Paraxanthine provides greater improvement in cognitive function than caffeine after performing a 10-km run.

RATIONALE: Intense exercise promotes fatigue and can impair cognitive function, particularly toward the end of competition when decision-making is often critical for success. For this reason, athletes often ingest caffeinated energy drinks prior to or during exercise to help them maintain focus, reaction time, and cognitive function during competition. However, caffeine habituation and genetic sensitivity to caffeine (CA) limit efficacy. Paraxanthine (PX) is a metabolite of caffeine reported to possess nootropic properties. This study examined whether ingestion of PX with and without CA affects pre- or post-exercise cognitive function. METHODS: 12 trained runners were randomly assigned to consume in a double-blind, randomized, and crossover manner 400 mg of a placebo (PL); 200 mg of PL + 200 mg of CA; 200 mg of PL + 200 mg of PX (ENFINITY®, Ingenious Ingredients); or 200 mg PX + 200 mg of CA (PX+CA) with a 7-14-day washout between treatments. Participants donated fasting blood samples and completed pre-supplementation (PRE) side effects questionnaires, the Berg-Wisconsin Card Sorting Test (BCST), and the Psychomotor Vigilance Task Test (PVTT). Participants then ingested the assigned treatment and rested for 60 minutes, repeated tests (PRE-EX), performed a 10-km run on a treadmill at a competition pace, and then repeated tests (POST-EX). Data were analyzed using General Linear Model (GLM) univariate analyses with repeated measures and percent changes from baseline with 95% confidence intervals. RESULTS: BCST correct responses in the PX treatment increased from PRE-EX to POST-EX (6.8% [1.5, 12.1], p = 0.012). The error rate in the PL (23.5 [-2.8, 49.8] %, p = 0.078) and CA treatment (31.5 [5.2, 57.8] %, p = 0.02) increased from PRE-EX values with POST-EX errors tending to be lower with PX treatment compared to CA (-35.7 [-72.9, 1.4] %, p = 0.059). POST-EX perseverative errors with PAR rules were significantly lower with PX treatment than with CA (-26.9 [-50.5, -3.4] %, p = 0.026). Vigilance analysis revealed a significant interaction effect in Trial #2 mean reaction time values (p = 0.049, ηp2 = 0.134, moderate to large effect) with POST-EX reaction times tending to be faster with PX and CA treatment. POST-EX mean reaction time of all trials with PX treatment was significantly faster than PL (-23.2 [-43.4, -2.4] %, p = 0.029) and PX+CA (-29.6 [-50.3, -8.80] %, p = 0.006) treatments. There was no evidence that PX ingestion adversely affected ratings of side effects associated with stimulant intake or clinical blood markers. CONCLUSIONS: Results provide some evidence that pre-exercise PX ingestion improves prefrontal cortex function, attenuates attentional decline, mitigates cognitive fatigue, and improves reaction time and vigilance. Adding CA to PX did not provide additional benefits. Therefore, PX ingestion may serve as a nootropic alternative to CA.

Analyses of the onset mechanisms of cardio-stimulatory action by aciclovir.

Cardio-stimulatory actions of aciclovir have been considered to primarily depend on the sympathetically-mediated reflex resulting from its hypotensive effect. To further clarify onset mechanisms of the cardio-stimulatory actions, we initially studied them using isoflurane-anesthetized dogs under thorough ß1-adrenoceptor blockade with atenolol (1 mg/kg, i.v.) (n = 4). Aciclovir (20 mg/kg/10 min, i.v.) decreased mean arterial blood pressure by 10 mmHg, whereas it increased heart rate by 10 bpm and maximum upstroke velocity of ventricular pressure by 928 mmHg/s, and shortened AH interval by 2 ms, indicating that cardio-stimulatory actions were not totally abolished by ß1-adrenoceptor blockade. Then, unknown mechanisms of cardio-stimulatory action were explored. Since aciclovir has a similar chemical structure to theophylline, in silico molecular docking simulation was performed, indicating aciclovir as well as theophylline possesses strong likelihood of interactions with phosphodiesterase 1A, 1C and 3A. Indeed, aciclovir inhibited phosphodiesterase 1A derived from the bovine heart (n = 4), moreover it exerted positive chronotropic action on the atrial tissue preparation of rats along with an increase of tissue cyclic AMP concentration (n = 4). These results indicate that cardio-stimulatory actions of aciclovir could result from not only hypotension-induced, reflex-mediated increase of sympathetic tone but also its inhibitory effects on phosphodiesterase in the heart.

Effects of imidazolium ionic liquids on skin barrier lipids - Perspectives for drug delivery.

Ionic liquids (ILs) have great potential to facilitate transdermal and topical drug delivery. Here, we investigated the mechanism of action of amphiphilic ILs 1-methyl-3-octylimidazolium bromide (C8MIM) and 3-dodecyl-1-methylimidazolium bromide (C12MIM) in skin barrier lipid models in comparison to their complex effects in human skin. C8MIM incorporated in a skin lipid model was a better permeation enhancer than C12MIM for water and model drugs, theophylline and diclofenac. Solid state 2H NMR and X-ray diffraction indicated that both ILs prefer the cholesterol-rich regions in skin lipids without significantly perturbing their lamellar arrangement and that C8MIM induces the formation of an isotropic lipid phase to a greater extent compared to C12MIM. C12MIM applied topically to the lipid model or human skin as a pretreatment was more potent than C8MIM. When co-applied with the drugs to human skin, aqueous C12MIM was more potent than C8MIM in enhancing theophylline permeation, but neither IL affected (even decreased) diclofenac permeation. Thus, the IL's ability to permeabilize skin lipid barrier is strongly modulated by its ability to reach the site of action and its interactions with drug and solvent. Such an interplay is far from trivial and requires detailed investigation to realize the full potential of ILs.

T7 phage-assisted evolution of riboswitches using error-prone replication and dual selection.

Leveraging riboswitches, non-coding mRNA fragments pivotal to gene regulation, poses a challenge in effectively selecting and enriching these functional genetic sensors, which can toggle between ON and OFF states in response to their cognate inducers. Here, we show our engineered phage T7, enabling the evolution of a theophylline riboswitch. We have replaced T7's DNA polymerase with a transcription factor controlled by a theophylline riboswitch and have created two types of host environments to propagate the engineered phage. Both types host an error-prone T7 DNA polymerase regulated by a T7 promoter along with another critical gene-either cmk or pifA, depending on the host type. The cmk gene is necessary for T7 replication and is used in the first host type for selection in the riboswitch's ON state. Conversely, the second host type incorporates the pifA gene, leading to abortive T7 infections and used for selection in the riboswitch's OFF state. This dual-selection system, termed T7AE, was then applied to a library of 65,536 engineered T7 phages, each carrying randomized riboswitch variants. Through successive passage in both host types with and without theophylline, we observed an enrichment of phages encoding functional riboswitches that conferred a fitness advantage to the phage in both hosts. The T7AE technique thereby opens new pathways for the evolution and advancement of gene switches, including non-coding RNA-based switches, setting the stage for significant strides in synthetic biology.

Antifungal potential of the new copper(II)-theophylline/1,10-phenanthroline complex against drug-resistant Candida species.

Candida spp. are the commonest fungal pathogens worldwide. Antifungal resistance is a problem that has prompted the discovery of novel anti-Candida drugs. Herein, 25 compounds, some of them containing copper(II), cobalt(II) and manganese(II) ions, were initially evaluated for inhibiting the growth of reference strains of Candida albicans and Candida tropicalis. Eight (32%) of the compounds inhibited the proliferation of these yeasts, displaying minimum inhibitory concentrations (MICs) ranging from 31.25 to 250 µg/mL and minimum fungicidal concentration (MFCs) from 62.5 to 250 µg/mL. Drug-likeness/pharmacokinetic calculated by SwissADME indicated that the 8 selected compounds were suitable for use as topical drugs. The complex CTP, Cu(theo)2phen(H2O).5H2O (theo = theophylline; phen = 1,10-phenanthroline), was chosen for further testing against 10 medically relevant Candida species that were resistant to fluconazole/amphotericin B. CTP demonstrated a broad spectrum of action, inhibiting the growth of all 20 clinical fungal isolates, with MICs from 7.81 to 62.5 µg/mL and MFCs from 15.62 to 62.5 µg/mL. Conversely, CTP did not cause lysis in erythrocytes. The toxicity of CTP was evaluated in vivo using Galleria mellonella and Tenebrio molitor. CTP had no or low levels of toxicity at doses ranging from 31.25 to 250 µg/mL for 5 days. After 24 h of treatment, G. mellonella larvae exhibited high survival rates even when exposed to high doses of CTP (600 µg/mL), with the 50% cytotoxic concentration calculated as 776.2 µg/mL, generating selectivity indexes varying from 12.4 to 99.4 depending on each Candida species. These findings suggest that CTP could serve as a potential drug to treat infections caused by Candida species resistant to clinically available antifungals.

Creation of Architecturally Minimal Transcriptionally Activating Riboswitches Responsive to Theophylline Reveals an Unconventional Design Strategy.

Riboswitches are noncoding RNA switches that are largely utilized in bacteria and play a significant role in synthetic biology. Nonetheless, their natural counterparts possess lengthy sequences and intricate structures, posing challenges for their modular integration into complex gene circuits. Consequently, it is imperative to develop simplified synthetic riboswitches that can be effortlessly incorporated into gene circuits. The conventional approach to generate synthetic riboswitches entails tedious library construction and extensive screening, which frequently yields suboptimal performance. To overcome this obstacle, alternative methods are urgently needed. In this study, we created a novel approach to designing a diverse set of transcription-activating riboswitches that exhibit high performance and broad compatibility. The strategy involved starting with a synthetic theophylline RNA aptamer and designing an expression platform that forms a transcriptional terminator in its inactive state but switches to an antiterminator when it is activated. Several sequences were designed, constructed, and subjected to virtual screening, resulting in the identification of two transcription-activating riboswitches. These riboswitches were then engineered to reduce the basal leakage and increase the activation level through extending the hairpin region using a screened random sequence. These architecturally minimal synthetic riboswitches were highly adapted to different constitutive promoters in a modular manner, generating a differentially responsive output to theophylline. As a proof-of-principle, the synthetic riboswitches were applied to rewire a synthetic quorum-sensing circuit (QSC). The reprogrammed QSC successfully modulated the temporal responsive profile against the activation. This strategy is expected to expand the variety of high-performance riboswitches that are responsive to different ligands, thereby further facilitating the design of complex genetic circuits.

Pre-mating administration of theophylline could prevent the transgenerational effects of maternal morphine dependence on offspring anxiety behavior: The role of dopamine receptors.

Opioid addiction causes some molecular alterations in the brain reward pathway, such as changes in gene expression that may be transferred to the next generation via epigenetic mechanisms such as histone acetylation. This study aimed to evaluate the effect of theophylline as an HDAC (Histone deacetylases) activator on D1 and D2 dopamine receptor expression in the nucleus accumbens (NAc) and anxiety behavior in the offspring of morphine-dependent female rats. Female rats were exposed to escalating doses of morphine for six days and were then treated with theophylline (20 mg/kg) or saline for 10 days before mating with normal male rats. Male and female offspring were tested for anxiety behavior using an elevated plus maze apparatus. Besides, the expression of D1 and D2 dopamine receptors in the NAc was evaluated by real-time PCR (polymerase chain reaction). Results showed that offspring of morphine-dependent female rats had increased expression of both D1 and D2 receptors in the NAc, as well as decreased anxiety behavior, compared to control offspring. However, the mentioned effects were returned to normal levels in the offspring whose morphine-dependent mothers had received theophylline for 10 days before mating. It is concluded that theophylline may be therapeutically effective in minimizing the adverse consequences of maternal morphine dependence on offspring behavior by restoring normal dopamine receptor expression levels and modulating anxiety. To completely comprehend the underlying mechanisms of this phenomenon, more research is required.

Theophylline-induced endothelium-dependent vasodilation is mediated by increased nitric oxide release and phosphodiesterase inhibition in rat aorta.

This study aimed to examine the endothelial dependence of vasodilation induced by the phosphodiesterase inhibitor theophylline in isolated rat thoracic aortas and elucidate the underlying mechanism, with emphasis on endothelial nitric oxide (NO). The effects of various inhibitors and endothelial denudation on theophylline-induced vasodilation, and the effect of theophylline on vasodilation induced by NO donor sodium nitroprusside, cyclic guanosine monophosphate (cGMP) analog bromo-cGMP, and ß-agonist isoproterenol in endothelium-denuded aorta were examined. The effects of theophylline and sodium nitroprusside on cGMP formation were also examined. We examined the effect of theophylline on endothelial nitric oxide synthase (eNOS) phosphorylation and intracellular calcium levels. Theophylline-induced vasodilation was greater in endothelium-intact aortas than that in endothelium-denuded aortas. The NOS inhibitor, NW-nitro-L-arginine methyl ester; non-specific guanylate cyclase (GC) inhibitor, methylene blue; and NO-sensitive GC inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a] quinoxalin-1-one inhibited theophylline-induced vasodilation in endothelium-intact aortas. Theophylline increased the vasodilation induced by sodium nitroprusside, bromo-cGMP, and isoproterenol. Theophylline increased cGMP formation in endothelium-intact aortas, and sodium nitroprusside-induced cGMP formation in endothelium-denuded aortas. Moreover, theophylline increased stimulatory eNOS (Ser1177) phosphorylation and endothelial calcium levels, but decreased the phosphorylation of inhibitory eNOS (Thr495). These results suggested that theophylline-induced endothelium-dependent vasodilation was mediated by increased endothelial NO release and phosphodiesterase inhibition.

Glutathione-Mediated Neuroprotective Effect of Purine Derivatives.

Numerous basic studies have reported on the neuroprotective properties of several purine derivatives such as caffeine and uric acid (UA). Epidemiological studies have also shown the inverse association of appropriate caffeine intake or serum urate levels with neurodegenerative diseases such as Alzheimer disease (AD) and Parkinson's disease (PD). The well-established neuroprotective mechanisms of caffeine and UA involve adenosine A2A receptor antagonism and antioxidant activity, respectively. Our recent study found that another purine derivative, paraxanthine, has neuroprotective effects similar to those of caffeine and UA. These purine derivatives can promote neuronal cysteine uptake through excitatory amino acid carrier protein 1 (EAAC1) to increase neuronal glutathione (GSH) levels in the brain. This review summarizes the GSH-mediated neuroprotective effects of purine derivatives. Considering the fact that GSH depletion is a manifestation in the brains of AD and PD patients, administration of purine derivatives may be a new therapeutic approach to prevent or delay the onset of these neurodegenerative diseases.

Theophylline Prevents Dexamethasone-Induced Atrophy in C2C12 Myotubes.

Skeletal muscle mass is maintained by a balance between the synthesis and degradation of muscle proteins, the collapse of which causes muscle wasting. The prevention of muscle wasting improves the quality of life and extends a healthy life. The methyl xanthine theophylline showed strong preventive activity against dexamethasone-induced muscle atrophy, as determined using the expression level of myosin heavy chain in C2C12 myotubes. Mechanistically, theophylline inhibited the expression of ubiquitin ligases MuRF1 and Cbl-b, but not that of atrogin-1. Furthermore, theophylline inhibits glucocorticoid receptor translocation to the nucleus. A pull-down assay using a theophylline probe revealed that theophylline and dexamethasone competitively interacted with the glucocorticoid receptor, suggesting an antagonistic activity of theophylline on glucocorticoid receptors. Additionally, theophylline inhibited the dexamethasone-induced phosphorylation of p38 and FoxO3a in C2C12 myotubes. These findings suggest that theophylline is an effective food ingredient in the prevention of glucocorticoid-induced skeletal muscle atrophy.

Theophylline reverses oxycodone's but not fentanyl's respiratory depression in mice while caffeine is ineffective against both opioids.

RATIONALE: The opioid epidemic remains a pressing public health crisis in the United States. Most of these overdose deaths are a result of lethal respiratory depression. In recent years the increasing incidence of opioid-involved overdose deaths has been driven by fentanyl, which is more resistant to adequate reversal by naloxone (NARCAN ®) than semi-synthetic or classical morphinan predecessors like oxycodone and heroin. For this and other reasons (e.g., precipitating withdrawal) non-opioidergic pharmacotherapies to reverse opioid-depressed respiration are needed. Methylxanthines are a class of stimulant drugs including caffeine and theophylline which exert their effects primarily via adenosine receptor antagonism. Evidence suggests methylxanthines can stimulate respiration by enhancing neural activity in respiratory nuclei in the pons and medulla independent of opioid receptors. This study aimed to determine whether caffeine and theophylline can stimulate respiration in mice when depressed by fentanyl and oxycodone. METHODS: Whole-body plethysmography was used to characterize fentanyl and oxycodone's effects on respiration and their reversal by naloxone in male Swiss Webster mice. Next, caffeine and theophylline were tested for their effects on basal respiration. Finally, each methylxanthine was evaluated for its ability to reverse similar levels of respiratory depression induced by fentanyl or oxycodone. RESULTS AND CONCLUSIONS: Oxycodone and fentanyl dose-dependently reduced respiratory minute volume (ml/min; MVb) that was reversible by naloxone. Caffeine and theophylline each significantly increased basal MVb. Theophylline, but not caffeine, completely reversed oxycodone-depressed respiration. In contrast, neither methylxanthine elevated fentanyl-depressed respiration at the doses tested. Despite their limited efficacy for reversing opioid-depressed respiration when administered alone, the methylxanthines safety, duration, and mechanism of action supports further evaluation in combination with naloxone to augment its reversal of opioid-depressed respiration.

Formulation and optimization of theophylline-loaded enteric-coated spanlastic nanovesicles for colon delivery; Ameliorate acetic acid-induced ulcerative colitis.

Treatment of colon diseases presents one of the most significant obstacles to drug delivery due to the inability to deliver sufficient drug concentration selectively to the colon. The goal of the proposed study was to develop, optimize, and assess an effective colon target delivery system of theophylline-based nanovesicles (TP-NVs) surrounded by a biodegradable polymeric shell of chitosan (CS) and Eudragit L100 (EL100) for the treatment of ulcerative colitis (UC). TP-loaded nanovesicles were fabricated using the ethanol injection method and coated with CS and EL100, respectively. We used a 32-factorial design approach to optimize the concentration of CS and EL100 to minimize particle size (PS) and maximize the cumulative amount of theophylline released (CTR) after 24 h. The optimized formulation was described using transmission electron microscopy (TEM), differential scanning calorimetry (DSC), and in vitro release. In-vivo quantification of theophylline in the gastrointestinal tract and in-vivo targeting potential in a rat model of acetic acid-induced colitis were also thoroughly evaluated. The characteristics of the optimal formula predicted by the 32-factorial design approach corresponded exceptionally well with the measured PS of 271.3 nm, the zeta potential of -39.9 mV, and CTR of 3.95, and a 99.93% after 5 and 24 h, respectively. Notably, the in vivo results in the rat model of colitis showed that the formulation with an optimized coat significantly improved theophylline distribution to the colon and markedly decreased the expression of interleukin-6 and ulcerative lesions compared to a pure theophylline solution. These outcomes elucidated the feasibility of a 32-factorial design to detect the crucial interactions between the study's components. Our findings suggested that enteric-coated nanovesicles formulations with optimal coat compositions of 0.2693% (w/v) and 0.75% (w/v) of CS and EL100, respectively, were promising carriers for colonic delivery of theophylline, a rate-limiting step in the treatment of UC.

Theophylline for the management of respiratory disorders in adults in the 21st century: A scoping review from the American College of Clinical Pharmacy Pulmonary Practice and Research Network.

Theophylline is an oral methylxanthine bronchodilator recommended as alternate therapy for the treatment of asthma and chronic obstructive pulmonary disease (COPD). However, it is not generally recommended for the treatment of other respiratory disorders such as obstructive sleep apnea (OSA) or hypoxia. Most clinical practice guidelines rely on evidence published prior to the year 2000 to make these recommendations. This scoping review aimed to gather and characterize evidence describing theophylline for the management of respiratory disorders in adults between January 1, 2000 and December 31, 2020. Databases searched included Ovid MEDLINE, Embase, CINAHL Complete, Scopus, and International Pharmaceutical Abstracts. This review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) extension for scoping reviews. Studies were included if they were published in English, theophylline was used for any respiratory disorder, and the study outcomes were disease- or patient-oriented. After removal of duplicates, 841 studies were screened and 55 studies were included. Results aligned with current clinical guideline recommendations relegating theophylline as an alternative therapy for the treatment of respiratory disorders, in favor of inhaled corticosteroids and inhaled bronchodilators. This scoping review identified the need for future research including: theophylline versus other medications deemed alternative therapies for asthma and COPD, meta-analyses of low-dose theophylline, and studies evaluating evidence-based patient-oriented outcomes for OSA, hypoxia, ventilator-induced diaphragmatic dysfunction, and spinal cord injury-related pulmonary function.

Massively Parallel Double-Selection Workflow for the Evolution of Molecular Switches Based on Surface-Display in Escherichia coli.

Microbial surface display of proteins is a versatile method for a wide range of biotechnological applications. Herein, the use of a surface display system in E. coli for the evolution of a riboswitch from an RNA aptamer is presented. To this end, a streptavidin-binding peptide (SBP) is displayed at the bacterial surface, which can be used for massively parallel selection using a magnetic separation system. Coupling gene expression from a riboswitch library to the display of SBP hence allows selection of library members that express strongly in the presence of a ligand. As excessive SBP expression leads to bacterial growth inhibition, it can be used to negatively select against leaky riboswitches expressing in the absence of ligand. Based on this principle, we devise a double selection workflow that enables quick selection of functional riboswitches with a comparatively low screening workload. The efficiency of our protocol by re-discovering a previously isolated theophylline riboswitch from a library was demonstrated, as well as a new riboswitch that is similar in performance, but slightly more responsive at low theophylline concentrations. Our workflow is massively parallel and can be applied to the screening or pre-screening of large molecular libraries.

Theophylline-Induced Relaxation Is Enhanced after Testosterone Treatment via Increased KV1.2 and KV1.5 Protein Expression in Guinea Pig Tracheal Smooth Muscle.

Theophylline is a drug commonly used to treat asthma due to its anti-inflammatory and bronchodilatory properties. Testosterone (TES) has been suggested to reduce the severity of asthma symptoms. This condition affects boys more than girls in childhood, and this ratio reverses at puberty. We reported that guinea pig tracheal tissue chronic exposure to TES increases the expression of ß2-adrenoreceptors and enhances salbutamol-induced K+ currents (IK+). Herein, we investigated whether the upregulation of K+ channels can enhance the relaxation response to methylxanthines, including theophylline. Chronic incubation of guinea pig tracheas with TES (40 nM, 48 h) enhanced the relaxation induced by caffeine, isobutylmethylxanthine, and theophylline, an effect that was abolished by tetraethylammonium. In tracheal myocytes, chronic incubation with TES increased theophylline-induced IK+; flutamide reversed this effect. The increase in IK+ was blocked by 4-aminopyridine by ~82%, whereas iberiotoxin reduced IK+ by ~17%. Immunofluorescence studies showed that chronic TES exposure increased the expression of KV1.2 and KV1.5 in airway smooth muscle (ASM). In conclusion, chronic exposure to TES in guinea pig ASM promotes upregulation of KV1.2 and KV1.5 and enhances theophylline relaxation response. Therefore, gender should be considered when prescribing methylxanthines, as teenage boys and males are likely to respond better than females.

Theophylline Attenuates BLM-Induced Pulmonary Fibrosis by Inhibiting Th17 Differentiation.

Idiopathic pulmonary fibrosis (IPF) is a chronic and refractory interstitial lung disease. Although there are two approved drugs for IPF, they were not able to completely cure the disease. Therefore, the development of new drugs is required for the effective treatment of IPF. In this study, we investigated the effect of theophylline, which has long been used for the treatment of asthma, on pulmonary fibrosis. The administration of theophylline attenuated the fibrotic changes of lung tissues and improved mechanical pulmonary functions in bleomycin (BLM)-induced pulmonary fibrosis. Theophylline treatment suppressed IL-17 production through inhibiting cytokines controlling Th17 differentiation; TGF-ß, IL-6, IL-1ß, and IL-23. The inhibition of IL-6 and IL-1ß by theophylline is mediated by suppressing BLM-induced ROS production and NF-κB activation in epithelial cells. We further demonstrated that theophylline inhibited TGF-ß-induced epithelial-to-mesenchymal transition in epithelial cells through suppressing the phosphorylation of Smad2/3 and AKT. The inhibitory effects of theophylline on the phosphorylation of Smad2/3 and AKT were recapitulated in BLM-treated lung tissues. Taken together, these results demonstrated that theophylline prevents pulmonary fibrosis by inhibiting Th17 differentiation and TGF-ß signaling.

Systematic Comparison and Rational Design of Theophylline Riboswitches for Effective Gene Repression.

Riboswitches are promising regulatory tools in synthetic biology. To date, 25 theophylline riboswitches have been developed for regulation of gene expression in bacteria. However, no one has systematically evaluated their regulatory effects. To promote efficient selection and application of theophylline riboswitches, we examined 25 theophylline riboswitches in Escherichia coli MG1655 and found that they varied widely in terms of activation/repression ratios and expression levels in the absence of theophylline. Of the 20 riboswitches that activate gene expression, only one exhibited a high activation ratio (63.6-fold) and low expression level without theophylline. Furthermore, none of the five riboswitches that repress gene expression were more than 2.0-fold efficient. To obtain an effective repression system, we rationally designed a novel theophylline riboswitch to control a downstream gene or genes by premature transcription termination. This riboswitch allowed theophylline-dependent downregulation of the TurboRFP reporter in a dose- and time-dependent manner. Its performance profile exceeded those of previously described repressive theophylline riboswitches. We then introduced as the second part a RepA tag (protein degradation tag) coding sequence fused at the 5'-terminal end of the turborfp gene, which further reduced protein level, while not reducing the repressive effect of the riboswitch. By combining two tandem theophylline riboswitches with a RepA tag, we constructed a regulatory cassette that represses the expression of the gene(s) of interest at both the transcriptional and posttranslational levels. This regulatory cassette can be used to repress the expression of any gene of interest and represents a crucial step toward harnessing theophylline riboswitches and expanding the synthetic biology toolbox. IMPORTANCE A variety of gene expression regulation tools with significant regulatory effects are essential for the construction of complex gene circuits in synthetic biology. Riboswitches have received wide attention due to their unique biochemical, structural, and genetic properties. Here, we have not only systematically and precisely characterized the regulatory properties of previously developed theophylline riboswitches but also engineered a novel repressive theophylline riboswitch acting at the transcriptional level. By introducing coding sequences of a tandem riboswitch and a RepA protein degradation tag at the 5' end of the reporter gene, we successfully constructed a simple and effective regulatory cassette for gene regulation. Our work provides useful biological components for the construction of synthetic biology gene circuits.

A purine derivative, paraxanthine, promotes cysteine uptake for glutathione synthesis.

Purine derivatives such as caffeine and uric acid have neuroprotective activities and are negatively correlated with the incidence of both Alzheimer's disease and Parkinson's disease. We have reported that an increment of intracellular glutathione (GSH) via cysteine uptake in neuronal cells is one of the mechanisms by which caffeine and uric acid confer neuroprotection. Here, we investigated whether caffeine metabolites such as paraxanthine, theophylline, theobromine, 1,7-dimethyluric acid and monomethylxanthines would increase cysteine uptake in mouse hippocampal slices. The metabolites were administered to hippocampal slices for 30 min at doses of 0, 10, or 100 µM, and then cysteine was added for 30 min. Paraxanthine, a major metabolite of caffeine, increased cysteine content in the slices, whereas the other metabolites did not. In vitro treatment with paraxanthine promoted cysteine uptake and increased GSH in HEK293 cells. The paraxanthine-induced cysteine uptake was inhibited by an excitatory amino-acid carrier-1 (EAAC1) inhibitor, and H2O2-induced cell damage was prevented by the paraxanthine treatment of SH-SY5Y cells. These results suggest that paraxanthine, an active metabolite of caffeine, acts to increase intracellular GSH levels via EAAC1 leading to neuroprotection.

The analysis of methylxanthine fractions obtained from Camellia sinensis cultivated in Turkey and effects on the in vitro inhibition of CYP2D6 enzyme.

Tea is a worldwide consumed herbal beverage and it was aimed in this study to reveal the major fractions of green and black tea in order to enlighten the in vitro inhibition potency on the well-known drug metabolizing enzyme CYP2D6 activity. Methylxanthine fractions were extracted from green and black tea and a yield of 0.265 g (1.06%) for 25 g of dried black tea and 0.302 g (1.2%) for 25 g of green tea was calculated. High-performance liquid chromatography analysis represented that the major components of the methylxanthine fractions were caffeine, theobromine, and theophylline. Methylxanthine content of black tea was 368.25 ± 4.6 µg/ml caffeine, 89.30 ± 2.3 µg/ml theobromine, and 3.40 ± 0.5 µg/ml theophylline, whereas that of green tea was 176.50 ± 3.7 µg/ml caffeine, 53.85 ± 1.4 µg/ml theobromine, and 2.06 ± 0.7 µg/ml theophylline. The results of concentration-dependent inhibition studies were 76% green tea, 75% black tea, and 55% caffeine at concentration of 10 mg/ml. The inhibition rates of green and black tea on CYP2D6 activity were 76% and 75%, respectively, where that of quinidine, the well-known inhibitor of CYP2D6, was 82%. Our results indicate that green and black tea is very likely to modify the CYP2D6 enzyme activity.