Effects of caffeine, methylliberine, and theacrine on vigilance, marksmanship, and hemodynamic responses in tactical personnel: a double-blind, randomized, placebo-controlled trial.

Background: Tactical athletes require fast reaction times (RT) along with high levels of vigilance and marksmanship performance. Caffeine has been shown to improve these measures but also results in increased blood pressure and jitteriness. Research on other purine alkaloids, such as methylliberine and theacrine, has suggested they do not increase blood pressure or jitteriness to the same extent, but their impact on tactical performance is unknown. Methods: A between-subjects, randomized, placebo-controlled design was used to test the effects of placebo (PLA), 300 mg caffeine (CAF), and a combination of 150 mg caffeine, 100 mg methylliberine, and 50 mg theacrine (CMT) on RT and marksmanship along with hemodynamic and arousal measures following a sustained vigilance task in tactical personnel (n = 48). Following consumption of the supplement, participants underwent a 150-min protocol consisting of two rounds. Each round began with leisurely reading followed by a 30-min vigilance task before beginning two trials of movement and marksmanship tasks. Hemodynamics and felt arousal were assessed throughout the protocol. Composite Z-scores were calculated for overall performance measures at each timepoint, and mixed-effects models were used to assess differences in RT, accuracy, and composite Z-scores along with hemodynamics and felt arousal. An α-level of 0.05 was used to determine statistical significance, and Cohen's d was used to quantify effect sizes. Results: A Group-by-Time interaction for vigilance RT (P = 0.038) indicated improvements for both CAF and CMT from round 1 to round 2 (P < 0.01) while PLA did not change (P = 0.27). No Group main effects or Group-by-Time interactions were found for movement or marksmanship performance (P > 0.20). Group main effects for systolic (SBP; P = 0.001) and diastolic blood pressure (DBP; P = 0.028) indicated higher SBP in CAF (P = 0.003, d= 0.84) and CMT (P = 0.007, d= 0.79) compared to PLA but only higher DBP in CAF (P = 0.025, d= 0.74). No Group-by-Time interaction or Group main effect was found for felt arousal (P > 0.16). Conclusions: These findings suggest similar benefits on RT during a vigilance task between CAF, containing 300 mg caffeine, and CMT above PLA, though CAF resulted in slightly less favorable hemodynamic changes. This study is the first to provide data showing similar efficacy of combined caffeine, methylliberine, and theacrine compared to double the caffeine dose consumed alone on vigilance RT but without a significant rise in DBP above PLA in tactical personnel.

Attenuation of Tumor Development in Mammary Carcinoma Rats by Theacrine, an Antagonist of Adenosine 2A Receptor.

Caffeine has been reported to induce anti-tumor immunity for attenuating breast cancer by blocking the adenosine 2A receptor. Molecular modeling showed that theacrine, a purine alkaloid structurally similar to caffeine, might be an antagonist of the adenosine 2A receptor equivalent to or more effective than caffeine. Theacrine was further demonstrated to be an effective antagonist of the adenosine 2A receptor as its concurrent supplementation significantly reduced the elevation of AMPK phosphorylation level in MCF-7 human breast cells induced by CGS21680, an agonist of adenosine 2A receptors. In an animal model, the development of mammary carcinoma induced by 7,12-Dimethylbenz[a]anthracene in Sprague-Dawley rats could be attenuated by daily supplement of theacrine of 50 or 100 mg/kg body weight. Both expression levels of cleaved-caspase-3/pro-caspase-3 and granzyme B in tumor tissues were significantly elevated when theacrine was supplemented, indicating the induction of programmed cell death in tumor cells might be involved in the attenuation of mammary carcinoma. Similar to the caffeine, significant elevation of interferon-γ and tumor necrosis factor-α was observed in the serum and tumor tissues of rats after the theacrine supplement of 50 mg/kg body weight. Taken together, theacrine is an effective antagonist of adenosine 2A receptors and possesses great potential to be used to attenuate breast cancer.

Identification of Co-Expressed Genes Related to Theacrine Synthesis in Tea Flowers at Different Developmental Stages.

Jiaocheng kucha is the first reported tea germplasm resource which contains theacrine founded in Fujian Province. Currently, the anabolic mechanism of theacrine within tea leaves is clear, but there are few studies focused on its flowers. In order to further explore the mechanism of theacrine synthesis and related genes in flowers, current study applied Jiaocheng kucha flowers (JC) as test materials and Fuding Dabaicha flowers (FD) as control materials to make transcriptome sequencing, and determination of purine alkaloid content in three different developmental periods (flower bud stage, whitening stage and full opening stage). The results showed that the flower in all stages of JC contained theacrine. The theacrine in the flower bud stage was significantly higher than in the other stages. The differentially expressed genes (DEGs) at three different developmental stages were screened from the transcriptome data, and were in a total of 5642, 8640 and 8465. These DEGs related to the synthesis of theacrine were primarily annotated to the pathways of purine alkaloids. Among them, the number of DEGs in xanthine synthesis pathway was the largest and upregulated in JC, while it was the smallest in caffeine synthesis pathway and downregulated in JC. Further weighted gene co-expression network (WGCNA) indicated that ADSL (CsTGY03G0002327), ADSL (CsTGY09G0001824) and UAZ (CsTGY06G0002694) may be a hub gene for the regulation of theacrine metabolism in JC. Our results will contribute to the identification of candidate genes related to the synthesis of theacrine in tea flowers, and explore the molecular mechanism of theacrine synthesis in JC at different developmental stages.

Theacrine, a Potent Antidepressant Purine Alkaloid from a Special Chinese Tea, Promotes Adult Hippocampal Neurogenesis in Stressed Mice.

Daily intake of tea has been known to relate to a low risk of depression. In this study, we report that a special variety of tea in China, Camellia assamica var. kucha (kucha), possesses antidepressant effects but with less adverse effects as compared to traditional tea Camellia sinensis. This action of kucha is related to its high amount of theacrine, a purine alkaloid structurally similar to caffeine. We investigated the antidepressant-like effects and mechanisms of theacrine in chronic water immersion restraint stress and chronic unpredictable mild stress mice models. PC12 cells and primary hippocampal neural stem cells were treated with stress hormone corticosterone (CORT) to reveal the potential antidepression mechanism of theacrine from the perspective of adult hippocampus neurogenesis. Results of behavioral and neurotransmitter analysis showed that intragastric administration of theacrine significantly counteracted chronic stress-induced depression-like disorders and abnormal 5-hydroxytryptamine (5-HT) metabolism with less central excitability. Further investigation from both in vivo and in vitro experiments indicated that the antidepressant mechanism of theacrine was associated with promoting adult hippocampal neurogenesis, via the modulation of the phosphodiesterase-4 (PDE4)/cyclic adenosine monophosphate (cAMP)/cAMP response-element binding (CREB)/brain-derived neurotrophic factor (BDNF)/tropomyosin-related kinase B (TrkB) pathway. Collectively, our findings could promote the prevalence of kucha as a common beverage with uses for health care and contribute to the development of theacrine as a potential novel antidepressant medicine.

A Theacrine-Based Supplement Increases Cellular NAD+ Levels and Affects Biomarkers Related to Sirtuin Activity in C2C12 Muscle Cells In Vitro.

There is evidence in rodents to suggest that theacrine-based supplements modulate tissue sirtuin activity as well as other biological processes associated with aging. Herein, we examined if a theacrine-based supplement (termed NAD3) altered sirtuin activity in vitro while also affecting markers of mitochondrial biogenesis. The murine C2C12 myoblast cell line was used for experimentation. Following 7 days of differentiation, myotubes were treated with 0.45 mg/mL of NAD3 (containing ~2 mM theacrine) for 3 and 24 h (n = 6 treatment wells per time point). Relative to control (CTL)-treated cells, NAD3 treatments increased (p < 0.05) Sirt1 mRNA levels at 3 h, as well as global sirtuin activity at 3 and 24 h. Follow-up experiments comparing 24 h NAD3 or CTL treatments indicated that NAD3 increased nicotinamide phosphoribosyltransferase (NAMPT) and SIRT1 protein levels (p < 0.05). Cellular nicotinamide adenine dinucleotide (NAD+) levels were also elevated nearly two-fold after 24 h of NAD3 versus CTL treatments (p < 0.001). Markers of mitochondrial biogenesis were minimally affected. Although these data are limited to select biomarkers in vitro, these preliminary findings suggest that a theacrine-based supplement can modulate select biomarkers related to NAD+ biogenesis and sirtuin activity. However, these changes did not drive increases in mitochondrial biogenesis. While promising, these data are limited to a rodent cell line and human muscle biopsy studies are needed to validate and elucidate the significance of these findings.

Theacrine and strictinin, two major ingredients for the anti-influenza activity of Yunnan Kucha tea.

ETHNOPHARMACOLOGICAL RELEVANCE: Kucha tea plant (Camellia assamica var. kucha Chang et Wang) is regarded as a mutant variety of wild Pu'er tea plant found in few mountain areas of Yunnan, China. Its fresh young leaves and shoots are picked by the indigenous aborigines in these local areas to prepare an herbal tea for the treatment of common cold empirically. MATERIALS AND METHODS: Two extra compounds of relative abundance were detected in Kucha tea in comparison with Pu'er tea, and their chemical structures were identified as chlorogenic acid and theacrine. These two compounds as well as two major compounds, strictinin and caffeine, in Kucha tea were evaluated for their cytotoxicity and inhibitory effects on human influenza virus A/Puerto Rico/8/34 by analyzing viral protein expression and progeny production. RESULTS: No or low cytotoxicity was detected for the four Kucha compounds when their concentrations were below 100 µM. Expression of viral NS1 protein was significantly inhibited by chlorogenic acid, theacrine or strictinin, but not caffeine at a concentration of 100 µM. The relative inhibitory potency was detected as chlorogenic acid < theacrine < strictinin, and both theacrine and strictinin displayed significant inhibition at a concentration of 50 µM. According to a plaque assay, viral progeny production was significantly reduced by theacrine or strictinin, but not by chlorogenic acid or caffeine under the same concentration of 100 µM. CONCLUSION: It is suggested that theacrine and strictinin are two major ingredients responsible for the anti-influenza activity of Yunnan Kucha tea traditionally used for the treatment of common cold.

Theacrine, a purine alkaloid from kucha, protects against Parkinson's disease through SIRT3 activation.

BACKGROUND: Oxidative damage of dopaminergic neurons is the fundamental causes of Parkinson's disease (PD) that has no standard cure at present. Theacrine, a purine alkaloid from Chinese tea Kucha, has been speculated to benefit the neurodegeneration in PD, through similar actions to its chemical analogue caffeine, albeit excluding side effects. Theacrine has nowadays gained a lot of interest for its multiple benefits, while the investigations are weak and insufficient. HYPOTHESIS/PURPOSE: It is well-known that tea has a wide range of functions, especially in the prevention and treatment of neurodegenerative diseases. Theacrine is an active monomer compound in Camellia assamica var. kucha Hung T. Chang & H.S.Wang (Kucha), which appears to be effective and safe in PD therapy. The aim of this study is to examine its actions in diverse PD models and explore the mechanisms. STUDY DESIGN: For determination of theacrine's effects, we employed diverse oxidative damage-associated PD models, including 6-OHDA-treated rats, MPTP-treated mice/zebrafish and MPP+-treated SH-SY5Y cells, and using caffeine, selegiline and depranyl as positve control. For investigation and verification of the mechanisms, we utilized approaches testing mitochondrial function-related parameters and enzyme activity as well as applied gene knockdown and overexpression. METHODS: We employed behavioral tests including spontaneous activity, pole, swimming, rotarod and gait, immunohistochemistry, HPLC, flow cytometry, immunohistochemistry, Western blot, gene knockdown by siRNA and overexpression by plasmid in this study. RESULTS: Theacrine is demonstrated to retrieve the loss of dopaminergic neurons and the damages of behavioral performance in multiple animal models of PD (6-OHDA-treated rats and in MPTP-treated mice and zebrafish). The followed data of MPP+-treated SH-SY5Y cells indicate that theacrine relieves apoptosis resulted from oxidative damage and mitochondrial dysfunction. Further investigations illustrate that theacrine activates SIRT3 directly. It is of advantage to prevent apoptosis through SIRT3-mediated SOD2 deacetylation that reduces ROS accumulation and restores mitochondrial function. This concept is elaborated by 3TYP that inhibits SIRT3 enzyme activity and knockdown/overexpression of SIRT3 gene, demonstrating a crucial role of SIRT3 in theacrine-benefited dopaminergic neurons. CONCLUSION: Theacrine prevents apoptosis of dopaminergic neurons through directly activating SIRT3 which deacetylating SOD2 and restoring mitochondrial functions.

Baiyacha, a wild tea plant naturally occurring high contents of theacrine and 3″-methyl-epigallocatechin gallate from Fujian, China.

Baiyacha (BYC) is a kind of wild tea plant growing and utilizing in the remote mountain area of Fujian province, Southeastern China. However, scientific studies on this plant remain limited. Our results showed that BYC exhibits the typical morphological characteristics of Camellia gymnogyna Chang, a closely related species of C. sinensis (L.) O. Kuntze, which was not found in Fujian before. Chemical profiling revealed that parts of BYC plants are rich in purine alkaloids and catechins, especially featuring high levels of theacrine and 3″-methyl-epigallocatechin gallate (EGCG3″Me), chemical compounds with multiple biological activities that are rarely observed in regular tea plants. The contents of EGCG3″Me and theacrine in BYC both increased with the leaf maturity of tea shoots, whereas the caffeine content decreased significantly. The obtained results provide abundant information about the morphology and chemical compounds of BYC and may be used for tea production, breeding, and scientific research in the future.

Novel insight into theacrine metabolism revealed by transcriptome analysis in bitter tea (Kucha, Camellia sinensis).

Kucha (Camellia sinensis) is a kind of unique wild tea resources in southwest China, containing sizeable amounts of theacrine (1,3,7,9-tetramethyluric acid) and having a special bitter taste both in fresh leaves and made tea. Theacrine has good healthy function locally. But the molecular mechanism of theacrine metabolism in Kucha was still unclear. In order to illuminate the biosynthesis and catabolism of theacrine in Kucha plants, three tea cultivars, C. sinensis 'Shangyou Zhongye' (SY) with low-theacrine, 'Niedu Kucha 2' (ND2) with middle-theacrine and, 'Niedu Kucha 3' (ND3) with high-theacrine, were used for our research. Purine alkaloid analysis and transcriptome of those samples were performed by High Performance Liquid Chromatography (HPLC) and RNA-Seq, respectively. The related gene expression levels of purine alkaloid were correlated with the content of purine alkaloid, and the results of quantitative real-time (qRT) PCR were also confirmed the reliability of transcriptome. Based on the data, we found that theacrine biosynthesis is a relatively complex process, N-methyltransferase (NMT) encoded by TEA024443 may catalyze the methylation at 9-N position in Kucha plant. Our finding will assist to reveal the molecular mechanism of theacrine biosynthesis, and be applied to selection and breeding of Kucha tea cultivars in the future.

Identification and characterization of N9-methyltransferase involved in converting caffeine into non-stimulatory theacrine in tea.

Caffeine is a major component of xanthine alkaloids and commonly consumed in many popular beverages. Due to its occasional side effects, reduction of caffeine in a natural way is of great importance and economic significance. Recent studies reveal that caffeine can be converted into non-stimulatory theacrine in the rare tea plant Camellia assamica var. kucha (Kucha), which involves oxidation at the C8 and methylation at the N9 positions of caffeine. However, the underlying molecular mechanism remains unclear. Here, we identify the theacrine synthase CkTcS from Kucha, which possesses novel N9-methyltransferase activity using 1,3,7-trimethyluric acid but not caffeine as a substrate, confirming that C8 oxidation takes place prior to N9-methylation. The crystal structure of the CkTcS complex reveals the key residues that are required for the N9-methylation, providing insights into how caffeine N-methyltransferases in tea plants have evolved to catalyze regioselective N-methylation through fine tuning of their active sites. These results may guide the future development of decaffeinated drinks.

Safety of Short-Term Supplementation with Methylliberine (Dynamine®) Alone and in Combination with TeaCrine® in Young Adults.

Methylliberine (Dynamine®; DYM) and theacrine (Teacrine®; TCR) are purine alkaloids purported to have similar neuro-energetic effects as caffeine. There are no published human safety data on DYM, and research on TCR is limited. The purpose of this study was to examine the effect of four weeks of DYM supplementation with and without TCR on cardiovascular function and blood biomarkers. One-hundred twenty-five men and women (mean age 23.0 yrs, height 169.7 cm, body mass 72.1 kg; n = 25/group) were randomly assigned to one of five groups: low-dose DYM (100 mg), high-dose DYM (150 mg), low-dose DYM with TCR (100 mg + 50 mg), high-dose DYM with TCR (150 mg + 25 mg) , and placebo. Regardless of group and sex, significant main effects for time were noted for heart rate, systolic blood pressure, and QTc (p < 0.001), high-density lipoproteins (p = 0.002), mean corpuscular hemoglobin (p = 0.018), basophils (p = 0.006), absolute eosinophils (p = 0.010), creatinine (p = 0.004), estimated glomerular filtration rate (p = 0.037), chloride (p = 0.030), carbon dioxide (p = 0.023), bilirubin (p = 0.027), and alanine aminotransferase (p = 0.043), among others. While small changes were found in some cardiovascular and blood biomarkers, no clinically significant changes occurred. This suggests that DYM alone or in combination with TCR consumed at the dosages used in this study does not appear to negatively affect markers of health over four weeks of continuous use.

Theacrine alleviates chronic inflammation by enhancing TGF-ß-mediated shifts via TGF-ß/SMAD pathway in Freund's incomplete adjuvant-induced rats.

Rheumatoid arthritis is a chronic and systemic autoimmune disease, which affects approximately 1% of the adult population worldwide. The present study investigated the therapeutic effect of theacrine (TC) on arthritis and its mechanisms in Freund's incomplete adjuvant (FIA)-induced SD rats. Rats were randomly divided into 5 groups: i) healthy control; ii) model; iii) positive control with methotrexate (MTX); iv) treatment with 12.5 mg/kg TC; and v) treatment with 25.0 mg/kg TC. The apparent scores, including changes in body weights, degree of paw swelling and arthritis indicators, were analyzed to evaluate the anti-chronic inflammatory effect of TC. The levels of interleukin (IL)-6 and transforming growth factor-ß (TGF-ß) in serum were measured by enzyme-linked immunosorbent assay. The protein and RNA expression levels of the critical factors in rats were measured to elucidate the mechanisms responsible for chronic inflammation and to verify molecular indexes of chronic inflammatory conditions. TC notably suppressed the severity of FIA-induced rat by attenuating the apparent scores, animal weight and inflammatory indexes in the 25 mg/kg TC group compared with the FIA rat model. Furthermore, TC significantly decreased the levels of IL-6 and increased the levels of TGF-ß. Histopathological examinations indicated that TC rescued the synovial hyperplasia and inflammatory cell infiltration in joint tissues. In addition, TC enhanced TGF-ß-mediated shifts in inflammatory marker expression in joint tissue. Overall, the present study demonstrated that TC exerted a superior anti-arthritic effect via the suppression of IL-6 and the activation of TGF-ß by the TGF-ß/SMAD pathway.

The effects of a caffeine-like supplement, TeaCrine®, on muscular strength, endurance and power performance in resistance-trained men.

BACKGROUND: TeaCrine® is the synthetic version to naturally occurring theacrine (1, 3, 7, 9-tetramethyluric acid) found in the leaves of Camellia kucha tea plants. A few studies have examined the effects of TeaCrine® on cognitive perception, but no research exists examining its effects on resistance exercise performance. The purpose of this study was to determine the efficacy of TeaCrine®, a caffeine-like compound, on maximal muscular strength, endurance, and power performance in resistance-trained men. METHODS: Twelve resistance-trained men participated in a randomized, double-blind, cross-over designed study. Each participant performed one-repetition maximum (1RM) bench press, 1RM squat, bench press repetitions to failure (RTF) at 70% 1RM, squat RTF at 70% 1RM, and 2-km rowing time trial 90 min after consumption of: (1) Caffeine 300 mg (CAFF300); (2) TeaCrine® 300 mg (TEA300); (3) TeaCrine® + Caffeine (COMBO; 150 mg/150 mg); (4) Placebo 300 mg (PLA). Power and velocity were measured using a TENDO Power Analyzer. Visual analogue scales for energy, focus, motivation to exercise, and fatigue were administered at baseline and 90 min post-treatment ingestion (pre-workout). Rating of perceived exertion was assessed after bench press RTF and squat RTF. RESULTS: There were no differences between groups for 1RM, RTF, and power in the bench press and squat exercises. Only CAFF300 resulted in significant increases in perceived energy and motivation to exercise vs. TEA300 and PLA (Energy: + 9.8%, 95% confidence interval [3.3-16.4%], p < 0.01; + 15.3%, 95% CI [2.2-28.5%], p < 0.02; Motivation to exercise: + 8.9%, 95% CI [0.2-17.6%], p = 0.04, + 14.8%, 95% CI [4.7-24.8%], p < 0.01, respectively) and increased focus (+ 9.6%, 95% CI [2.1-17.1%], p = 0.01) vs. TEA300, but there were no significant differences between CAFF300 and COMBO (Energy + 3.9% [- 6.9-14.7%], Focus + 2.5% [- 6.3-11.3%], Motivation to exercise + 0.5% [- 11.6-12.6%]; p > 0.05). CONCLUSION: Neither TEA300, CAFF300, COMBO, or PLA (when consumed 90 min pre-exercise) improved muscular strength, power, or endurance performance in resistance-trained men. Only CAFF300 improved measures of focus, energy, and motivation to exercise.

Theacrine attenuates epithelial mesenchymal transition in human breast cancer MDA-MB-231 cells.

Theacrine, a purine alkaloid structurally similar to caffeine, has recently become of interest as a potential therapeutic compound. Here, we investigated the antimetastatic potential of theacrine on human breast cancer MDA-MB-231 cells. We observed that theacrine can reverse epithelial-to-mesenchymal transition (EMT), which resulted in a decrease in the levels of mesenchymal markers (Fibronectin, Vimentin, N-cadherin, Twist, and Snail) and an increase in the levels of epithelial markers (Occludin and E-cadherin) in the cells. Additionally, theacrine attenuates TGF-ß-induced EMT, cell adhesion, migration, and invasion in MDA-MB-231 cells. Overall, our results suggest that theacrine may inhibit the breast cancer cell metastasis by reversing the EMT process.

The effects of TeaCrine® and caffeine on endurance and cognitive performance during a simulated match in high-level soccer players.

BACKGROUND: Theacrine (1,3,7,9-tetramethyluric-acid) is a pure alkaloid with a similar structure to caffeine and acts comparably as an adenosine receptor antagonist. Early studies have shown non-habituating effects, including increases in energy and focus in response to Teacrine®, the compound containing pure theacrine. The purpose of this study was to determine and compare the effects of Teacrine® and caffeine on cognitive performance and time-to-exhaustion during a simulated soccer game in high-level male and female athletes. METHODS: Male and female soccer players (N = 24; MAge = 20.96 ± 2.05y, MMaleVO2max = 55.31 ± 3.39 mL/O2/kg, MFemaleVO2max = 50.97 ± 3.90 mL/O2/kg) completed a 90-min simulated treadmill soccer match over four randomized sessions (TeaCrine®, caffeine, TeaCrine® + caffeine, placebo). Cognitive testing at halftime and end-of-game including simple reaction time (SRT), choice RT (CRT), and cognitive-load RT with distraction questions (COGRT/COGRTWrong) was performed, with a run time-to-exhaustion (TTE) at 85% VO2max following end-of-game cognitive testing. Session times and pre-exercise nutrition were controlled. RM-MANOVAs with univariate follow-ups were conducted and significance was set at P < 0.05. RESULTS: TTE trended towards significance in TeaCrine® and TeaCrine® + caffeine conditions compared to placebo (P < 0.052). A condition main effect (P < 0.05) occurred with faster CRT in caffeine and TeaCrine® + caffeine compared to placebo. COGRTWrong showed a significant time main effect, with better accuracy at end-of-game compared to halftime (P < 0.05). A time x condition interaction in SRT (P < 0.05) showed placebo improved from halftime to end-of-game. CONCLUSIONS: The 27-38% improvements in TTE reflect increased performance capacity that may have important implications for overtime scenarios. These findings suggest TeaCrine® favorably impacts endurance and the combination with caffeine provides greater benefits on cognitive function than either supplement independently.

Theacrine protects against nonalcoholic fatty liver disease by regulating acylcarnitine metabolism.

OBJECTIVE: Acylcarnitine metabolism disorder contributes significantly to the pathogenesis of nonalcoholic fatty liver disease (NAFLD). There are, however, few ideal medications for NAFLD, which work by targeting acylcarnitine metabolism. The aim of this study was to investigate the protective effects of theacrine, a rare purine alkaloid isolated from Camellia assamica var. kucha, against acylcarnitine metabolism disorder in NAFLD. METHODS: The pharmacological activities of theacrine were studied using high-fat diet (HFD)-fed ApoE-/- and C57BL/6J mice models. Oleate-treated HepG2 and L-02 cells were used to investigate the molecular mechanism of theacrine on acylcarnitine metabolism. The target of theacrine was confirmed in vitro as the blockade of sirtuin 3 (SIRT3) and protein kinase A. RESULTS: Theacrine inhibits hepatic steatosis and liver inflammation and improves energy expenditure in HFD-fed mice. Theacrine ameliorates acylcarnitine metabolism disorder in HFD-fed mice and oleate-treated hepatocytes by improving fatty acid oxidation. The underlying mechanism involves theacrine's activation of the mitochondrial deacetylase SIRT3 and consequently, the increased activity of long-chain acyl coenzyme A dehydrogenase (LCAD) through deacetylation. CONCLUSION: Theacrine promotes acylcarnitine metabolism in NAFLD through the SIRT3/LCAD signaling pathway. The target of theacrine's activities on NAFLD is identified as SIRT3.

Molecular and thyroid hormone binding properties of lamprey transthyretins: The role of an N-terminal histidine-rich segment in hormone binding with high affinity.

Transthyretin (TTR) is a plasma thyroid hormone (TH) binder that emerged from an ancient hydroxyisourate hydrolase by gene duplication. To know how an ancient TTR had high affinity for THs, molecular and TH binding properties of lamprey TTRs were investigated. In adult serum, the lipoprotein LAL was a major T3 binder with low affinity. Lamprey TTRs had an N-terminal histidine-rich segment, and had two classes of binding sites for 3,3',5-triiodo-L-thyronine (T3): a high-affinity and a low-affinity site. Mutant TTRΔ3-11, lacking the N-terminal histidine-rich segment, lost the high-affinity T3 binding site. [125I]T3 binding to wild type TTR and mutant TTRΔ3-11, was differentially modulated by Zn2+. Zn2+ contents of wild type TTR were 7-10/TTR (mol/mol). Our results demonstrate that lamprey TTR is a Zn2+-dependent T3 binder. The N-terminal histidine-rich segment may be essential for neo-functionalization (i.e., high-affinity T3 binding activity) of an ancient TTR after gene duplication.

Online Raman spectroscopy for structural biology on beamline ID29 of the ESRF.

Raman spectroscopy can probe the structure and conformations of specific chemical groups within proteins and may thus be used as a technique complementary to X-ray crystallography. This combined approach can be decisive in resolving ambiguities in the interpretation of enzymatic or X-ray induced processes. Here, we present an online Raman setup developed at the European Synchrotron that allows for interleaved Raman spectra acquisition and X-ray diffraction measurements with fast probe exchange and simple alignment while maintaining a high sensitivity over the entire spectral range. This device has been recently employed in the study of a covalent intermediate in the O2-dependent breakdown of uric acid by the cofactor-free enzyme urate oxidase and to monitor its decay induced by X-ray exposure.

Characteristics of the brown hagfish Paramyxine atami transthyretin: Metal ion-dependent thyroid hormone binding.

Transthyretin (TTR) is a vertebrate-specific protein involved in thyroid hormone distribution in plasma, and its gene is thought to have emerged by gene duplication from the gene for the ancient TTR-related protein, 5-hydroxyisourate hydrolase, at some early stage of chordate evolution. We investigated the molecular and hormone-binding properties of the brown hagfish Paramyxine atami TTR. The amino acid sequence deduced from the cloned hagfish TTR cDNA shared 33-50% identities with those of other vertebrate TTRs but less than 24% identities with those of vertebrate and deuterostome invertebrate 5-hydroxyisourate hydrolases. Hagfish TTR, as well as lamprey and little skate TTRs, had an N-terminal histidine-rich segment, allowing purification by metal-affinity chromatography. The affinity of hagfish TTR for 3,3',5-triiodo-L-thyronine (T3) was 190 times higher than that for L-thyroxine, with a dissociation constant of 1.5-3.9nM at 4°C. The high-affinity binding sites were strongly sensitive to metal ions. Zn2+ and Cu2+ decreased the dissociation constant to one-order of magnitude, whereas a chelator, o-phenanthroline, increased it four times. The number of metal ions (mainly Zn2+ and Cu2+) was approximately 12/TTR (mol/mol). TTR was also a major T3-binding protein in adult hagfish sera and its serum concentration was approximately 8µM. These results suggest that metal ions and the acquisition of N-terminal histidine-rich segment may cooperatively contribute to the evolution toward an ancient TTR with high T3 binding activity from either 5-hydroxyisourate hydrolase after gene duplication.

A comparative analysis of chemical compositions in Camellia sinensis var. puanensis Kurihara, a novel Chinese tea, by HPLC and UFLC-Q-TOF-MS/MS.

Camellia sinensis var. puanensis Kurihara (Puan tea) is a kind of ancient tea plant newly found in Jiangxipo and the surrounding areas of Puan County (Guizhou, China). People there always believe that drinking Puan tea is beneficial to the promotion of health and prevention of diseases. However, detailed information on its compositions has not been reported. Therefore, in this study, the varieties and contents of purine alkaloids and polyphenols in Puan tea were identified and determined by HPLC and UFLC-Q-TOF-MS/MS. Our results showed that theacrine, but not caffeine, was the dominated purine alkaloid detected in Puan tea. Meanwhile, Puan tea contained B-type procyanidin dimer, trimer and dimer monogallate, which were not detected in Camellia sinensis, Camellia ptilophylla and Camellia assamica var. kucha. The obtained results could support the local uses of Puan tea in health and nutrition and contribute to the research of tea variety.