bluuebunny/crossref_metadata_2025_split huggingface dataset:
Biological Activity of Resveratrol‐Hydroxycinnamic Acid Ester Conjugates
Authors: Urbaniak Alicja Joanna , Kacprzak Karol , Senol Fatma Sezer , Orhan Ilkay Erdogan , Radominska‐Pandya Anna 
Published: November 2023

Resveratrol (3,5,4′‐trihydroxy‐trans‐stilbene, tRV) is one of the most biologically active polyphenols of natural origin. It exhibits many beneficial properties namely antioxidant, anti‐inflammatory, cancer preventing, and cardio‐protective. Despite that, the wide application of tRV, is limited by its poor water solubility, low stability, and consequently, weak bioavailability when administered orally. To overcome this deficiency, our group has developed novel resveratrol‐based derivatives by conjugation with other bioactive compounds such as hydroxycinnamic acid derivatives (p‐, m‐coumaric, ferulic and sinapinic). It is anticipated that conjugation of tRV will result in derivatives with lowered metabolism, higher stability, and altered molecular targets. The developed synthetic method is a four step procedure and leads to the high‐yield production of compounds with high purity. In this work, tRV esters and their unconjugated counterparts were tested in vitro for their biological activity. Experiments were carried out to check their potency to inhibit cholinoesterases, tyrozynaze, elastaze, and collagenase; moreover, their ability as antioxidants and metal chelators were investigated. Resveratryl tri‐p‐coumarate, resveratryl tri‐m‐coumarate, and resveratryl triferulate express significant inhibitory activity towards butyrylcholinesterase, IC50= 29.57 ± 3.33 μg/ml, IC50=32.40±1.29 μg/ml, IC50=36.89 ± 1.05 μg/ml respectively. These values were similar to the IC50 values of reference compound galanthamine HBr. Inhibitory activity towards acetylcholinesterase were slightly lower. tRV conjugates with p‐coumaric and ferulic acid were the most potent in elastase inhibition assay; however, the tyrosynase inhibition study showed more of an effect by resveratryl tri‐p‐coumarate and resveratryl trisinapinate. The unconjugated tRV exhibited the strongest collagenase inhibiting property. tRV ester with sinapinic acid exhibited the highest metal chelating activity. Unconjugated tRV, sinapinic, and ferulic acids exhibited the highest antioxidant activity in both 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) and Ferric‐Reducing Antioxidant Power (FRAP) assays. The properties of free radical scavengers were diminished with the conjugated compounds; however, synthetic esters of those compounds may still be considered as good free radical scavengers.Support or Funding Information[NIH/NIDA DA039143 to ARP, Kosciuszko Foundation's Exchange Program to the US to AU]
The ProS Peptide of Porcine Cathelicidins Is an Active Component of Swine Concentrated Animal Feeding Operations (CAFOs) Dust that Mobilizes Calcium and Stimulates Proliferation in Human Airway Smooth Muscle Cells
Authors: Toews Myron L. , Schulte Nancy A. , Tu Yaping , Panettieri Reynold A. , Romberger Debra J. , Tian Geng 
Published: November 2023

Components in the dust from swine concentrated animal feeding operations (CAFOs) can induce inflammatory lung disease with features of chronic obstructive lung disease. An aqueous hogbarn dust extract (HDE) induces pro‐inflammatory responses in both airway cells in culture and in mouse airways in vivo, thus providing a starting point for identifying the dust molecules that are involved. Proteomics analysis of an EGF receptor‐activating peak from gel filtration of HDE revealed multiple fragments from the ProS peptide precursor of a subset of porcine anti‐bacterial cathelicidins. Antibodies against ProS confirmed its presence in HDE. The full‐length ProS peptide, without the N‐terminal signal peptide and the C‐terminal anti‐bacterial peptide, stimulated proliferation of human airway smooth muscle (HASM) cells assessed as [3H]thymidine incorporation. Stimulation was concentration‐dependent, with a maximal stimulation of 2‐ to 3‐fold, greater than that with EGF and somewhat less than with the G protein‐coupled receptor (GPCR) mitogen lysophosphatidic acid (LPA). ProS stimulation was blocked by the EGF receptor inhibitor AG1478 and the phospholipase C inhibitor U73122, but not by the Gq inhibitor YM‐254890 or the matrix metalloprotease inhibitor GM6001. ProS also stimulated a concentration‐dependent increase in intracellular calcium that was not blocked by AG1478 but was blocked by U73122 and by the Gq inhibitor YM‐254890. These data indicate that ProS is likely to be a novel Gq‐coupled GPCR agonist that mobilizes calcium in HASM cells via Gq activation but stimulates HASM cell proliferation through a Gq‐independent pathway involving EGF receptor activation. Whether and how the phospholipase C‐dependent calcium response may be related to the phospholipase C‐dependent proliferation response remains to be established. Further studies are in progress to identify the specific receptor and signaling pathways involved. The simultaneous formation of both an anti‐bacterial peptide to fight infection and the ProS peptide to activate EGF receptors to initiate cell proliferation and repair suggests a unique dual role for the cathelicidin precursor protein.Support or Funding InformationFunded by NIOSH 1RO1‐0H008539‐09 to DJR and UNMC Institutional Funds to MLT
Investigation of Pyrrolizidine Alkaloid‐Induced Pulmonary Toxicity
Authors: Lian Wei , Ma Jiang , Wong Chi‐Wai , Zhu Lin , Lin Ge 
Published: November 2023

IntroductionPyrrolizidine alkaloids (PAs) are widely present in plants, and about half of them had been reported to induce toxicity in different organs. The previous study has proved that PAs exert their toxicity by metabolic activation to form dehydro‐PAs (DHPAs), which interact with proteins to form pyrrole‐protein adducts leading to toxicity. Monocrotaline (MCT) and retrorsine (RTS) are two representative toxic PAs. MCT is known to cause pneumotoxicity, while other PAs, including RTS, induced pneumotoxicity is unknown. The present study aimed to investigate whether other PAs can also cause pneumotoxicity and the mechanism initiating PA‐induced pneumotoxicity.MethodsMCT and RTS in rat liver and lung microsomal incubation were tested to compare the metabolic site of these two PAs. Then, the transport of DHPAs in blood after a single dose of MCT or RTS in rats was studied by using LC‐MS/MS analysis. DHPAs in blood fractions were detected by addition of glutathione (GSH) to form pyrrole‐GSH conjugate, and the addition of saline was used as the control. The biomarker of PA‐induced toxicity, pyrrole‐protein adducts in liver and lung of MCT or RTS treated rats were also determined. The histological study was performed to examine RTS‐induced pneumotoxicity in rats.ResultsThe microsomal incubation results showed that DHPAs were primarily generated in liver. Comparing with the control group, significant increase in the formation of pyrrole‐GSH conjugate was found in GSH‐added red blood cells (RBC) and plasma of both PA‐treated rats. Further, hepatic pyrrole‐protein adducts level in RTS‐treated rats was found about 3‐folds of that in MCT‐treated rats, while pyrrole‐protein adducts levels in lung were similar in both PA‐treated groups. Mild to severe vascular medial hypertrophy was observed along with the time after RTS treatment from 2 days to 4 weeks.These findings proved that RTS could cause pneumotoxicity. Furthermore, it provided, for the first time, that for both PAs, 1) DHPAs were present in the blood circulation; 2) DHPAs were transported by not only RBC as demonstrated previously but also by plasma; and 3) the transport of DHPAs by both RBC and plasma may be common for different toxic PAs rather than solely for MCT.ConclusionsOur study delineated the mechanism initiating PA‐induced pneumotoxicity via migration of DHPAs in blood circulation, and also revealed a potential risk of PA‐induced pulmonary toxicity.Support or Funding InformationThis study was supported by RGC HKSAR (GRF Project No. 469712 and 14111816), CUHK Direct Grant (No. 4054215 and 4054302) and CUHK One‐off Funding for Joint Lab/Research Collaboration (Project Code: 3132968)
Downregulation of intestinal P450 enzymes in a mouse model of ulcerative colitis and the impact on P450‐mediated drug metabolism
Authors: Fan Xiaoyu , Ding Xinxin , Zhang Qing‐Yu 
Published: November 2023

Intestinal cytochrome P450 enzymes (P450s) play an essential role in the first‐pass metabolism of orally administered drugs and other xenobiotics. The expression of intestinal P450s can be regulated by many environmental or pathophysiological factors, including tissue inflammation. In the present study, we have explored the impact of inflammation on the expression of intestinal P450s in a mouse model of dextran sulfate sodium (DSS)‐induced colitis. Several P450 enzymes, including CYP1A1, CYP2B, CYP2C, and CYP3A, were downregulated at both mRNA and protein levels in mice treated with DSS (2.5% in drink water for 7 days), along with increased cytokine levels in colon epithelium. The impact of this downregulation on P450‐mediated drug metabolism was also studied. In addition, the expression of several nuclear receptors that are potentially involved in the regulation of intestinal P450 expression was compared between control and DSS‐treated groups. Whereas no difference was detected in the expression of PXR, CAR, or FXR, the expression of several FXR target genes was significantly decreased by the DSS treatment. Interestingly, the expression of VDR and its target genes was also suppressed by the DSS treatment. Thus, both FXR and VDR may be involved in the downregulation of intestinal P450s in DSS‐induced colitis. Further studies on the mechanism of P450 downregulation in the intestine by DSS‐induced colitis are warranted.Support or Funding InformationSupported in part by NIH grant GM082978
Transcriptional Regulation of Disease‐Drug Interactions
Authors: Xie Wen 
Published: November 2023

Drug metabolism and disposition are critical in maintaining the chemical and functional homeostasis of xenobiotics and endobiotics. Accumulating evidence suggests that many hepatic and systemic diseases can affect drug metabolism and disposition by regulating the expression and/or activity of DMEs and transporters in the liver. This presentation will focus on the recent progress in describing and understanding the hepatic injury and sepsis responsive regulation of DMEs in animal models. Liver ischemia and reperfusion (I/R) will be used as a typical example of a hepatic injury model, whereas lipopolysaccharides (LPS) and cecal ligation and puncture (CLP) will be used as the sepsis models. Our results showed that liver I/R and sepsis can affect liver metabolism through the transcriptional regulation of DMEs in the liver. In the meantime, the expression and/or activity of DMEs can affect the disease manifestations of I/R and sepsis. It is hoped that understanding the disease effect on drug metabolism will facilitate the efficient and safe use of drugs in the clinic.Support or Funding InformationThis work is supported in part by the NIH grant ES023438.
Comparison of expression profiles of drug‐metabolizing enzymes between human liver and the hepatic cell lines HepG2, Hep3B, and HuH7: a parallel reaction monitoring (PRM)‐based targeted proteomics study
Authors: Shi Jian , Wang Xinwen , Zhu Hao‐Jie 
Published: November 2023

BackgroundSeveral human hepatic cell lines have been widely used as an in vitro model for drug metabolism related studies because of their availability and immortalization. However, the validity of this model is a subject of debate as the expression patterns of drug‐metabolizing enzymes (DMEs) in these cells could differ significantly from that of human livers. In the present study, we systematically quantified the basal protein expression of 61 clinically important DMEs in the human liver‐derived cell lines HepG2, Hep3B, and HuH7, and compared the expression profiles to that in human livers.MethodsWith high resolution and high mass accuracy, parallel reaction monitoring (PRM) is a new powerful quantitative proteomics tool, providing wider dynamic range and higher selectivity than selected reaction monitoring (SRM). A PRM‐based targeted proteomics assay was developed for relative quantification of DMEs in the microsomes prepared from human livers and the hepatic cell lines HepG2, Hep3B, and HuH7. The 45 targeted enzymes include 14 cytochromes P450 enzymes (CYPs), 9 UDP‐glucuronosyltransferases (UGTs), and 38 other enzymes relevant to clinical drug metabolism.ResultsWith the exception of aldehyde dehydrogenase 1 family member A1 (ALDH1A1), glutathione s‐transferase omega‐1 (GSTO1), and catechol‐O‐methyltransferase (COMT), most of the DMEs are expressed at a substantially lower level relative to that in human livers. In addition, the expression levels of different enzymes vary markedly among the three hepatic cell lines.ConclusionsProtein expression profiles of DMEs differ significantly between human livers and the hepatic cell lines HepG2, Hep3B, and HuH7. Appropriate caution must be exercised when considering these cell lines for drug metabolism studies, and interpreting the data from the studies based on these cell lines.
Physical Interactions of Cytochrome b5 with Human Cytochrome P450 Enzymes
Authors: Bart Aaron G , Scott Emily E 
Published: November 2023

Human cytochrome P450 enzymes metabolize drugs, detoxify environmental compounds, and synthesize many endogenous biomolecules. Binding of the small heme protein cytochrome b5 (b5) can increase, inhibit, or have no effect on P450 catalysis, depending on the specific P450 and/or substrate. Although some evidence points to allosteric modulation of P450 conformations, it is unclear how b5 produces disparate outcomes for different P450 enzymes. Solution NMR was used as a high‐resolution approach to systematically map the surfaces of b5 interacting with several different human P450 enzymes and ascertain the relative affinities of these protein/protein interactions.NMR analysis of b5 interactions with the xenobiotic‐metabolizing cytochrome P450 enzymes CYP2A6, CYP2D6, and CYP3A4 suggests they interact with varying b5 surfaces. Addition of CYP2A6 caused dramatic differential line‐broadening of resonances for b5 residues in alpha helices 3 and 4, while titrating with CYP3A4 resulted in differential broadening corresponding to residues in alpha helices 2 and 4. In contrast, titrations with CYP2D6 caused minimal line‐broadening of b5 resonances, suggesting a weaker interaction. Only when an excess of CYP2D6 was present were line‐broadening effects observed, primarily for residues in b5 alpha helix 4. To evaluate the importance of these different b5 surfaces in complex formation, key anionic b5 residues were mutated. Consistent with the above observations, the D65N mutation in b5 helix 3 prevented formation of the b5/CYP2A6 complex, but not the b5/CYP3A4 complex. Conversely, the E49Q mutation in b5 alpha helix 2 significantly reduced formation of the b5/CYP3A4 complex, but not the b5/CYP2A6 complex. Ongoing studies include additional human P450 enzymes, but the data already in hand demonstrate that three different P450 enzymes interact in distinct ways with cytochrome b5. These varying P450/b5 interaction modes and relative affinities may underlie or contribute to the differential effects b5 has on P450 catalysis. Thus, functional studies are used to validate the relevance of these P450/b5 interactions using parallel evaluation of substrate oxidation.Overall, this study provides new insights on protein/protein interactions that modulate the function of a clinically important enzyme superfamily.Support or Funding InformationNational Institutes of Health R37 GM076343, T32 GM08545 Dynamic Aspects of Chemical Biology
Tissue Specific Regulation of CYP26 Enzymes
Authors: Tripathy Sasmita , Isoherranen Nina 
Published: November 2023

all‐trans‐retinoic acid (atRA) regulates many biological processes via RAR and PPAR mediated pathways. The metabolism and clearance of atRA is mediated by the CYP26A1 and CYP26B1 enzymes, that both appear to be critical retinoic acid hydroxylases during fetal development and adult life contributing to tissue specific regulation of atRA concentrations and signaling. CYP26A1 and CYP26B1 have different tissue specific expression both in adults and during development, and distinct switches occur between fetal expression patterns and adult tissues for both CYP26A1 and CYP26B1. In human fetal tissues CYP26A1 is exclusively expressed in brain, whereas CYP26B1 is not present in brain but found in other tissues. In adults, CYP26A1 is predominantly expressed in liver, whereas CYP26B1 is predominantly found in cerebellum and ubiquitously expressed in many other tissues such as adipose, testes, lymphocytes and kidney. While both CYP26A1 and CYP26B1 expression is believed to be regulated by atRA, the mechanisms that lead to distinct tissue specific expression and regulation of the CYP expression are not known. The aim of this study was to examine the tissue specific regulation of CYP26A1 and CYP26B1 by RAR and PPAR using various models of human liver, adipose and immune cells that reflect tissues specifically expressing CYP26A1 or CYP26B1. Our data revealed that constitutively CYP26A1 is predominantly expressed in liver cells, whereas CYP26B1 is found in adipocytes and lymphocytes. Similarly, CYP26A1 was highly inducible by atRA in liver cells while CYP26B1 was induced in adipocytes and lymphocytes. siRNA mediated knock down of nuclear receptors showed that CYP26A1 mRNA expression is solely regulated by RARα/β, whereas CYP26B1 expression is mainly regulated by PPARγ. These data suggest that the tissue specific expression of CYP26 enzymes is regulated by different nuclear receptors acting on the CYP26 promoter and that the two CYP26 enzymes will respond differently to endogenous and xenobiotic regulators of CYP expression. In conclusion, these findings may explain the different phenotypes observed after loss of CYP26A1 or CYP26B1 activity.Support or Funding InformationFunding: NIH R01 GM111772
Physical complexes formed by CYP1A2 and CYP2B4 are associated with altered P450 activity
Authors: Connick John Patrick , Reed James Robert , Backes Wayne L 
Published: November 2023

It has been demonstrated that the presence of one P450 can affect the activity of another. Previous studies have shown, for example, that when CYP1A2, CYP2B4, and stoichiometrically‐limiting concentrations of NADPH‐cytochrome P450 reductase (CPR) were incorporated into phospholipid vesicles, CYP1A2 activity was higher and CYP2B4 activity was lower than observed in single‐P450 systems. These data are inconsistent with a model of simple competition among P450s for CPR binding but could be caused by physical interactions among the pair of P450s. The goal of this work was to determine how physical interactions between CYP1A2 and CYP2B4 might play a role in modulating the P450‐CPR interaction and P450 activity. Various pairs of P450 and CPR constructs were tagged with one each of GFP and Renilla luciferase and transfected into HEK293T cells. Complexes between these proteins were detected by measuring energy transfer between the tags. A complex between CYP1A2 and CYP2B4 was detected both in the presence and absence of cotransfected, untagged CPR. Interactions of both P450s with CPR were detected, and untagged CYP1A2 was shown to disrupt the CPR‐CYP2B4 interaction while untagged CYP2B4 had no effect on the CPR‐CYP1A2 interaction. These data are consistent with a model in which CPR preferentially binds to the CYP1A2 moiety of a CYP1A2‐CYP2B4 complex. Previous work has also shown—using BRET, crosslinking, and kinetic measurements—that CYP1A2 forms homomeric complexes associated with inhibition of CYP1A2 activity at subsaturating CPR. Here it is shown using BRET that these complexes were disrupted by the presence of either CPR or CYP2B4. The activity of GFP‐tagged CYP1A2 and CYP2B4 co‐expressed in HEK293T cells was measured and shown to be consistent with data from the prior experiments on purified proteins. These findings explain the observed effects of CYP1A2 and CYP2B4 on each other's activity.Support or Funding InformationNIH ES004344NIH ES013648Louisiana Board of Regents
Induction of Apoptosis and Generation of Reactive Oxygen Species by Novel Chalcone Derivatives
Authors: Stang Christopher R.T. , Adams Bryce R. , Faler Ashton D. , Schuler Brian R. , Eichel Amanda M. , Dick Matt J. , Orahood Cassandra B. , Whited Tawna L. , Rao P.S.S. , Dudley Richard W. , Khupse Rahul S. , Schneider Ryan A. 
Published: November 2023

The presence of alpha‐beta unsaturated systems in naturally occurring flavonoids has been attributed to a diverse array of physiological activities. We previously reported on the anti‐cancer activity of several chalcone and flavanone derivatives from our small flavonoid library. Here we present the anti‐cancer effects of our two newest chalcone derivatives, RK15 and RK19, in four diverse human cancer cell lines: A‐172 glioblastoma, A375 melanoma, HT‐29 colon carcinoma, and MCF7 breast adenocarcinoma. The XTT cell proliferation assay revealed that RK15 was the most potent chalcone derivative in our library with IC50 values of 3, 4, and 6 μM respectively in A375, A‐172, and HT‐29 cells. Interestingly, RK15 was much less potent in MCF7 cells, with an IC50 of 85 μM. The second chalcone derivative, RK19, was much less potent than RK15, with IC50 values ranging from 56 to 95 μM in these four cell lines.We investigated the mechanism of cell death elicited by these compounds using the Caspase‐Glo 3/7 assay. RK19 induced executioner caspase‐3/7 activity in A‐172, A375, and HT‐29 cells. An increase in caspase‐3/7 activity was also noted following RK15 treatment in A‐172 cells but interestingly not in HT‐29 cells. A reversal experiment using the pan‐caspase inhibitor Z‐VAD‐FMK was utilized to confirm the results from the Caspase‐Glo 3/7 assay. Since others have shown that chalcones can generate reactive oxygen species (ROS), we hypothesized that RK15 may lead to apoptosis through ROS generation. A DCFDA plate‐based assay was utilized to measure ROS generation following RK15 treatment. RK15 modestly increased ROS levels in A375 and HT‐29 cells, but not in A‐172 cells. These results suggest that though RK15 may increase ROS in some instances, an alternate mechanism of action is likely involved in the observed apoptotic effect.