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Abstract

Several studies have demonstrated the remarkable properties of microbiota and their metabolites in the pathogenesis of several inflammatory diseases. 10-Hydroxy-cis-12-octadecenoic acid (HYA), a bioactive metabolite generated by probiotic microorganisms during the process of fatty acid metabolism, has been studied for its protective effects against epithelial barrier impairment in the intestines. Herein, we examined the effect of HYA on gingival epithelial barrier function and its possible application for the prevention and treatment of periodontal disease. We found that GPR40, a fatty acid receptor, was expressed on gingival epithelial cells; activation of GPR40 by HYA significantly inhibited barrier impairment induced by Porphyromonas gingivalis, a representative periodontopathic bacterium. The degradation of E-cadherin and beta-catenin, basic components of the epithelial barrier, was prevented in a GPR40-dependent manner in vitro. Oral inoculation of HYA in a mouse experimental periodontitis model suppressed the bacteria-induced degradation of E-cadherin and subsequent inflammatory cytokine production in the gingival tissue. Collectively, these results suggest that HYA exerts a protective function, through GPR40 signaling, against periodontopathic bacteria-induced gingival epithelial barrier impairment and contributes to the suppression of inflammatory responses in periodontal diseases.

Protective effect of HYA-induced GPR40 signaling on epithelial barrier impairment

To examine a possible role of bioactive metabolites in gingival epithelial barrier function, we performed an in vitro permeability assay using fluorescein isothiocyanate (FITC) -conjugated dextran after the optimization of HYA concentration (Supplementary Fig. ). The epithelial barrier impairment induced by P. gingivalis was significantly inhibited by pretreatment with HYA but not HYB (Fig. ). Pretreatment with the selective GPR40 antagonist GW1100 partially diminished the protective property of HYA, indicating a GPR40-dependent mechanism (Fig. ). Taken together, these results demonstrate that HYA prevents P. gingivalis-induced barrier impairment, via its activation of GPR40 signaling.

HYA reduces local inflammatory cytokine production in gingival tissue in vivo

In order to examine the clinical relevance of HYA in periodontitis, an in vivo study was carried out using a mouse experimental periodontal disease model. After validation of epithelial GPR40 expression in the gingiva, periodontitis was induced by applying a ligature on the molars with repeated oral inoculation of P. gingivalis (Supplementary Figs ,). The mRNA expression levels of inflammatory cytokines such as IL-1β, TNF-α and IL-6 in gingival tissues of the ligated HYA-treated group were significantly decreased in comparison with those of the ligated sham-treated group, with a tendency of suppression of alveolar bone destruction (Fig. ). The ligated HYA-treated group showed a higher immunofluorescence intensity for E-cadherin in the epithelium when compared to that of the ligated sham-treated group (Fig. ). No differences of E-cadherin intensity were observed between the unligated groups (Supplementary Fig. ). In consistent with in vitro study, no alteration of E-cadherin mRNA expression was observed for all groups (Supplementary Fig. ). Collectively, these results indicate that HYA reinforces gingival epithelial barrier function by inhibiting E-cadherin degradation, resulting in the suppression of periodontal inflammatory responses and subsequent alveolar bone destruction in vivo.

Reagents and antibodies

10-hydroxy-cis-12-octadecenoic acid (HYA) and 10-hydroxyoctadecanoic acid (HYB) were synthesized according to methods published previously. Anti-GPR40 antibody was purchased from GeneTex Inc. (San Antonio, TX, USA). Anti-E-cadherin, anti-Beta-catenin, anti-ERK and anti-phosphorylated ERK antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Rabbit anti-mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Cell Signaling Technology) and peroxidase-labeled anti-rabbit IgG antibody (Cell Signaling Technology) were used for Western blotting experiments. A selective GPR40 antagonist, GW1100, was purchased from Cayman Chemical (Ann Arbor, MI, USA).

Abstract

Flavin-dependent enzymes catalyze many oxidations, including formation of ring structures in natural products. The gene cluster for biosynthesis of fumisoquins, secondary metabolites structurally related to isoquinolines, in the filamentous fungus Aspergillus fumigatus harbors a gene that encodes a flavoprotein of the amine oxidase family, termed fsqB (fumisoquin biosynthesis gene B). This enzyme catalyzes an oxidative ring closure reaction that leads to the formation of isoquinoline products. This reaction is reminiscent of the oxidative cyclization reported for berberine bridge enzyme and tetrahydrocannabinol synthase. Despite these similarities, amine oxidases and berberine bridge enzyme–like enzymes possess distinct structural properties, prompting us to investigate the structure–function relationships of FsqB. Here, we report the recombinant production and purification of FsqB, elucidation of its crystal structure, and kinetic analysis employing five putative substrates. The crystal structure at 2.6 Å resolution revealed that FsqB is a member of the amine oxidase family with a covalently bound FAD cofactor. N-methyl-dopa was the best substrate for FsqB and was completely converted to the cyclic isoquinoline product. The absence of the meta-hydroxyl group, as e.g. in l-N-methyl-tyrosine, resulted in a 25-fold lower rate of reduction and the formation of the demethylated product l-tyrosine, instead of a cyclic product. Surprisingly, FsqB did not accept the d-stereoisomer of N-methyltyrosine, in contrast to N-methyl-dopa, for which both stereoisomers were oxidized with similar rates. On the basis of the crystal structure and docking calculations, we postulate a substrate-dependent population of distinct binding modes that rationalizes stereospecific oxidation in the FsqB active site.

Production, purification, and characterization of FsqB

FsqB was expressed in Escherichia coli yielding 1 mg of FsqB from 1 g of pellet after purification by nickel–nitrilotriacetic acid affinity chromatography. For crystallization, the protein was further purified by means of size-exclusion chromatography. The UV-visible absorption spectrum of FsqB possesses the typical features of an FAD-containing protein with absorption maxima at 460 and 370 nm (A). The bathochromic shift of the long wavelength absorption maximum by ∼10 nm compared with free FAD is most likely due to the monocovalent attachment of the 8α-methyl group to cysteine residue 414. Similar UV-visible absorption spectra were previously reported for human dimethylglycine dehydrogenase (hDMGDH) () and N-methyl-tryptophan oxidase (), which feature the same covalent linkage. The extinction coefficient of the FAD bound to FsqB was calculated to 12,350 m−1 cm−1. Consumption of N-methyl-dopa was investigated from pH 4 to 9 using an oxygen sensor to monitor the reaction yielding a bell-shaped profile with an optimum at pH 7.6. Photoreduction of FsqB led to the formation of a stable flavin semiquinone with an absorption maximum at 396 nm. Further reduction of the flavin semiquinone to the hydroquinone could not be achieved by light. The flavin semiquinone was reoxidized very slowly by molecular oxygen reaching completion after ∼70 min (B).

Kinetic parameters of FsqB

Following product analysis, we also determined pre–steady-state parameters in the stopped-flow apparatus for N-methyl-dopa, rac-N-methyl-meta-tyrosine, and l-N-methyl-tyrosine. The highest rate of flavin reduction was observed with (racemic) N-methyl-dopa, whereas (racemic) N-methyl-meta-tyrosine and l-N-methyl-tyrosine were 10–25 times slower (the kred values given in were measured at a single fixed concentration of 2.5 mm). A more detailed analysis of the reduction of FsqB using rac-N-methyl-dopa or the l-stereoisomer yielded limiting rates of 51 ± 3 and 21 ± 3 s−1, as well as dissociation constants of 9.2 ± 0.7 and 8.0 ± 1.3 mm, respectively (A and ). Thus, the limiting rate for l-N-methyl-dopa is only ∼2.5-fold lower than for the racemate, indicating that both stereoisomers are accepted by the enzyme. Furthermore, this result also suggests that the enzyme has a preference for d-N-methyl-dopa (commercially not available). To distinguish the rate of reduction for the d- and l-stereoisomer, we repeated the pre–steady-state measurements at a lower concentration of rac-N-methyl-dopa (i.e. 0.2 mm) and obtained a clear biphasic behavior with observed rates of 1.63 ± 0.04 and 0.61 ± 0.01 s−1, respectively, presumably reflecting the rate of reduction for the two stereoisomers in the racemic mixture. This 2.5-fold difference is similar to the difference observed for the racemate compared with the l-stereoisomer (), supporting our conclusion that the d-stereoisomer is a slightly better substrate than the l-stereoisomer. Notably, at substrate concentrations above 0.2 mm, the reaction showed essentially monophasic behavior because the fast-reacting enantiomer outcompetes the slower one. Interestingly, reduction of the FAD cofactor was not observed with d-N-methyl-tyrosine, suggesting that FsqB exhibits strict stereo-preference for the l-N-methyl-tyrosine stereoisomer (S configuration) in contrast to what was found with N-methyl-dopa.

Kinetic parameters of FsqB

Steady-state kinetics were determined for N-methyl-dopa (racemate and the l-stereoisomer), l-N-methyl-tyrosine, and rac-N-methyl-meta-tyrosine using oxygen depletion to monitor enzyme turnover. As shown in B, enzyme activity was a function of substrate concentration reaching saturation at ≈8 s−1. Similarly, the Km for the racemate and the l-enantiomer of N-methyl-dopa differed only slightly, i.e. 0.16 ± 0.01 and 0.27 ± 0.02 mm, respectively. For l-N-methyl-tyrosine and rac-N-methyl-meta-tyrosine, saturation was reached at ≈4 and ≈3.5 s−1, respectively. The Km for these substrates was 2.1 ± 0.2 and 1.6 ± 0.6 mm, respectively. summarizes the kinetic parameters for all substrates oxidized by FsqB. As judged by the resulting kcat/Km values, N-methyl-dopa (racemate and the l-enantiomere) is more efficiently oxidized by FsqB than l-N-methyl-tyrosine and rac-N-methyl-meta-tyrosine ().

Abstract

Two new thermophilic branched chain amino acid transaminases have been identified within the genomes of different hyper-thermophilic archaea, Geoglobus acetivorans, and Archaeoglobus fulgidus. These enzymes belong to the class IV of transaminases as defined by their structural fold. The enzymes have been cloned and over-expressed in Escherichia coli and the recombinant enzymes have been characterized both biochemically and structurally. Both enzymes showed high thermostability with optimal temperature for activity at 80 and 85°C, respectively. They retain good activity after exposure to 50% of the organic solvents, ethanol, methanol, DMSO and acetonitrile. The enzymes show a low activity to (R)-methylbenzylamine but no activity to (S)-methylbenzylamine. Both enzymes have been crystallized and their structures solved in the internal aldimine form, to 1.9 Å resolution for the Geoglobus enzyme and 2.0 Å for the Archaeoglobus enzyme. Also the Geoglobus enzyme structure has been determined in complex with the amino acceptor α-ketoglutarate and the Archaeoglobus enzyme in complex with the inhibitor gabaculine. These two complexes have helped to determine the conformation of the enzymes during enzymatic turnover and have increased understanding of their substrate specificity. A comparison has been made with another (R) selective class IV transaminase from the fungus Nectria haematococca which was previously studied in complex with gabaculine. The subtle structural differences between these enzymes has provided insight regarding their different substrate specificities.

Structural Studies

The GEO1900 holoform (internal aldimine form; GEO1900_holo) and the unproductive complex of holoenzyme with the keto substrate AKG-(GEO1900_AKG) have been determined to 1.9 and 2.2 Å resolution, respectively, in the trigonal space group P3221. There are three GEO1900 subunits per asymmetric unit, which form a tight hexamer with 32 point group symmetry with their symmetry mates. The AF0933 structure has been determined in the holoform (AF0933_holo) and in complex with the inhibitor gabaculine (GABC) (AF0933_GABC) to 2.1 and 2.0 Å resolution, respectively, in the orthorhombic P212121 space group. The asymmetric unit contains a hexameric AF0933 molecule which is very similar to GEO1900 hexamer observed in the crystal. All of the structures have been refined to low R-factors with good stereochemical parameters as shown in Table . In the GEO1900 structures two N-terminal amino acids which are not present in AF0933, were not defined in the electron density. The interdomain loop residues 121–129 could be fully built into the subunits A and B in GEO1900_AKG structure. The bound ligand AKG was found only in subunit A and was clearly defined in the electron density (Figure ). The interdomain loop could only be fully built in one subunit (D) out of the six subunits in both the AF0933_holo and AF0933_GABC structures. The crystal contacts in AF0933 appear to be contributing to the lower disorder of the loop in the D subunit. The inhibitor, gabaculine was not present in the electron density in the subunit F of AF0933, therefore this subunit was built as an internal aldimine in the AF0933_GABC structure. In other subunits the gabaculine-PLP adduct was modeled with partial occupancy in the range of 0.6–0.7. There are no residues in a cis conformation in the well-defined regions of the GEO1900 and AF0933 structures. Residue Glu249 is a Ramachandran plot outlier (Ramakrishnan and Ramachandran, ) in all subunits of both AF0933 structures. The corresponding Glu251 has similar main chain angles in the GEO1900 structures, but lies in a generously allowed region of the Ramachandran plot (Laskowski et al., ). The fungal class IV TAms from Aspergillus and Nectria species (Łyskowski et al., ; Sayer et al., ; Thomsen et al., ) have a Gly residue at this position. Many residues in both of the GEO1900 and AF0933 structures have been modeled with multiple conformations of some amino acid side chains. The residues Gln107 and Asn108 were modeled with alternative conformations of the main chain in most subunits of the AF0933_holo structure and in the subunits E and F of the AF0933_GABC structure.

ORAC antioxidant assay

The oxygen radical absorbance capacity for each MAA was carried out using the ORAC Antioxidant Assay Kit (Zenbio, North Carolina, USA) according to manufacturer's instructions. Trolox standards were prepared in the assay buffer (0–100 μM) along with serial dilutions of each MAA (500-0 μM) and an ascorbic acid positive control (0–100 μM). 150 μl of the fluorescein working solution was added to central wells of a 96 well plate, with 25 μl of each of the standards or MAA in duplicate, and the plate incubated at 37 °C for at least 15 min. The 2,2′-azobis(2-amidinopropane) dihydrochloride [AAPH; sold as 2,2′-azobis(2-methylpropanimidamine) dihydrochloride] working solution was added to each well (25 μl) to initiate the reaction. Fluorescence was measured in a preheated incubation chamber (37 °C) using a Spectra Max 384 Plus spectrophotometer with excitation/emission = 485/530 nm immediately (t = 0) and then every minute for 30 min. Standard curves were generated for each compound and the area under the curve calculated. Each MAA tested was expressed as a Trolox equivalent concentration.

MAAs Porphyra-334 and shinorine compete with Keap1-Nrf2 interaction in vitro

The MAAs porphyra-334 and shinorine together with eight selected antioxidants (ascorbic acid, tBHQ, caffeic acid, chlorogenic acid, curcumin, quercetin, EGCG, trans-resveratrol) plus the known electrophilic Nrf2 activator sulforaphane were evaluated for their interaction with the Kelch-repeat domain of the Keap1 protein using both in vitro FP and thermal shift assays. In the FP assay, the test compounds competed with a fluorescein labelled peptide (FITC-[β-Ala]-DEETGEF-OH) based on the high affinity ETGE motif that binds Nrf2 to the Kelch-repeat domain (). The protein-protein interaction of the FITC-[β-Ala]-DEETGEF-OH peptide had a KD of 96 nM in the presence of the Kelch-repeat domain protein and the unlabelled Ac-DEETGEF-OH peptide competed with this interaction with an IC50 of 5.4 μM []. Both porphyra-334 and shinorine gave nearly equivalent ligand-receptor estimated IC50 values of ∼100 μM, requiring a much greater concentration in the presence of excess Kelch-repeat domain protein than the binding of the native peptide sequence []. In contrast, no significant interactions were detected between the eight known antioxidants or sulforaphane and the Kelch-repeat domain protein.Fig. 3Measurement of specific, non-reactive binding of MAAs to the Kelch-repeat domain of Keap1. Ligand-receptor binding was measured using a fluorescence polarization assay in comparison to a high affinity, labelled synthetic peptide. The MAAs porphyra-334 and shinorine gave moderate IC50 values of ∼100 μM, whereas no significant interactions were detected between the Keap1 Kelch-repeat domain and any of the eight selected antioxidants or known electrophilic Nrf2 activator sulforaphane. The figure shows results for 100 μM concentration of MAAs and known antioxidants. Error bars represent one standard deviation of the mean, based on 3 experimental replicates, and statistically significant results (based on p-value of two sided t-test ≤ 0.05 adjusted for multiple testing based on Bonferroni correction) are marked with an asterisk.Fig. 3

MAAs Porphyra-334 and shinorine compete with Keap1-Nrf2 interaction in vitro

The MAAs and eight selected antioxidants were then tested as ligands of the Kelch-repeat domain protein using the thermal shift (differential scanning fluorimetry, DSF) assay. The assays involved treatment of the Kelch-repeat domain protein with the test compounds of interest, followed by heating of the samples to denature the protein. Ligands that bind and stabilise the Kelch-repeat domain protein result in higher denaturation (Tm) temperatures for the complex compared to the free protein. An unlabelled version of the peptide (Ac-DEETGEF-OH) used in the FP assay was used as the positive control in the thermal shift assays against which binding of the test compounds to the Kelch-repeat domain were compared. When used at a 50 μM concentration, the Ac-DEETGEF-OH peptide had a ΔTm relative to the unbound protein of 3.91 ± 0.04 °C. Both porphyra-334 and shinorine also demonstrated elevated ΔTm values to the Kelch-repeat domain protein, albeit when used at 100 μM (porphyra-334 ΔTm = 0.93 ± 0.02 °C, shinorine ΔTm = 0.64 ± 0.11 °C). However, no significant protein-ligand interactions were detectable when the eight antioxidants or the electrophilic Nrf2 activator sulforaphane were tested. The thermal shift assays reflected the results of the FP assays for binding of MAAs and test substrates to the Kelch-repeat domain protein of Keap1.

Porphya-334 and shinorine are in-vitro antioxidants

Scavenging of the DPPH free radical is a common assay used to assess the antioxidant activity of a compound. Ascorbic acid is a well-known DPPH free-radical scavenger and is commonly used as a positive control, which was used also for comparison in this study. The IC50 value for ascorbic acid was 24.5 ± 1.1 μM in the methanol reaction medium. The free radical scavenging activities of MAAs were markedly lower compared to that of ascorbic acid. The IC50 values were: 185.2 ± 3.2 μM for porphyra-334 (equivalent to 13.23% of the activity of ascorbic acid) and 399.0 ± 1.1 μM for shinorine (equivalent to 6.14% of the activity of ascorbic acid).

Abstract

Natural antioxidant gamma-tocotrienol (GT3), a vitamin E family member, provides intestinal radiation protection. We seek to understand whether this protection is mediated via mucosal epithelial stem cells or sub-mucosal mesenchymal immune cells. Vehicle- or GT3-treated male CD2F1 mice were exposed to total body irradiation (TBI). Cell death was determined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Villus height and crypt depth were measured with computer-assisted software in tissue sections. Functional activity was determined with an intestinal permeability assay. Immune cell recovery was measured with immunohistochemistry and Western blot, and the regeneration of intestinal crypts was assessed with ex vivo organoid culture. A single dose of GT3 (200 mg/kg body weight (bwt)) administered 24 h before TBI suppressed cell death, prevented a decrease in villus height, increased crypt depth, attenuated intestinal permeability, and upregulated occludin level in the intestine compared to the vehicle treated group. GT3 accelerated mesenchymal immune cell recovery after irradiation, but it did not promote ex vivo organoid formation and failed to enhance the expression of stem cell markers. Finally, GT3 significantly upregulated protein kinase B or AKT phosphorylation after TBI. Pretreatment with GT3 attenuates TBI-induced structural and functional damage to the intestine, potentially by facilitating intestinal immune cell recovery. Thus, GT3 could be used as an intestinal radioprotector.

2.1. Experimental Model, GT3 Treatment, Irradiation, Tissue Procurement, and Euthanasia

All animal studies were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal protocol (AUP #3823) was approved by the Institutional Animal Care and Use Committee of the University of Arkansas for Medical Sciences (UAMS). Male CD2F1 mice were obtained from Charles River Laboratories (Houston, TX); mice between 8 and 12 weeks of age were used for experiments. They were housed in conventional cages in a pathogen-free environment with controlled humidity, temperature, and a 12–12 light–dark cycle with free access to drinking water and standard chow (Teklad, Madison, WI, USA).

2.1. Experimental Model, GT3 Treatment, Irradiation, Tissue Procurement, and Euthanasia

GT3 was prepared freshly before administration with 5% Tween-80 (Fisher Scientific, Waltham, MA, USA) in saline. A single dose of 100 µL vehicle or GT3 (200 mg/kg body weight (bwt)) was administered subcutaneously 24 h before TBI.

Abstract

Background: Vascular diseases are multifactorial and several risk factors may have synergetic effect on the global vascular risk. Among patients with diabetes, we investigated whether vitamin B6 species differ according to global cardiovascular risk. Methods: The present observational study included 122 patients with type 2 diabetes (mean (SD) age = 69.9 (9.1) years; 50% men). Concentrations of vitamin B6 vitamers were measured. Classical blood biomarkers and risk factors were used to compute a multivariate risk score. Results: Plasma concentrations of 4-pyridoxic acid were higher in patients with high risk versus those with low risk scores (48.2 (63.7) vs. 31.9 (15.0) nmol/L; p = 0.031). Plasma pyridoxine was significantly lowered in patients at high risk (2.8 (28.4) vs. 38.1 (127.8) nmol/L; p = 0.003). PAr index (4-pyridoxic acid/pyridoxal + pyridoxal 5′-phosphate) (1.05 (0.07) vs. 0.84 (0.06); p = 0.017) and the ratio of 4-pyridoxic acid/pyridoxine (7.0 (4.8) vs. 3.9 (3.2); p < 0.001) were higher in patients at high risk. After adjustment for cystatin C and C-reactive protein, only pyridoxine and 4-pyridoxic acid/pyridoxine ratio remained significantly different according to vascular risk scores. 4-Pyridoxic acid/pyridoxine ratio was the best marker to discriminate between patients according to their risk scores—area under the curve (AUC) (95% confidence intervals (CI)) = 0.72 (0.62–0.81). 4-Pyridoxic acid/pyridoxine ratio was directly related to plasma levels of soluble vascular cell adhesion molecule 1. Conclusion: Vitamin B6 metabolism was shifted in patients with multiple vascular risk factors. The catabolism to 4-pyridoxic acid was enhanced, whereas the catabolism to pyridoxine was lowered. High 4-Pyridoxic acid/pyridoxine ratio is independently associated with global cardiovascular risk.

2.2. Exposure Definition

The main exposure in the current study is a multivariate cardiovascular risk score that has been developed and validated in patients with diabetes []. This score is computed from common clinical measurements available in primary care settings, such as age, sex, BMI, total cholesterol, high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), systolic blood pressure, diastolic blood pressure, and HbA1c (, ). The score estimates the two-year risk of hospitalization due to a cardiovascular event in patients with type 2 diabetes []. The present observational study used already established global risk scores and it was not planned to be prospective or to prove if subjects would attain the outcome predicted by the risk scores in the future.

3.2. Plasma Vitamin B6 Species in Patients with Type 2 Diabetes According to Cardiovascular Hospitalization Risk

The plasma concentrations of vitamin B6 forms are shown in in the whole group and in subgroups according to risk scores. 4-Pyridoxic acid was higher in patients in the high-risk group compared to those in the low-risk group (mean = 48.2 vs. 31.9 nmol/L: p = 0.031). The differences in concentrations of 4-pyridoxic acid was not significant between the two risk categories after adjusting for serum cystatin C (p = 0.168) or for cystatin C and CRP (p = 0.166). The percentage of plasma 4-pyridoxic acid of total vitamin B6 (i.e., the sum of vitamin B6 forms) was higher in the high-risk group, even after adjustment for cystatin C and CRP (p = 0.005). Plasma concentration of pyridoxine was significantly lower in the high-risk group compared to that in the low-risk group (12.8 vs. 38.1 nmol/L; p = 0.003). This difference remained significant after adjustment for cystatin C (p = 0.007) or for cystatin C and CRP (p = 0.010). Plasma concentrations of pyridoxal, PLP, pyridoxamine, and pyridoxamine phosphate, or the sum of all vitamin B6 forms did not differ significantly between the risk categories. Thus, 4-pyridoxic acid (elevated by 51%) and pyridoxine (lowered by 66%) were the vitamin B6 forms with marked changes in patients with high vascular risk scores compared to those with low risk scores.

Methods

Twenty-seven patients with recurrent or progressive primary brain tumor (based on clinical and MRI/CT data) were studied using dynamic 18F-Fluciclovine brain imaging for up to 4 h. Of these, 16 patients also had 11C-Methionine brain scans. Visual findings, semi-quantitative analyses and pharmacokinetic modeling of a subset of the 18F-Fluciclovine images was conducted. The information derived from these analyses were compared to data from 11C-Methionine and to contrast-enhanced MRI.

Results

18F-Fluciclovine was positive for all 27 patients, whereas contrast MRI was indeterminate for three patients. Tumor 18F-Fluciclovine SUVmax ranged from 1.5 to 10.5 (average: 4.5 ± 2.3), while 11C-Methionine’s tumor SUVmax ranged from 2.2 to 10.2 (average: 5.0 ± 2.2). Image contrast was higher with 18F-Fluciclovine compared to 11C-Methionine (p < 0.0001). This was due to 18F-Fluciclovine’s lower background in normal brain tissue (0.5 ± 0.2 compared to 1.3 ± 0.4 for 11C-Methionine). 18F-Fluciclovine uptake in both normal brain and tumors was well described by a simple one-compartment (three-parameter: Vb,k1,k2) model. Normal brain was found to approach transient equilibrium with a half-time that varied greatly, ranging from 1.5 to 8.3 h (mean 2.7 ± 2.3 h), and achieving a consistent final distribution volume averaging 1.4 ± 0.2 ml/cc. Tumors equilibrated more rapidly (t1/2ranging from 4 to 148 min, average 57 ± 51 min), with an average distribution volume of 3.2 ± 1.1 ml/cc. A qualitative comparison showed that the rate of normal brain uptake of 11C-Methionine was much faster than that of 18F-Fluciclovine.

Conclusion

Tumor uptake of 18F-Fluciclovine correlated well with the established brain tumor imaging agent 11C-Methionine but provided significantly higher image contrast. 18F-Fluciclovine may be particularly useful when the contrast MRI is non-diagnostic. Based on the data gathered, we were unable to determine whether Fluciclovine uptake was due solely to recurrent tumor or if inflammation or other processes also contributed.

Patients

This prospective clinical study was performed after Institutional Review Board approval (Memorial Sloan-Kettering Cancer Center IRB#03–028). Twenty-seven patients who underwent 18F-Fluciclovine brain PET for suspicion of recurrent or progressive gliomas between August 2004 and January 2008 were recruited under two different protocols (16 in the first, 11 in the second) with different funding sources (NCI and an internal source). The inclusion criteria, however, were identical and the data acquisition procedures were very similar with the primary difference being that the 11C-methionine study was left off of the second protocol. All patients fasted for at least 4 h before PET imaging. Written informed consent was obtained from all patients.

Synthesis of 18F-Fluciclovine and 11C-methionine

Clinical-grade 18F-labeled Fluciclovine and 11C-labeled methionine were produced by the Radiochemistry and Molecular Imaging Probes Core at MSKCC.

18F-Fluciclovine and 11C-methionine PET image acquisition

Following a 1-min IV infusion of 370 MBq of 18F-Fluciclovine, dynamic images of the brain were acquired in 2D mode (i.e. with septa) for 45 min on a GE Advance PET (first 21 patients) or a GE Discovery STE PET/CT (last 6 patients) scanner. The patients were subsequently removed from the scanner but returned up to three times for 20-min static images of the brain between 90 and 240 min post injection. No adverse effects were reported following the injection of 18F- Fluciclovine.

Introduction

In this study, we present high-resolution crystal structures of ThiL from the pathogen Acinetobacter baumannii (AbThiL) bound to substrate Thiamine monophosphate (TMP) and substrate analog adenylyl-imidodiphosphate (AMPPNP), and bound to its products TPP and adenosine diphosphate (ADP). The structures highlight both global structural differences and differences regarding ligand binding between AbThiL and comparable structures from Aquifex aeolicus (AaThiL). Anomalous diffraction could also confirm a plasticity of AbThiL with regards to metal identity in the active site. The structures further support proposals made on the reaction mechanism for AaThiL.

Crystallization

Crystals grown in the presence of 5 mM AMPPNP/TMP/MgCl2 had many growth defects. Hence, co-crystals of AbThiL with 5 mM AMPPNP/MgCl2 from an optimization screen based on MCSG1 G4 (19% w/v PEG 3350, 200 mM K/Na-tartrate, 100 mM HEPES/NaOH pH 7.5) were soaked overnight with 5 mM AMPPNP/TMP/MgCl2. Crystals were harvested in two steps with reservoir with 20% v/v EG, 5 mM AMPPNP/TMP/MgCl2 added as the cryo protectant.

Abstract

Bixin is a hydrophobic carotenoid present in the integument of the seeds of Bixa orellana. Microencapsulation was applied to obtain water dispersible formulations and protect the colorant against degradation. Microencapsulated systems were obtained by spray-drying a mild alkaline bixin dispersion with different encapsulating materials. The encapsulation trials were performed with and without native carbohydrates of the integument in addition to the main encapsulant. It was possible to dry dispersions with up to 10% bixin counted on total solids. All the studied systems were characterized by colorimetry, UV-vis spectroscopy, Scanning Electron Microscopy, light microscopy, turbidometric sedimentation analyses and laser light diffraction analyses. All the systems showed aqueous dispersibility but displayed differences in their transparency, UV-vis spectra and physical stability at pH 3. The results show that the native carbohydrates enhance the encapsulation efficiency of other encapsulating materials. The chemical composition of this native carbohydrate fraction shows the presence of polysaccharides containing arabinose, galactose and glucose as monomers. Starch was identified enzymatically. The native carbohydrates allowed the encapsulation of bixin in its native microcrystalline form, resulting in a multilayer structure after spray-drying. In addition, the colorant particles displayed dispersibility under acidic aqueous conditions suggesting that they are stabilized by the native carbohydrates after the microcapsules are dissolved.