). CONCLUSION: 18F-FAMT is an important pre-treatment diagnostic modality and its accumulation is a good predictor of disease-free survival (DFS) in patients with operable esophageal cancer.

A pulse-chase epitope labeling to study cellular dynamics of newly synthesized proteins: a novel strategy to characterize NPC biogenesis and ribosome maturation/export.

The vast number of cellular proteins performs their roles within macromolecular assemblies and functional cell networks. Hence, an understanding of how multiprotein complexes are formed and modified during biogenesis is a key problem in cell biology. Here, we describe a detailed protocol for a nonradioactive pulse-chase in vivo-labeling approach. The method is based on the incorporation of an unnatural amino acid (O-methyl-tyrosine) by the nonsense suppression of an amber stop codon that quickly fuses an affinity tag of choice to a protein of interest. This affinity tag could be used to directly isolate the newly synthesized proteins and hence allows for the characterization of early complex biogenesis intermediates. Combined with a tetracycline controllable riboswitch in the 5'-UTR of the respective mRNA, this approach became a versatile tool to study dynamic protein assembly within cellular networks (Stelter et al., 2012). In the context of this volume, this method notably provides a suitable approach to study NPC, ribosome and mRNP biogenesis, or nuclear protein translocation. This chapter includes detailed protocols to track newly synthesized, epitope pulsed-chased proteins by western blot, their assembly within complexes using immunoprecipitation, and their subcellular localization using indirect immunofluorescence or subcellular fractionation. While these protocols use budding yeast as model system, this method can be adapted to other model systems.

Reactive oxygen species are physiological mediators of the noradrenergic signaling pathway in the mouse supraoptic nucleus.

Free radicals are essential for the vasopressin (AVP) response to plasmatic hyperosmolarity. Noradrenergic afferents are the major projections on the supraoptic nucleus (SON) of the hypothalamus and stimulate the expression of AVP via a nitric oxide (NO) pathway. In this study, we investigated the mechanisms linking free radicals and noradrenaline (NA)-induced regulation of AVP. Analysis of Tg8 transgenic mice, invalidated for the monoamine oxidase-A gene and with consequently high levels of brain monoamines and AVP in the SON, showed that free radicals are more abundant in their SON than in that of wild-type mice (WT). Antioxidant superoxide dismutase 1 and 2 and catalase enzyme activities were also higher in these mice than in WT. This may explain the observed absence of cytotoxicity that would otherwise be associated with such high level of free radicals. Treatment of Tg8 mice with α-MPT, a blocking agent for NA synthesis, decreased both the production of free radicals and the AVP levels in the SON. Furthermore, incubation of ex vivo slices including the SON with NA increased the production of free radicals and AVP levels in wild-type mice. When NA was associated with α-lipoic acid, an antioxidant blocking the production of free radicals, AVP remained at its control level, indicating that free radicals are required for the effect of NA on the expression of AVP. In slices incubated with SNP, a producer of NO, free radicals and AVP levels increased. When NA was associated with L-NAME (a NO synthase blocker), the levels of free radicals and AVP were the same as in controls. Thus, the noradrenaline-NO pathway, which stimulates the expression of vasopressin, involves free radicals. This study provides further evidence of the physiological importance of free radicals, which should no longer be considered solely as cytotoxic factors.