A fluorescence turn "on-off" imaging probe for sequential detection of Al3+ and L-Cysteine in HeLa cells.

At this "Aluminum Age", exposure to aluminum (metallic or ionic form) is inevitable and inestimable. The presence of aluminum in biological systems is evident but more often aluminum toxicity is less understood. Therefore, the presence of biologically reactive aluminum needs to be identified and quantified. Alongside metals, L-cysteine, an essential amino acid, plays a pivotal role in the homeostasis of cellular oxidative and reductive stress. However, excess (<7g) could be lethal and can lead to death. Thus, in-situ selective detection of aluminum and L-cysteine is of larger interest. Here we report a fluorogenic probe (R) for the sequential selective detection and quantification of Al3+ and L-cysteine in a semi-aqueous medium (3:7; water: DMSO). The probe (R) was synthesized by a one-step acid-mediated condensation reaction between pyridine-3,4-diamine and 2-hydroxy-1-napthaldehyde. The synthesized probe was characterized using 1H and 13C NMR, and HR-Mass spectroscopic techniques. The probe (R) is non-emissive in nature, but on recognition of Al3+, the probe R showed "turn-on" emission (bright yellow colour) showing two emission maxima (522 nm and 547 nm), and no naked eye observable color change. Other competing cations do not show any noticeable fluorescence outcome. The R + Al3+ ensemble can specifically detect L-cysteine among all the essential amino acids by showing a fluorescence "turn-off" response. The sensing mechanism of Al3+ is obeying the chelation-enhanced fluorescence (CHEF) effect. The binding constant of R + Al3+ is 0.3 × 104 M-1. The limit of detection (LoD) for Al3+ and L-cysteine are 2.02 × 10-7 M and 0.5 × 10-5 M respectively. The probe (R) can show maximum efficiency within the pH range (7.0-10.0). The probe is found non-toxic (>80 % cell viability with 15 µM concentration) and employed for the in-vitro fluorescence imaging in the HeLa cell.

Enhancing osteogenic differentiation of diabetic tendon stem/progenitor cells through hyperoxia: Unveiling ROS/HIF-1α signalling axis.

Diabetic calcific tendinopathy is the leading cause of chronic pain, mobility restriction, and tendon rupture in patients with diabetes. Tendon stem/progenitor cells (TSPCs) have been implicated in the development of diabetic calcified tendinopathy, but the molecular mechanisms remain unclear. This study found that diabetic tendons have a hyperoxic environment, characterized by increased oxygen delivery channels and carriers. In hyperoxic environment, TSPCs showed enhanced osteogenic differentiation and increased levels of reactive oxygen species (ROS). Additionally, hypoxia-inducible factor-1a (HIF-1a), a protein involved in regulating cellular responses to hyperoxia, was decreased in TSPCs by the ubiquitin-proteasome system. By intervening with antioxidant N-acetyl-L-cysteine (NAC) and overexpressing HIF-1a, we discovered that blocking the ROS/HIF-1a signalling axis significantly inhibited the osteogenic differentiation ability of TSPCs. Animal experiments further confirmed that hyperoxic environment could cause calcification in the Achilles tendon tissue of rats, while NAC intervention prevented calcification. These findings demonstrate that hyperoxia in diabetic tendons promotes osteogenic differentiation of TSPCs through the ROS/HIF-1a signalling axis. This study provides a new theoretical basis and research target for preventing and treating diabetic calcified tendinopathy.

Hydrogen sulfide induces Ca2+ influx in the principal cells of rat cortical collecting ducts.

Hydrogen sulfide (H2S) acts as a gas-signaling agent in various tissues. Although it has been reported that endogenous enzymes that generate H2S are expressed abundantly in the kidney, few reports examine cellular responses to H2S in renal tubular epithelial cells. In this study, we investigated the effects of NaHS, an H2S donor, and l-cysteine, a substrate for H2S production, on the principal cells of rat cortical collecting ducts (CCDs). NaHS increased the intracellular Ca2+ concentration ([Ca2+]i) in the principal cells. The removal of extracellular Ca2+ largely attenuated the [Ca2+]i response. The TRPV4 channel blocker significantly inhibited the effect of NaHS. Extracellular administration of l-cysteine also elicited a rise in [Ca2+]i. Prior treatment of CCDs with AOAA, an inhibitor of H2S production enzyme, l-cysteine-induced [Ca2+]i response was significantly reduced. These results suggest that not only exogenous H2S but also endogenously produced H2S triggers the extracellular influx pathway of Ca2+ in the principal cells of rat CCDs.

S-Allyl-L-cysteine (SAC) inhibits copper-induced apoptosis and cuproptosis to alleviate cardiomyocyte injury.

Cardiomyocyte injury is closely related to various myocardial diseases, and S-Allyl-L-cysteine (SAC) has been found to have myocardial protective effects, but its mechanism is currently unclear. Meanwhile, copper also has various physiological functions, and this study found that copper inhibited cell viability in a concentration and time-dependent manner, and was associated with multiple modes of death. Elesclomol plus CuCl2 (ES + Cu) significantly inhibited cell viability, and this effect could only be blocked by copper chelator TTM, indicating that "ES + Cu" induced cuproptosis in cardiomyocytes. SAC reduced the inhibitory effects of high concentration copper and "ES + Cu" on cell viability in a concentration and time-dependent manner, indicating that SAC plays a cardioprotective role under stress. Further mechanism study showed that high concentration of copper significantly induced cardiomyocyte apoptosis and increased the levels of LDH, MDA and ROS, while SAC inhibited the apoptosis and injury of cardiomyocytes induced by copper. "ES + Cu" significantly increased intracellular copper levels and decreased the expression of FDX1, LIAS, Lip-DLST and Lip-DLAT; FDX1 siRNA did not affect the expression of LIAS, but further reduced the expression of Lip-DLST and Lip-DLAT; SAC did not affect the expression of these genes, but enhanced the effect of "ES + Cu" in down-regulating these gene expression and restored intracellular copper levels. In addition, "ES + Cu" reduced ATP production, weakened the activity of mitochondrial complex I and III, inhibited cell viability, and increased the contents of injury markers LDH, MDA, CK-MB and cTnI, while SAC significantly improved mitochondrial function injury and cardiomyocyte injury induced by "ES + Cu". Therefore, SAC can inhibit apoptosis and cuproptosis to play a cardioprotective role.

Targeting threonine deaminase with chiral Au NPs: A novel strategy for E. coli inhibition.

Bacterial infections are becoming a significant threat to global human health due to the growing prevalence of biofilm-related infections and the rise in antibiotic resistance. D/l-cysteine functionalized chiral gold nanoparticles (D/P-Au NPs or L/P-Au NPs) have demonstrated a potent antibacterial effect against E. coli, while the mechanism remains to be elucidated through additional research. Threonine deaminase (TD) is a crucial enzyme involved in branched-chain amino acid (BCAA) biosynthesis in E. coli and is involved in cysteine's antimicrobial effects. This study investigated the interaction between chiral Au NPs (D/P-Au NPs or L/P-Au NPs) and TD as well as its effect on enzyme activity. It demonstrates that chiral Au NPs interact with TD through hydrophobic forces, forming a ground state complex that induces changes in the secondary structure of TD and reduces enzyme activity in a concentration-dependent manner. We found that the exogenous supplementation of isoleucine and valine (2 mg/mL) significantly reduced the antibacterial activity of chiral Au NPs, especially for L/P-Au NPs. The proteomics results indicate that the expression of ilvA and ilvB was down-regulated after L/P-Au NPs treatment, which would interfere with the synthesis of BCAAs. These results demonstrate that chiral Au NPs cause cell death of E. coli partly due to inhibition of TD enzyme activity and the synthesis of branched-chain amino acids.

Production of L-serine and its derivative L-cysteine from renewable feedstocks using Corynebacterium glutamicum: advances and perspectives.

L-serine and its derivative L-cysteine have broad industrial applications, and their direct fermentative production from renewable biomass is gaining increasing attention. Corynebacterium glutamicum is an extensively studied and well-established industrial microorganism, which is a predominant microbial host for producing amino acids. In this review, updated information on the genetics and molecular mechanisms underlying L-serine and L-cysteine production using C. glutamicum is presented, including their synthesis and degradation pathways, and other intracellular processes related to their production, as well as the mechanisms underlying substrate import and product export are also analyzed. Furthermore, metabolic strategies for strain improvement are systematically discussed, and conclusions and future perspectives for bio-based L-serine and L-cysteine production using C. glutamicum are presented. This review can provide a thorough understanding of L-serine and L-cysteine metabolic pathways to facilitate metabolic engineering modifications of C. glutamicum and development of more efficient industrial fermentation processes for L-serine and L-cysteine production.

Crystal to Hydrogel Transformation in S-Benzyl-L-Cysteine-Containing Cyclic Dipeptides - Nanostructure Elucidation and Applications.

Cyclic dipeptides (CDPs) are crucial building blocks for a range of functional nanomaterials due to their simple chemical structure and high molecular stability. In this investigation, we synthesized a set of S-benzyl-L-cysteine-based CDPs (designated as P1-P6) and thoroughly examined their self-assembly behavior in a methanol-water solvent to elucidate the relationship between their structure and gelation properties. The hydrophobicity of the amino acids within the CDPs was gradually increased. The present study employed a comprehensive array of analytical techniques, including NMR, FT-IR, AFM, thioflavin-T, congo-red CD, X-ray crystallography, and biophysical calculations like Hirshfield Surface analysis and DFT analysis. These methods revealed that in addition to hydrogen bonding, the hydrophobic nature of the amino acid side chain significantly influences the propensity of CDPs to form hydrogels. Each CDP yielded distinct nanofibrillar networks rich in ß-sheet structures, showcasing unique morphological features. Moreover, we explored the practical application of these CDP-based hydrogels in water purification by utilizing them to remove harmful organic dyes from contaminated water. This application underscores the potential of CDPs in addressing environmental challenges, offering a promising avenue for the future development of these materials in water treatment technologies.

A multifactorial study of in situ antioxidant activity of modified GrO in myocardial reperfusion injury using the Langerdorff model.

Carbon nanomaterials possess antioxidant properties that can be applied in biomedicine and clinics for the development of new highly effective treatments against oxidative stress-induced diseases like ischemic heart disease. We previously reported the usage of graphene oxide (GrO) as a precursor for the elaboration of such prototypes. The promising findings led to the development of two new modifications of GrO: nitrogen-doped (N-GrO) and l-cysteine functionalized (S-GrO) derivatives as possible antioxidant agents in ischemia-reperfusion (I/R) conditions. In this study, the cardioprotective and antioxidant potential of modified GrO as a pre-treatment in rats was evaluated for the first time. In Langendorff isolated rat heart I/R model, the left ventricle developed pressure (LVDP), the end-diastolic pressure (EDP), the maximal (dP/dtmax) and minimal (dP/dtmin) value of the first derivative of LVDP, and heart rate (HR) were measured. The oxidative-nitrosative markers, in particular, the rate of O2*- and H2O2 generation, the content of malonic dialdehyde, diene conjugates, and leukotriene as well as cNOS and iNOS activity were estimated. Obtained results show a significant restoration of cadiodynamic parameters at the reperfusion period. Simultaneously, all samples significantly reduced the rate of reactive oxygen species (ROS) and lipid peroxidation markers in cardiac homogenates and preserved cNOS activity at the preischemic level. This evidence makes GrO derivatives promising candidates for the correction of reperfusion disorders affecting myocardial function.

Nitric oxide and ion channels mediate L-cysteine-induced inhibition of colonic smooth muscle contraction.

Previous studies have suggested that L-cysteine regulates gut motility through hydrogen sulfide. However, the mechanisms involved in the L-cysteine-induced response have not been extensively studied. This study aimed to investigate the underlying mechanisms of action of L-cysteine on spontaneous contraction of rat colon. Longitudinal and circular muscle strips from rat middle colon were prepared to measure the spontaneous contractile activities of colon in an organ bath system. Whole-cell voltage-clamp techniques were applied to record the currents of L-type voltage-dependent Ca2+ channels (VDCCs) and voltage-gated K+ channels (Kv) in isolated smooth muscle cells (SMCs) from colon. L-cysteine inhibited the spontaneous contraction of longitudinal and circular muscle strips from the rat colon in a concentration-dependent manner. The inhibition induced by L-cysteine was significantly decreased by inhibitors of H2S synthesis (p < 0.05). Furthermore, the suppression induced by L-cysteine was partially attenuated by tetrodotoxin, L-NNA and glibenclamide (p < 0.05). Whole-cell voltage-clamp recordings showed that L-cysteine caused a remarkable reduction in the peak currents of VDCCs and significantly increased the membrane currents of Kv channels in isolated SMCs (p < 0.05). We concluded that L-cysteine inhibits the contractile activities of smooth muscle strips from the rat colon. The relaxation in response to L-cysteine may be in part mediated by a nitrergic pathway and by inhibiting the VDCCs in combination with a direct activation of the KV channels and KATP channels.

Impact of the Acetyl Group on Cysteine: A Study of N-Acetyl-Cysteine through Rotational Spectroscopy.

Herein, we report a spectroscopic study of N-acetyl-L-cysteine, an important antioxidant drug, using Fourier-transform microwave techniques and in isolated conditions. Two conformers are observed, where most stable structure adopts a cis disposition, and the second conformer has a lower abundance and adopts a trans disposition. The rotational constants and the barriers to methyl internal rotation are determined for each conformer, allowing a precise conformation identification. The results show that the cis form adopts an identical structure in the crystal, solution, and gas phases. Additionally, the structures are contrasted against those of cysteine.

Engineering of the Lrp/AsnC-type transcriptional regulator DecR as a genetically encoded biosensor for multilevel optimization of L-cysteine biosynthesis pathway in Escherichia coli.

L-cysteine is an important sulfur-containing amino acid being difficult to produce by microbial fermentation. Due to the lack of high-throughput screening methods, existing genetically engineered bacteria have been developed by simply optimizing the expression of L-cysteine-related genes one by one. To overcome this limitation, in this study, a biosensor-based approach for multilevel biosynthetic pathway optimization of L-cysteine from the DecR regulator variant of Escherichia coli was applied. Through protein engineering, we obtained the DecRN29Y/C81E/M90Q/M99E variant-based biosensor with improved specificity and an 8.71-fold increase in dynamic range. Using the developed biosensor, we performed high-throughput screening of the constructed promoter and RBS combination library, and successfully obtained the optimized strain, which resulted in a 6.29-fold increase in L-cysteine production. Molecular dynamics (MD) simulations and electrophoretic mobility shift analysis (EMSA) showed that the N29Y/C81E/M90Q/M99E variant had enhanced induction activity. This enhancement may be due to the increased binding of the variant to DNA in the presence of L-cysteine, which enhances transcriptional activation. Overall, our biosensor-based strategy provides a promising approach for optimizing biosynthetic pathways at multiple levels. The successful implementation of this strategy demonstrates its potential for screening improved recombinant strains.

Mitofilin in cardiovascular diseases: Insights into the pathogenesis and potential pharmacological interventions.

The impact of mitochondrial dysfunction on the pathogenesis of cardiovascular disease is increasing. However, the precise underlying mechanism remains unclear. Mitochondria produce cellular energy through oxidative phosphorylation while regulating calcium homeostasis, cellular respiration, and the production of biosynthetic chemicals. Nevertheless, problems related to cardiac energy metabolism, defective mitochondrial proteins, mitophagy, and structural changes in mitochondrial membranes can cause cardiovascular diseases via mitochondrial dysfunction. Mitofilin is a critical inner mitochondrial membrane protein that maintains cristae structure and facilitates protein transport while linking the inner mitochondrial membrane, outer mitochondrial membrane, and mitochondrial DNA transcription. Researchers believe that mitofilin may be a therapeutic target for treating cardiovascular diseases, particularly cardiac mitochondrial dysfunctions. In this review, we highlight current findings regarding the role of mitofilin in the pathogenesis of cardiovascular diseases and potential therapeutic compounds targeting mitofilin.

NiCo2O4@SnS2nanosheets on carbon cloth as efficient bi-functional material for high performance supercapacitor and sensor applications.

Bi-functional materials provide an opportunity for the development of high-performance devices. Up till now, bi-functional performance of NiCo2O4@SnS2nanosheets is rarely investigated. In this work, NiCo2O4@SnS2nanosheets were synthesized on carbon cloth by utilizing a simple hydrothermal technique. The developed electrode (NiCo2O4@SnS2/CC) was investigated for the detection of L-Cysteine and supercapacitors applications. As a non-enzymatic sensor, the electrode proved to be highly sensitive for the detection of L-cysteine. The electrode exhibits a reproducible sensitivity of 4645.82µA mM-1cm-2in a wide linear range from 0.5 to 5 mM with a low limit of detection (0.005µM). Moreover, the electrode shows an excellent selectivity and long-time stability. The high specific surface area, enhanced kinetics, good synergy and distinct architecture of NiCo2O4@SnS2nanosheets produce a large number of active sites with substantial energy storage potential. As a supercapacitor, the electrode exhibits improve capacitance of 655.7 F g-1at a current density of 2 A g-1as compare to NiCo2O4/CC (560 F g-1). Moreover, the electrode achieves 95.3% of its preliminary capacitance after 10 000 cycles at 2 A g-1. Our results show that NiCo2O4@SnS2/CC nanosheets possess binary features could be attractive electrode material for the development of non-enzymatic biosensors as well as supercapacitors.

On-Off-On Fluorometric Detection of Hg(II) and L-Cysteine Using Red Emissive Nitrogen-Doped Carbon Dots for Environmental and Clinical Sample Analysis.

This research introduces a novel fluorescence sensor 'on-off-on' employing nitrogen-doped carbon dots (N-CDs) with an 'on-off-on' mechanism for the selective and sensitive detection of Hg(II) and L-cysteine (L-Cys). N-CDs was synthesized using citric acid as the carbon precursor and urea as the nitrogen source in dimethylformamide (DMF) solvent, resulting in red emissive characteristics under UV light. Comprehensive spectroscopic analyses, including UV-Vis, fluorescence, FT-IR, XRD, XPS, Raman, and Zeta potential techniques, validated the structural and optical characteristics of the synthesized N-CDs. The maximum excitation and emission of N-CDs were observed at 548 and 622 nm, respectively. The quantum yield of N-CDs was calculated to be 16.1%. The fluorescence of N-CDs effectively quenches upon the addition of Hg(II) due to the strong coordination between Hg(II) and the surface functionalities of N-CDs. Conversely, upon the subsequent addition of L-Cys, the fluorescence of N-CDs was restored. This restoration can be attributed to the stronger affinity of the -SH group in L-Cys towards Hg(II) relative to the surface functionalities of N-CDs. This dual-mode response enabled the detection of Hg(II) and L-Cys with impressive detection limits of 15.1 nM and 8.0 nM, respectively. This sensor methodology effectively detects Hg(II) in lake water samples and L-Cys levels in human urine, with a recovery range between 99 and 101%. Furthermore, the N-CDs demonstrated excellent stability, high sensitivity, and selectivity, making them a promising fluorescence on-off-on probe for both environmental monitoring of Hg(II) and clinical diagnostics of L-Cys.

Continuous Detection of Cobalt Ions and Pyrophosphates by NAC-CdTe Quantum Dots Fluorescence Probes.

In this work, a simple and sensitive N-acetyl-L-cysteine (NAC)-covered CdTe quantum dots (NAC-CdTe QDs) fluorescence probe for continuous detection of Co2+ and pyrophosphate ions (PPi, P2O74-) was synthesized. The fluorescence of the quantum dots was significantly quenched by Co2+ through the coordination of Co2+ and the carboxyl groups on the NAC-CdTe quantum dots. Interestingly, the combination of NAC-CdTe quantum dots with Co2+ yields a new fluorescence probe of Co2+-modified NAC-CdTe QDs (Co2+@NAC-CdTe). The addition of PPi restored the fluorescence due to the competition between PPi and carboxyl groups with Co2+ causing Co2+ to detach from the surface of Co2+@NAC-CdTe quantum dots. Thus, a sensitive and reversible detection of Co2+ and PPi had been successfully established. The Co2+@NAC-CdTe quantum dots fluorescence probe exhibits excellent selectivity and high sensitivity toward PPi detection with low detection limit of 0.28 µM. In addition, the novel fluorescence probe was successfully applied to detect the concentration of PPi in environmental water samples and in-vitro cells imaging.

Association of 1-bromopropane exposure with asthma prevalence: A Korean National health and Nutritional examination survey (2020-2021)-based study.

Exposure to 1-bromopropane (1-BP) is an emerging environmental and health concern due to its increasing environmental prevalence. Although the health effects of 1-BP exposure have been under-recognized, current evidence suggests the possibility of adverse pulmonary health effects due to 1-BP exposure. However, the association between 1-BP exposure and asthma prevalence remains unclear. Thus, we aimed to examine the association between 1-BP exposure and asthma prevalence in the general population. Using nationally representative data, we explored the potential impacts of indoor air quality (IAQ)-related behavioral factors on the level of 1-BP exposure. This study included 1506 adults from the 2020-2021 Korea National Health and Nutrition Examination Survey. The prevalence of asthma was based on self-reported physician-diagnosed asthma. Urinary N-acetyl-S-(n-propyl)-L-cysteine (BPMA) levels were measured as a biomarker of 1-BP exposure, using high-performance liquid chromatography-mass spectrometry. Multiple logistic regression models were performed to investigate the associations between urinary BPMA metabolite and asthma prevalence after adjusting for potential confounders. Log-linear multiple regression models were used to examine the association between IAQ-related behavior and urinary BPMA concentration. Forty-seven individuals with asthma and 1459 without asthma were included. Individuals in the highest quartile of urinary BPMA concentration had a 2.9 times higher risk of asthma than those in the lowest quartile (odds ratio [OR]: 2.85, 95% confidence interval [CI]: 1.02-7.98). The combination of natural and mechanical ventilation was associated with a reduced urinary BPMA concentration. Our findings suggest that 1-BP exposure is associated with the prevalence of asthma in adults and revealed higher urinary levels of BPMA in our study population compared to those in other countries. Given the emerging importance of IAQ, actively managing and modifying behavioral patterns to reduce 1-BP exposure in indoor environments could substantially attenuate the risk of asthma-related to 1-BP exposure.

Metabolic control analysis enabled the improvement of the L-cysteine production process with Escherichia coli.

L-cysteine is an amino acid with relevance to the pharmaceutical, food, feed, and cosmetic industry. The environmental and societal impact of its chemical production has led to the development of more sustainable fermentative L-cysteine production processes with engineered E. coli based on glucose and thiosulfate as sulphur source. Still, most of the published processes show low yields. For the identification of further metabolic engineering targets, engineered E. coli cells were withdrawn from a fed-batch production process, followed by in vivo metabolic control analysis (MCA) based on the data of short-term perturbation experiments, metabolomics (LC-MS), and thermodynamic flux analysis (TFA). In vivo MCA indicated that the activities of the L-cysteine synthases of the cells withdrawn from the production process might be limiting, and we hypothesised that the L-cysteine precursor O-acetylserine (OAS) might be exported from the cells faster than it took to transform OAS into L-cysteine. By increasing the expression of the L-cysteine synthases, either sulfocysteine synthase or L-cysteine synthase, which transform OAS into L-cysteine, an improvement of up to 70% in specific L-cysteine productivity and up to 47% in the final L-cysteine concentration was achieved in standardised fed-batch processes thereby increasing the yield on glucose by more than 85 to 9.2% (w/w). KEY POINTS: • Metabolic control analysis was applied to analyse L-cysteine production with E. coli • OAS export was faster than its transformation to L-cysteine • Overexpression of L-cysteine synthases improved L-cysteine productivity and yield.

Phenylephrine Enhances the Mitogenic Effect of S-Allyl-L-cysteine on Primary Cultured Hepatocytes through Protein Kinase C-Induced B-Raf Phosphorylation.

The co-mitogenic effects of the α1-adrenoceptor agonist phenylephrine on S-allyl-L-cysteine (SAC)-induced hepatocyte proliferation were examined in primary cultures of adult rat hepatocytes. The combination of phenylephrine (10-10-10-6 M) and SAC (10-6 M) exhibited a significant dose-dependent increase in the number of hepatocyte nuclei and viable cells compared to SAC alone. This combination also increased the progression of hepatocyte nuclei into the S-phase. The potentiating effect of phenylephrine on SAC-induced cell proliferation was counteracted by prazosin (an α1-adrenergic receptor antagonist) and GF109203X (selective protein kinase C (PKC) inhibitor). In addition, PMA (direct PKC activator) potentiated the proliferative effects of SAC similarly to phenylephrine. In essence, these findings suggest that PKC activity plays a crucial role in enhancing SAC-induced cell proliferation. Moreover, the effects of phenylephrine on SAC-induced Ras activity, Raf phosphorylation, and extracellular signal-regulated kinase 2 (ERK2) phosphorylation were investigated. Phenylephrine (or PMA) in combination with SAC did not augment Ras activity, but further increased ERK2 phosphorylation and its upstream B-Raf phosphorylation. These results indicate that PKC activation, triggered by stimulating adrenergic α1 receptors, further amplifies SAC-induced cell proliferation through enhanced ERK2 phosphorylation via increased B-Raf-specific phosphorylation in primary cultured hepatocytes.

Borderline Range Determined Using Data From Validation Study of Alternative Methods for Skin Sensitization: ADRA, IL-8 Luc Assay, and EpiSensA.

Most predictive models that use alternatives to animal experiments divide judgements into two classes with a cutoff value for each model. However, if the results of alternative methods are close to the cutoff values, the true result may be ambiguous because of variability in the data. Therefore, the OECD GL497 uses a judgement method that establishes a borderline range (BR) around a cutoff value using a statistical method. However, because there is no detailed description of how the BR is calculated, we clarified two specific points. The scale-constant correction method was used to calculate the median absolute deviation (MAD) around the median. In addition, the bottom-raised transformation method was used when the data were "0" because calculation of the BR requires that all data are logarithmic. Indeed, the BRs for the amino acid derivative reactivity assay (ADRA), interleukin-8 reporter gene assay (IL-8 Luc), and epidermal sensitization assay (EpiSensA) were calculated using data from each validation study. The results showed that the BR for ADRA and IL-8 Luc ranged from 4.1 to 5.9 and 1.25 to 1.57, respectively. Furthermore, the BRs of four genes (ATF3, GCLM, DNAJB4, and IL-8) evaluated using EpiSensA ranged from 10.71 to 21.02, 1.64 to 2.45, 1.61 to 2.52, and 3.11 to 5.16, respectively. The difference (deviation) between the lower and upper BR limits and cutoff value for each alternative method were comparable to those of the alternative methods listed in the guidelines (DPRA, KerarinoSens, and h-CLAT) and thus were considered as adequate.

The effect of L-cysteine on starch and protein degradation during barley germination.

OBJECTIVES: In order to investigate the impact of L-cysteine (L-Cys) on starch and protein degradation during barley germination. The amylase activities, degradation of macromolecules during germination were determined in this study. METHODS: Barley was germinated in petri dish for 0 to 5 days with different levels of L-Cys (0 mM, 2.5 mM, 5 mM, 10 mM). RESULTS: L-Cys addition increased the total limit dextrinase (LD) activities and decreased the LD inhibitor activities during whole germination stage. The activities of α-amylase, ß-amylase and free LD were increased with the addition of 2.5, 5 mM L-Cys at germination days 1 to 4. Due to higher amylase in malt with the addition of L-Cys, the non-fermentable sugars were reduced and the glucose, maltotriose were improved. Furthermore, the protein degradation analysis showed that low molecular weight protein increased and middle molecular weight protein decreased obviously in wort from the malt germinated with L-Cys, demonstrating that the L-Cys promote the protein degradation. Lastly, the filtration performance of malt with the addition of L-Cys during malting was better than the control. CONCLUSION: In conclusion, L-Cys can promote the degradation of storage material (starch, protein) during barley germination, leading to a better green malt quality.