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Discovery and Physiological Role

Lysine and ornithine are degraded in a similar manner in Clostridium sticklandii, a Gram-positive anaerobe. 5,6-LAM participates in the second step of the fermentation pathway of lysine in which lysine is converted to acetic acid, ammonia and butyric acid, while 4,5-OAM takes part in the first step of the fermentation pathway of ornithine, in which ornithine is converted to acetate, carbon dioxide, alanine and ammonia by clostridia []. Dr. Theressa Stadtman and coworkers first discovered and performed initial studies on 5,6-LAM [,]. Initially, it was assumed that there are two distinct enzymes [–] displaying 5,6-aminomuatase activity relating to two distinct substrates, d-lysine and l-β-lysine. Later, it was found that these are the same enzyme, which can accept two different substrates [,]. Dyer and Costilow [] reported OAM activity for the first time in crude extracts, and a subsequent study by Tsuda and Friedmann on cofactor requirements was performed using partially purified extracts []. Later, separation, purification and properties of 4,5-OAM was reported by Somack and Costilow [].

Pyridoxal-5′-Phosphate (PLP) Binding Site and Active Site Residues

d-Ornithine soaked 4,5-OAM crystals [] provided the glimpse of substrate bound structure, in which Lys629 has moved away from PLP and ornithine is bound to PLP through external aldimine. Hydrogen bonding and salt bridge interactions of Gln299, Arg297, His225, Tyr160, and His182 with carboxylate moiety of d-ornithine and electrostatic interaction between Glu81 and α-amine anchor the substrate in the active site (). The hydrogen bonding interaction of pyridine nitrogen with Ser162 is absent. Similar interactions are observed in the case of substrate analog dl-2,4-diaminobutyric acid (2,4-DAB) with few exceptions. His225 and Tyr160 do not make hydrogen bonds with the carboxylate while Ser162 and Lys629 make additional hydrogen bonds with pyridine nitrogen and phenolic oxygen, respectively ().

Abstract

Approximately half the proline residues in fibrillar collagen are hydroxylated. The predominant form is 4-hydroxyproline, which helps fold and stabilize the triple helix. A minor form, 3-hydroxyproline, still has no clear function. Using peptide mass spectrometry, we recently revealed several previously unknown molecular sites of 3-hydroxyproline in fibrillar collagen chains. In fibril-forming A-clade collagen chains, four new partially occupied 3-hydroxyproline sites were found (A2, A3, A4 and (GPP)n) in addition to the fully occupied A1 site at Pro986. The C-terminal (GPP)n motif has five consecutive GPP triplets in α1(I), four in α2(I) and three in α1(II), all subject to 3-hydroxylation. The evolutionary origins of this substrate sequence were investigated by surveying the pattern of its 3-hydroxyproline occupancy from early chordates through amphibians, birds and mammals. Different tissue sources of type I collagen (tendon, bone and skin) and type II collagen (cartilage and notochord) were examined by mass spectrometry. The (GPP)n domain was found to be a major substrate for 3-hydroxylation only in vertebrate fibrillar collagens. In higher vertebrates (mouse, bovine and human), up to five 3-hydroxyproline residues per (GPP)n motif were found in α1(I) and four in α2(I), with an average of two residues per chain. In vertebrate type I collagen the modification exhibited clear tissue specificity, with 3-hydroxyproline prominent only in tendon. The occupancy also showed developmental changes in Achilles tendon, with increasing 3-hydroxyproline levels with age. The biological significance is unclear but the level of 3-hydroxylation at the (GPP)n site appears to have increased as tendons evolved and shows both tendon type and developmental variations within a species.

Methods

Endogenous generation of electrophilic omega-3 PUFAs and their hydroxy precursors was evaluated by mass spectrometry in neutrophils isolated from healthy subjects, both at baseline and upon stimulation with calcium ionophore. For the clinical trial, participants were healthy adults 30–55 years of age with a reported EPA+DHA consumption of ≤300 mg/day randomly assigned to parallel groups receiving daily oil capsule supplements for a period of 4 months containing either 1.4 g of EPA+DHA (active condition, n = 24) or identical appearing soybean oil (control condition, n = 21). Participants and laboratory technicians remained blinded to treatment assignments.

Results

5-lypoxygenase-dependent endogenous generation of 7-oxo-DHA, 7-oxo-DPA and 5-oxo-EPA and their hydroxy precursors is reported in human neutrophils stimulated with calcium ionophore and phorbol 12-myristate 13-acetate (PMA). Dietary EPA+DHA supplementation significantly increased the formation of 7-oxo-DHA and 5-oxo-EPA, with no significant modulation of arachidonic acid (AA) metabolite levels.

Introduction

Herein, the endogenous formation of electrophilic α,β-unsaturated ketone derivatives of DHA, EPA and DPA is reported in neutrophils isolated from healthy subjects. Specifically, 5-LO-dependent generation of 7-oxo-DHA, 7-oxo-DPA, 5-oxoEPA and their hydroxy precursors is reported. Moreover, the effects of EPA and DHA consumption at a clinically-relevant dose (1.4 g/day) on the production of these ω-3 PUFA ketone derivatives was evaluated in healthy human subjects participating in a randomized, placebo-controlled clinical trial. There was a significant increase in levels of electrophilic ω-3 PUFA ketone derivatives in neutrophils under both basal and activated conditions, with no significant change in AA-derived metabolites. It is proposed that these reactive products transduce the salutary actions of ω-3 PUFAs by modifying the activity of electrophile-sensitive transcription factors that regulate the expression of inflammatory and metabolic-related genes. This report follows CONSORT guidelines for clinical trials .

Abstract

Dietary ω-3 polyunsaturated fatty acids (PUFAs) decrease cardiovascular risk via suppression of inflammation. The generation of electrophilic α,β-unsaturated ketone derivatives of the ω-3 PUFAs docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA) in activated human macrophages is catalyzed by cyclooxygenase-2 (Cox-2). These derivatives are potent pleiotropic anti-inflammatory signaling mediators that act via mechanisms including the activation of Nrf2-dependent phase 2 gene expression and suppression of pro-inflammatory NF-κB-driven gene expression. Herein, the endogenous generation of ω-3 PUFAs electrophilic ketone derivatives and their hydroxy precursors was evaluated in human neutrophils. In addition, their dietary modulation was assessed through a randomized clinical trial.

Introduction

Among oxygenated PUFAs, electrophilic α,β-unsaturated ketone derivatives are of interest because they a) are end-products of hydroxylated fatty acid metabolism, b) display reversible electrophilic reactivity with nucleophilic protein targets via Michael addition reaction and c) modulate the activity of critical inflammatory and host defense signaling pathways . In this regard, electrophilic PUFAs activate the Nrf2-dependent anti-oxidant response, are partial agonists of the nuclear lipid receptor PPARγ, and suppress pro-inflammatory NF-κB signaling at multiple levels , –. Recently, a method has been developed for the mass spectrometric detection and quantification of electrophilic fatty acids, based on the use of the β-mercaptoethanol (BME) as nucleophilic bait . Its use in profiling electrophilic fatty acid generation by activated macrophages led to the discovery of Cox-2-dependent generation of 13- and 17-oxo-derivatives of docosahexaenoic (DHA) and docosapentaenoic acid (DPA). These electrophilic ketone derivatives mediate pleitropic anti-inflammatory actions via modulation of transcriptional regulatory protein function thus representing a new class of endogenous lipid mediators.

1. Introduction

A dose-finding study of menatetrenone in Japan [] administered daily doses of 15, 45, 90, and 135 mg and revealed that 45 mg was the minimum effective dose for improving bone mass parameters evaluated by microdensitometry and/or single photon absorptiometry in postmenopausal women with osteoporosis. This optimal dose (45 mg/day) for the treatment of osteoporosis is about 150–180 times greater than the recommended daily dietary intake of vitamin K (250–300 μg) []. No toxic effects of menatetrenone (45 mg/day) have been reported []. High-dose vitamin K is needed to prevent fractures in postmenopausal women with osteoporosis []. However, the effect of menatetrenone on the skeleton remains a matter of controversy [,,,,,,,], and the role of menatetrenone in the treatment of osteoporosis therefore needs to be clarified. In clinical practice, menatetrenone is frequently used in combination with bisphosphonates.

3. Menatetrenone Monotherapy

Menatetrenone reduces serum ucOC concentrations [,]. The anti-fracture efficacy of menatetreone in postmenopausal osteoporosis has been shown in eight RCTs () [,,,,,,,]. Of the eight RCTs, six were performed in Japan, one in Indonesia, and one in China. One Japanese RCT was a phase IV trial (Osteoporotic Fracture (OF) study) conducted in a large sample of postmenopausal women with osteoporosis (n = 4378) []. The dose of menatetrenone was 45 mg/day in all these studies. Placebo controls were used in one RCT, non-treatment controls in four RCTs, calcium supplementation controls in two RCTs, and active controls (alfacalcidol) in one RCT. The number of study subjects ranged from 44–4378 in the menatetrenone and control groups, and the study periods ranged from 1–3 years.

5. Discussion

The present review revealed positive evidence for the effect of menatetrenone monotherapy on BMD and the incidence of fractures (mainly vertebral fractures) in postmenopausal women with osteoporosis. However, this evidence was derived from RCTs with small sample sizes. Thus, menatetrenone is used as a second-line medicine for the treatment of postmenopausal osteoporosis in Japan []. Knapen et al. [] reported that menatetrenone (45 mg/day) improved hip bone geometry, bone strength indices, and bone mineral content in healthy postmenopausal women, supporting the effect of menatetrenone on bone health.

4. Combined Menatetrenone and Alendronate Therapy

Hirao et al. [] conducted a RCT to examine the benefit of combined menatetrenone and alendronate therapy in postmenopausal women with osteoporosis. The increase in femoral neck BMD and the decrease in serum ucOC concentrations were greater in the alendronate plus menatetrenone group compared with the alendronate alone group.

Abstract

Background. In this retrospective analysis we assessed the role of [18F]-FACBC-PET/CT in the prostatic cancer staging. Procedure. 30 first [18F]-FACBC-PET/CT images of 26 patients (68.1 ± 5.8 years) were analyzed. PET/CT findings were compared with PSA concentrations, with PSA doubling times (PDT), and with correlative imaging. Results. On 16 [18F]-FACBC (53.3%) scans, 58 metabolically active lesions were found. 12 (20.7%) lesions corresponding to the local relapse were found in prostate/prostate bed and seminal vesicles, 9 (15.5%) lesions were located in regional lymph nodes, 10 (17.2%) were located in distal lymph nodes, and 26 (44.8%) metabolically active lesions were found in the skeleton. In one case, focal uptake was found in the brain, confirmed further on MRI as meningioma. The mean S-PSA level in patients with positive [18F]-FACBC findings was 9.5 ± 16.9 μg/L (0.54–69 μg/L) and in patients with negative [18F]-FACBC findings was 1.96 ± 1.87 μg/L (0.11–5.9 μg/L), but the difference was not statistically significant. However, the PSA doubling time (PDT) in patients with positive findings was significantly shorter than PDT in patients with negative findings: 3.25 ± 2.09 months (0.3–6 months) versus 31.2 ± 22.02 months (8–84 months), P < 0.0001. There was a strong positive correlation between PSA value and number of metabolically active lesions (R = 0.74) and a negative correlation between PDT and number of metabolically active lesions (R = −0.56). There was a weak negative correlation between PDT and SUVmax⁡ (R = −0.30). Conclusion. According to our preliminary clinical experience, [18F]-FACBC-PET may play a role in in vivo restaging of an active prostate cancer, especially in patients with a short S-PSA doubling time.

2.1. Patients

30 [18F]-FACBC-PET/CT scans of 26 patients (25 with confirmed prostatic cancer in the period 1999–2013) were analyzed: age ranging from 56 to 77 years (mean age 68.1 ± 5.8); Gleason score 5–9 (mean 7.1 ± 1.4); PSA value 0.11–69 μg/L (mean 7.9 ± 14.6). FACBC-PET/CT scans for restaging were done in 25 patients with confirmed prostatic cancer and in one patient with suspicion of prostate cancer.

2.2. Imaging PET/CT Protocol

Examination was done on Siemens Biograph PET Scanner, combined with low-dose CT. The injected activity dose of [18F]-anti-FACBC ranged from 230 to 320 MBq (mean 328 ± 56.8). Early imaging was performed by starting at 8.0 ± 3.0 min (range of 4–16 min) from pelvic region using 3 min per bed position imaging protocol followed by whole body imaging from the calvarium to the midthighs at 23.4 ± 6.3 min (range of 16–37 min) using 3 min per bed position. The tracer was purchased from Uppsala (Uppsala Imanet, Sweden), and the production has been described by Sörensen et al. [].

3. Results

On 16 [18F]-FACBC-PET scans (53.3%), a total of 58 metabolic active lesions were detected. In prostate, prostate bed and seminal vesicles focal uptake was found in 12 cases (20.7%), with SUVmax⁡ ranging from 3.7 to 7.2 (mean 5.07 ± 1.9). In local lymph nodes, 9 lesions (15.5%) were found with SUVmax⁡ 4.1–10.7 (mean 8.1 ± 2.6) and, in distal lymph nodes, 10 lesions (17.2%) with SUVmax⁡ 3.4–11.9 (mean 7.35 ± 2.7) were found. A total of 26 bone lesions (44.8%) with SUVmax⁡ 4.2–8.8 (mean 5.4 ± 0.7) were observed. In one case, there was increased tracer uptake in the brain with a SUVmax⁡ 8.1 (1.7%) confirmed later on MRI as meningioma. The findings are presented for each patient individually in .

Abstract

Djungarian hamster (P. sungorus) and Roborovskii hamster (P. roborovskii) belong to the same genus of phodopus. Roborovskii hamster shows high locomotor activity and low level of dopamine (DA) in the brain. Administration of L-tyrosine, a precursor of DA, decreases locomotor activity in Roborovskii hamsters. However, the amino acid metabolism in relation to the hyperactivity is not yet well known. In the present study, L- and D-amino acid concentrations in the brain, liver, and plasma in Djungarian and Roborovskii hamsters were investigated during day and night times to explain the possible difference in hyperactivity between them. Most of the examined amino acids were higher in the night time when hamsters are active compared to those in day time. L- and D-tyrosine concentrations were higher in the liver of Roborovskii hamsters than in Djungarian hamsters. Furthermore, brain concentration of D-tyrosine was higher in the Roborovskii than in Djungarian hamsters, but no significant difference was observed for L-tyrosine concentrations between the two species. These results suggest that the conversion of L-tyrosine to D-tyrosine in the brain of Roborovskii hamster may be higher than in Djungarian hamster, which may cause low DA concentration and hyperactivity in Roborovskii hamster. On the other hand, L- and D-serine, which are known as sedative factors, were lower in Roborovskii hamsters than Djungarian hamster. These results suggest that species-specific regulation in amino acid metabolism may contribute to hyperactivity in Roborovskii hamsters.

Animals and housing conditions

Male Djungarian hamsters (n = 14) and Roborovskii hamsters (n = 14), 3 weeks of age, were purchased from a local dealer similarly as reported previously (Kabuki et al. , ,[], ). We used the minimum number of animals in each group considering the statistical power of sample size and reared under controlled environments. They were housed in a group of three or four animals per cage and freely given access to a standard diet (MF; Oriental Yeast, Tokyo, Japan) and water. A 12-h day/night cycle was maintained throughout the experiment, with light on at 08:00 and off at 20:00. Room temperature was maintained at 23 ± 1°C and humidity at 60%. This study was conducted according to the guidelines for animal experiments in the Faculty of Agriculture and on the Graduate Course of Kyushu University, and to Law No. 105 and Notification No. 6 of the government.

Tissue preparation

After an acclimation period of 8 days, the brains of Djungarian hamsters and Roborovskii hamsters were obtained during the day time (14:00) when hamsters are resting and the night time (02:00) when hamsters are active (n = 7 in each group). Hamsters were decapitated under anesthesia with isoflurane (Escain®, Mylan, Osaka, Japan), and trunk blood was collected each time. The whole brains were carefully taken out from the skulls and placed on a cold glass dish. The livers were taken, and washed with saline. The plasma separated from brood, which was centrifuged at 4°C, 3000 × g for 20 min. The tissue samples were frozen in liquid nitrogen, and all the samples were stored at −80°C until analysis.

Results

In the brain, both L- and D-tyrosine as well as L- and D-serine levels were significantly (P < 0.05) higher at night than day time. D-Tyrosine level was significantly (P < 0.01) higher in Roborovskii than in Djungarian hamsters (Figure B), but no significant difference was found in L-tyrosine between the two species (Figure A). Both L- and D-serine levels were significantly (P < 0.01) higher in Djungarian than Roborovskii hamsters (Figure C,D).

Introduction

The aim of this study was to examine whether the two endogenous retinoids atRA and ddRA affect the mRNA expression profile differently in cultured human primary keratinocytes using oligonucleotide arrays. All genes causing ARCI were analyzed in detail. The expression profiles after retinoid treatment were examined both in proliferating and differentiated keratinocytes, as representatives for basal and suprabasal epidermal keratinocytes, respectively.

Materials

atRA and ddRA were obtained from Sigma-Aldrich (Stockholm, Sweden) and Roche AG (Basle, Switzerland), respectively. The compounds were dissolved in ethanol and the concentration and purity were determined by UV absorbance and HPLC analysis, respectively. Stock solutions of 1 mM were prepared. The purity of atRA and ddRA was 99.1 and 88.3 %, respectively. The two major impurities in ddRA were 13cRA (1.5 %) and atRA (10.2 %). All handling of retinoids was performed under yellow reduced light.

Cell culture and retinoid treatment

Primary keratinocytes from a lightly pigmented female donor (42 years) were used at passage 3 (Life Technologies, Stockholm, Sweden). The cells were maintained in EpiLife serum-free keratinocyte medium containing growth supplement (HKGS or HKGS kit) and gentamicin/amphotericin (all from Life Technologies). Proliferating cells in 6-well plates at 30–40 % confluency were switched to EpiLife medium without EGF for 5 h prior to starting treatment with retinoids. Keratinocytes at 80 % confluency were switched to EpiLife− supplemented with 1.5 mM CaCl2 for 4 days with the purpose to induce cellular differentiation. Cells were subsequently exposed to retinoids as above in EpiLife medium without rhEGF. Both proliferating and differentiated cells were exposed to 1 µM of atRA, ddRA or vehicle (ethanol) for 4 and 24 h. All treatments were performed in triplicates. The cells were washed with PBS twice and frozen at −70 °C until further processing.

atRA and ddRA regulate gene expression most profoundly in differentiating keratinocytes

We compared the transcriptional profiles in atRA- and ddRA-treated keratinocytes with vehicle-treated keratinocytes. In cells exposed to one of the two retinoids, the affected RNA transcripts were few in numbers in proliferating keratinocytes as compared to differentiated cells (see Table ). The genes altered in response to atRA exposure is shown as a heat map (Online Resource 4). However, the number of altered transcripts was in the same range after treatments with both retinoids, and most of the transcripts were significantly altered by both retinoids. The top ten-induced and -suppressed annotated transcripts after atRA exposure are shown in (Online Resource 5). The expression of some genes in atRA- vs. vehicle-treated differentiating cells was verified by qPCR (Fig. b–i).

Gene expression was not regulated differently by atRA and ddRA

Although the major number of retinoid-regulated transcripts was induced or suppressed equally efficiently by atRA and ddRA, there were also transcripts that were significantly altered by only one of the retinoids when comparing to vehicle-treated controls. However, when comparing the expression profiles generated by the two retinoids with each other, no genes were significantly altered (data not shown).

Abstract

Metabolic pathways in eubacteria and archaea often are encoded by operons and/or gene clusters (genome neighborhoods) that provide important clues for assignment of both enzyme functions and metabolic pathways. We describe a bioinformatic approach (genome neighborhood network; GNN) that enables large scale prediction of the in vitro enzymatic activities and in vivo physiological functions (metabolic pathways) of uncharacterized enzymes in protein families. We demonstrate the utility of the GNN approach by predicting in vitro activities and in vivo functions in the proline racemase superfamily (PRS; InterPro IPR008794). The predictions were verified by measuring in vitro activities for 51 proteins in 12 families in the PRS that represent ∼85% of the sequences; in vitro activities of pathway enzymes, carbon/nitrogen source phenotypes, and/or transcriptomic studies confirmed the predicted pathways. The synergistic use of sequence similarity networks3 and GNNs will facilitate the discovery of the components of novel, uncharacterized metabolic pathways in sequenced genomes.

Introduction

In this manuscript, we describe the use of a new bioinformatic strategy, genome neighborhood networks (GNNs), to discover the enzymes, transport systems, and transcriptional regulators that constitute metabolic pathways, thereby facilitating prediction of their individual in vitro activities and combined in vivo metabolic functions. As the first demonstration of its use, we applied this approach to the functionally diverse proline racemase superfamily (PRS) and predicted functions for >85% of its members. The predictions were verified using high-throughput protein expression and purification, in vitro enzyme activity measurements, microbiology (phenotypes and transcriptomics), and X-ray crystallography.

Abstract

The purpose of this study was to elucidate the role of gamma-tocotrienol (GT3)-mobilized progenitors in mitigating damage to mice exposed to a supralethal dose of cobalt-60 gamma-radiation. CD2F1 mice were transfused 24 h post-irradiation with whole blood or isolated peripheral blood mononuclear cells (PBMC) from donors that had received GT3 72 h prior to blood collection and recipient mice were monitored for 30 days. To understand the role of GT3-induced granulocyte colony-stimulating factor (G-CSF) in mobilizing progenitors, donor mice were administered a neutralizing antibody specific to G-CSF or its isotype before blood collection. Bacterial translocation from gut to heart, spleen and liver of irradiated recipient mice was evaluated by bacterial culture on enriched and selective agar media. Endotoxin in serum samples also was measured. We also analyzed the colony-forming units in the spleens of irradiated mice. Our results demonstrate that whole blood or PBMC from GT3-administered mice mitigated radiation injury when administered 24 h post-irradiation. Furthermore, administration of a G-CSF antibody to GT3-injected mice abrogated the efficacy of blood or PBMC obtained from such donors. Additionally, GT3-mobilized PBMC inhibited the translocation of intestinal bacteria to the heart, spleen, and liver, and increased colony forming unit-spleen (CFU-S) numbers in irradiated mice. Our data suggests that GT3 induces G-CSF, which mobilizes progenitors and these progenitors mitigate radiation injury in recipient mice. This approach using mobilized progenitor cells from GT3-injected donors could be a potential treatment for humans exposed to high doses of radiation.

Drug preparation and administration

GT3 formulation in 5% Tween-80 in saline was purchased from Yasoo Health, Inc. (Johnson City, TN, USA). Olive oil was used as vehicle control (equivalent to the quantity of GT3) in 5% Tween-80. The final GT3 concentration was adjusted to administer a dose of 200 mg/kg in 0.1 ml. Control mice received 0.1 ml of vehicle. Mice were administered GT3 or vehicle subcutaneously (sc) at the nape of the neck with a 23 G needle attached to a Luer-Lock syringe 72 h before harvesting blood. No infections or local reactions were noted at the site of injection. AMD3100 (commercially known as plerixafor or Mozobil, Sigma-Aldrich, St. Louis, MO, USA) was diluted to 1.25 mg/ml with phosphate-buffered saline (PBS) for a dose of 5 mg/kg . It has been reported that AMD3100 aids in mobilizing progenitors from bone marrow to peripheral circulation and improves the yield of progenitors when injected to mice 1 h prior to harvest , . All donor mice, including those receiving the vehicle (olive oil), also received AMD3100 sc in a volume of 0.1 ml using a 23 G needle 1 h before blood collection to enhance mobilization .