bluuebunny/crossref_metadata_2025 huggingface dataset:
Contractions Of The SHR Aorta To High Doses Of Epigallocatechin Gallate Are Due To Vasoconstrictor Prostanoids
Authors: LI ZHUOMING , KOO MARCEL W.L. , MAN RICKY Y.K. , VANHOUTTE PAUL M.
Published: June 2021

Objective

Regular consumption of green tea is believed to be beneficial for the cardiovascular system. However, high concentrations of epigallocatechin gallate (EGCG), a catechin of green tea, cause contractions of the aorta of spontaneously hypertensive rats (SHR). The present studies were designed to investigate the mechanisms underlying these EGCG‐induced contractions.

Methods

Isometric tension was measured in isolated aortic rings from 36‐weeks‐old male SHR. The release of prostanoids end‐products was measured with enzyme immunoassay kits. The intracellular reactive oxygen species (ROS) concentration was measured by confocal microscopy.

Results

From 10−6 to 10−4 M, EGCG induced concentration‐dependent contractions in preparations with and without endothelium. These contractions were abolished by cyclooxygenase (COX) inhibitor indomethacin or the thromboxane‐prostanoid (TP) receptor antagonist S18886, and reduced by antioxidants. The release of prostaglandin F1α, prostaglandin F2α and thromboxane B2 was significantly increased by 10−4 M EGCG. A burst of ROS was observed upon exposure to 10−4 M of EGCG.

Conclusions

The EGCG‐induced contractions involve the production of ROS, activation of COX, production of prostanoids and stimulation of TP receptors. This study indicates a possible adverse effect of EGCG administration and tea consumption at high concentrations.
Immunohistochemical studies of Vascular endothelial growth factor and tumor suppressor protein P53 in mammary tumor of mice under the effect of green tea
Authors: Amin Afaf Mosad Amin
Published: June 2021

This work aimed to study the effect of green tea extract on transplanted mammary tumor in Swiss mice through studying VEGF & P53. Male Swiss mice weighing 25–30 g were used in this work. They were divided into 3 equal groups 12 animals in each group. The first group was injected i.m with 1/106 MT cells/0.1 ml saline solution/mouse for 2 weeks. The second group was injected with tumor cells as in the 1st group and kept on 5mg green tea extract in 0.2ml dist .water/mouse via gavage for 2 weeks. The third group received 5mg GT extract in 0.2 ml distilled water/mouse daily for two weeks before tumor injection. Six animals from each group were sacrificed after 1 and 2 weeks from tumor injection. The tumor was measured by a caliper and its size was calculated using the formula: 3/4Π r3. The tumor specimens were processed for immunohistochemical studies of VEGF and P53.

The results of the present study showed that the angiogenic growth factor VEGF, the key signal used by oxygen deprived cells promote the growth of blood vessels which increase the ability of the mammary tumor to grow. Our observations have raised the possibility that animals received GT extract before tumor injection showed remarkable reduction in the size of the tumor. As regards P53, a tumor suppressor protein. Immunohistochemical stained sections of MT using monoclonal P53 antibody revealed its low expression in tumor cells and its over expression after application of GT extract suggesting its important role to arrest the cell cycle. This ceasing of the cell cycle forced the cell to self destruction by inducing apoptosis through p53 dependent pathway.
In Utero Exposure to Nicotine Reduces PKC epsilon Gene Expression in the Fetal Rat Heart
Authors: Lawrence Jennifer C. A. , Xiao Daliao , Zhang Lubo
Published: June 2021

Previous studies have demonstrated that fetal nicotine exposure reduces cardiac PKCε protein expression and increases heart susceptibility to ischemia/reperfusion injury in adult offspring. The present study hypothesizes that in utero exposure to nicotine increases methylation of the PKCε gene promotor and reduces PKCε expression in the fetal heart. Pregnant Sprague‐Dawley rats were treated with nicotine and saline as a control beginning on day four of gestation via subcutaneous minipumps. On day 21 of gestation the fetuses were removed and the fetal hearts isolated. The nicotine treatment signifciantly decreased the abundance of PKCε protein (control: 1.07±0.12; Nicotine: 0.56±0.11) and mRNA (Control: 0.04± 0.00; Nicotine: 0.02± 0.00). Previously eight transcription factor biniding sites have been identified in the promotor region of the PKCε gene, which contain CpG sites (Stra13, PPARG, EGR1, E2F, MTF1 at 1398, SP1 at 1655, SP1 at 1733, and MTF1 at 1833). DNA was isolated from the fetal heart and treated with sodium bisulfite for methylation studies. The levels of methylation of each transcription factor binding site in the PKCε promotor region were determined by quantitative PCR following the bisulfite treatment. Fetal nicotine exposure significantly increased CpG methylation at both the EGR1 and MTF1 (−1398) binding sites in the PKCε promotor in the fetal heart. The results demonstrate that exposure to nicotine in utero causes epigentic modification of DNA methylation in the PKCε promotor resulting in PKCε gene repression in the developing heart and subsequent pathophysiological changes in cardiac function in offspring. (supported in part by NIH grant HL82779).
Amelioration By Tempol Of The Hypertensive And Baroreflex Depressant Effects Of Cyclosporine In Conscious Rats: Role Of Cardiac Autonomic Control
Authors: El‐Mas Mahmoud M , Omar Amal G , Helmy Mai M , Mohy El‐Din Mahmoud M
Published: June 2021

Previous studies including ours showed that cyclosporine (CSA) causes hypertension and baroreflex dysfunction. This study tested the hypothesis that increased oxidative damage mediates CSA effects. Experiments were undertaken in rats to investigate the effects of individual and combined 7‐day treatments with CSA (25 mg/kg/day) and tempol (superoxide dismutase mimetic, 100 mg/kg/day) on BP, reflex HR responses to peripherally mediated pressor and depressor responses, and biomarkers of oxidative stress. The role of cardiac autonomic control in cyclosporine‐tempol interaction was also investigated. CSA elevates blood pressure and reduced reflex bradycardic (phenylephrine) and tachycardic (sodium nitroptrusside) responses. The ability of muscarinic (atropine) or β‐adrenoceptors (propranolol) blockade to reduce reflex HR responses was reduced in CSA‐treated rats, suggesting impairment by CSA of cardiac autonomic control. Concurrent administration of tempol abolished CSA‐induced hypertension, baroreflex dysfunction, and autonomic impairment. Tempol also reversed the CSA‐induced increases in aortic and brainstem nitrite/nitrate and malondialdehyde and decreases in aortic superoxide dismutase. These findings implicate oxidative stress, at peripheral and central sites, in CSA‐induced hypertension and baroreflex dysfunction.
Orphanin FQ/Nociceptin Activates Nuclear Factor Kappa B In Neuronal Cells
Authors: Donica Courtney L , Awwad Hibah O , Wu Huaqing , Standifer Kelly M
Published: June 2021

Endogenous neuropeptide orphanin FQ/nociceptin (OFQ/N) and its receptor, opioid receptor like‐1 (ORL1; or Nociceptin orphanin peptide receptor, NOP), have been shown to play a modulatory role throughout the body on nociceptive sensitivity, including motor function, spatial learning, smooth muscle tone, cardiac function and the immune system. ORL1 is a G protein coupled receptor (GPCR) that couples to Gi/o proteins and modulates expression and release of inflammatory mediators from immune cells and in the CNS. Inhibitory GPCRs have been shown to activate the immune system regulator, NFκB. The NFκB family consists of several subunits, including p105 and p50. When activated, NFκB translocates to the nucleus and can modify transcription. To determine if OFQ/N modulates NFκB activity, SH‐SY5Y human neuroblastoma cells were treated with OFQ/N and assessed for changes in nuclear accumulation, DNA binding and protein expression. OFQ/N increases the nuclear accumulation of NFκB at 30 min and increases the DNA binding of NFκB by 1 hr as determined by electromobility shift assay (EMSA). Additionally, immunoblot analysis of SH‐SY5Y cell lysates indicates that NFκB p50 protein expression is up‐regulated by 2 hr. This suggests that OFQ/N may modulate immune system function by activating NFκB. These studies were supported by DA017380 and OCAST grant HR08‐152 to KMS.
Atrial Natriuretic Peptide (ANP) Activates Pancreatic Islet Phosphatidylinositol‐3′‐Kinase (PI3K)/Akt/FoxO1a/Cyclin D2 Signaling
Authors: You Hui , Laychock Suzanne G
Published: June 2021

A link has been made between ANP and diabetes mellitus since hyperglycemia and diabetes are associated with elevated ANP levels. Natriuretic peptide receptor (NPR)‐A and NPR‐B mRNAs were detected in insulin secreting INS‐1E insulinoma cells and isolated rat pancreatic islets, but only NPR‐A protein was expressed on islet α‐and β‐cells. Culture of INS‐1E cells and islets with ANP (0.01–1 μM) increased cell proliferation, DNA biosynthesis and cyclin D2 mRNA levels. An analogue of cGMP, the second messenger of NPR‐A, also elevated DNA biosynthesis and cyclin D2 mRNA levels. Enhanced DNA biosynthesis and cyclin D2 mRNA induced by ANP were inhibited by LY294002, an inhibitor of PI3K. ANP and 8‐bromo‐cGMP also stimulated the phosphorylation of Akt (387±10% of control (P<0.05)) and its downstream target forkhead box O1a (FoxO1a) (175±11% of control (P<0.05)), a transcriptional repressor of cyclin D2; LY294002 inhibited these responses. The transcription of pancreas duodenum homeobox‐1 (PDX‐1) and glucokinase regulated by FoxO1a was increased 2‐ to 3‐fold with ANP present. The evidence suggests that NPR‐A stimulation results in activation of a growth promoting signaling pathway in the pancreatic β‐cell that includes PI3K/Akt/FoxO1a/cyclin D2. The activation of PI3K/Akt by ANP or 8‐bromo‐cGMP promotes cyclin D2, PDX‐1 and glucokinase transcription by phosphorylating and restricting FoxO1a activity.
Endothelin‐1‐induced COX‐2 Expression in Mouse Brain Endothelial Cells
Authors: Chan Hui‐ju , Yang Chuen‐Mao
Published: June 2021

Endothelin‐1 (ET‐1) is a pro‐inflammatory peptide which plays important role in the pathogenesis of brain inflammatory diseases. This process may be due to the COX‐2 expression and PGE2 synthesis induced by ET‐1. However, the molecular mechanisms underlying ET‐1‐induced COX‐2 expression in mouse brain endothelial cells which contributes to inflammatory responses were largely unknown. Here, Western blotting, RT‐PCR and EIA analyses showed that ET‐1‐induced COX‐2 expression and PGE2 synthesis was attenuated by pretreatment with the inhibitors of ETB receptor (BQ788), MEK1/2 (U0126), p38 MAPK (SB202190), and JNK (SP600125), and transfection with siRNA of ERK1, p38, and JNK2. In addition, ET‐1 stimulated phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK which was attenuated by the inhibitors of MEK1/2 (U0126), p38 MAPK (SB202190), and JNK (SP600125), and transfection with siRNA of ERK1, p38, and JNK2. Furthermore, ET‐1‐induced COX‐2 expression and PGE2 synthesis was inhibited by a selective NF‐κB inhibitor (Bay11‐7082). The activation of NF‐κB was consistent with IκB‐α degradation in plasma and NF‐κB translocation into nucleus in these cells. NF‐κB translocation was blocked by Bay11‐7082, U0126, SB202190, and SP600125. These results suggested that in mouse brain endothelial cells, ET‐1‐induced COX‐2 expression was mediated through phosphorylation of MAPKs and NF‐κB pathways.
The SWIM domain of MEKK1 is required for complex formation with Jun that controls Fra‐2 ubiquitination
Authors: Cuevas Bruce Daniel , Duellman Tyler
Published: June 2021

The MAPK kinase kinase (MKKK) MEKK1 is unique among kinases in that it contains E3 ubiquitin ligase activity. MEKK1 has been reported to mediate ubiquitination of c‐Jun, thus requiring MEKK1/Jun complex formation, but the nature of that complex has not been described. The objective of this study was to determine if Jun forms a complex with MEKK1, and if so, characterize that interaction. Here we report that MEKK1 directly associates with the DNA‐binding region (DBD) of Jun, and this complex requires the MEKK1 SWIM domain, the function of which was previously unknown. We also demonstrate that direct interaction between the SWIM domain and AP‐1 proteins is limited to Jun proteins, and this requires amino acids conserved in the DBD of Jun, but not Fos proteins. Finally, we demonstrate that the Fos family protein Fra‐2 associates with the MEKK1 complex via Jun, and that Jun is required for MEKK1‐dependent Fra‐2 ubiquitination. Altogether, our data show that, in addition to regulating AP‐1 phosphorylation through control of MAPK activity, MEKK1, Jun, and Fra‐2 form a multi‐protein complex that regulates Fra‐2 ubiquitination.

This work was supported by a grant from the National Institutes of Health.
Pharmacological and toxicological study of two analogues of amphotericin by using in vitro models
Authors: León‐Buitimea Angel , Morales‐Nava Rosmarbel , Fernández‐Zertuche Mario , Ortega‐Blake Ivan , Reyes‐Esparza Jorge , Rodriguez‐Fragoso Lourdes
Published: June 2021

The aim of present study was to investigate the pharmacological and toxicological effects of amphotericin B and two analogues (AMB0‐A1 and AMB0‐A2) on in vitro models. For study we used epithelial kidney cells in order to evaluate changes in cell proliferation, cell cycle and identify the cell death. Cells were treated with AmB or analogues in three concentrations (1, 10 and 100μM). The hemolytic activity was tested in human red blood cells. For pharmacological study we cultured C. albicans. Yeasts were treated with AmB or analogues and the inhibitory effect was analyzed by using Flow Citometry. Both did not affect renal cells and red blood cells at 1 and 10μM after 24h treatment. However, they reduced cell viability (17 y 11% respectively, (p<0.05) as compared with AmB. The AmB and analogues induced changes in cell proliferation, cell cycle and cell death in a dose‐dependent manner. Both analogues showed similar anti‐mycotic effect against C. albicans yeasts, at 10 and 100 μM after 24h treatment, p< 0.05. In conclusion, both analogues displayed anti‐mycotic activity against Candida albicans and had less toxic effects than AmB. Analogues might have a potential use as a new alternative in the treatment of the systemic fungal infections and/or as a base for further studies.
Identification of novel chemosensitivity nodes using siRNA synthetic lethal screens
Authors: Kitchens Carolyn A , McDonald Peter R , Thaker Nikhil G , Shun Tong Ying , Pollack Ian F , Lazo John S
Published: June 2021

Understanding the different modes of action of chemotherapeutic agents and how they synergize may lead to improved dosing regimens and novel combination treatments with molecularly targeted drugs. To identify chemosensitivity nodes for anticancer drugs, we implemented a short interfering RNA (siRNA) screen targeting 5,520 unique druggable genes in T98G human glioblastoma cells. In this screen we transiently transfected cells with targeting siRNAs for 48 hours, then treated with a sub‐lethal concentration of vinblastine (VBL), a specific anticancer agent, for an additional 48 hours. We analyzed cell viability using a resazurin fluorescent dye assay and identified gene products that sensitized T98G cells to VBL using two orthogonal statistical methods. The Median of the Absolute Deviations (MAD) method detected outliers using a MAD score threshold of ±3.5. Samples were ranked according to their viability ratio (cell viability with VBL and siRNA to cell viability with siRNA only) and the top 2.5 percent were selected as hits. The cell viability of samples treated with VBL and siRNA versus siRNA alone were compared using a two‐sample t‐test and samples with a p ≤ 0.01 were considered to be statistically different and therefore synergistic VBL candidates. These analysis methods have identified 65 gene products that sensitize cells to VBL, which are currently being investigated further.
Regulation of breast cancer resistance protein (BCRP/ABCG2) by microRNA‐328
Authors: Pan Yuzhuo , Morris Marilyn , Yu Aiming
Published: June 2021

Breast cancer resistance protein (BCRP/ABCG2) is a membrane transporter that affects pharmacokinetic properties of many anticancer drugs. However, ABCG2 post‐transcriptional regulation and role of microRNAs (miRNAs) in drug disposition remain elusive. Computational analyses indicated that microRNA‐328 (miR‐328) might readily target the 3′‐untranslated region (3′UTR) of ABCG2. We then noted that 1) the levels of miR‐328 and ABCG2 in MCF‐7 and MCF‐7/MX100 breast cancer cells were inversely related, and 2) the levels of miR‐328 could be rescued in MCF‐7/MX100 cells after transfection with a miR‐328‐expressing plasmid. Luciferase assays showed that ABCG2 3′‐UTR‐luciferase activity was decreased more than 50% in MCF‐7/MX100 cells after transfection with miR‐328‐expressing plasmid, the activity was increased over 100% in MCF‐7 cells by a miR‐328 antagomir. Furthermore, ABCG2 protein expression was down‐regulated when miR‐328 was over‐expressed, and ABCG2 protein expression was up‐regulated when miR‐328 was inhibited by selective antagomir, which altered intracellular mitoxantrone accumulation and cell sensitivity to mitoxantrone, Together, these findings suggest that miR‐328 targets ABCG2 3′UTR, controls ABCG2 protein expression, and affects drug disposition. This project was supported by UB Interdisciplinary Research Development Fund, and in part by NIH R01DA021172.
Low Dose Deguelin Suppresses In‐situ Oral Cancer Meatstasis through Down Regulation of Akt‐independent NF‐kB Signaling
Authors: Jan Yi‐Hua , Lai Tsung‐Ching , Chiu Tony , Chang Jeng‐Shou , Hsiao Michael
Published: June 2021

Deguelin, a rotenoid from Mundulea sericea, has a diversity of antitumor activities. Previous studies have shown deguelin inhibited tumor cell proliferation by inducing apoptosis through the inhibition of Akt signaling. However, the anti‐metastatic effects of deguelin have not been explored. In this study, we evaluated low dose deguelin (IC10) effects on oral cancer cells invasion/migration potentials in vitro and on in‐situ oral cancer animal model. Our results showed deguelin significantly inhibited the invasion and migration ability of oral cancer cells at low dose with no cytotoxic effects as evaluated by cell viability, proliferation and cell cycle distribution analyses. Western blot analysis showed low dose deguelin did not alter the phosphorylation level of AKT in SAS and CA922 cell lines. However, low dose deguelin suppressed the activation of IκB kinase and led to the sequential inhibition of IκB phosphorylation. Low dose deguelin also suppressed the NF‐κB reporter activity in a dose‐dependent and time‐dependent manner. Furthermore, in vivo study showed that low dose deguelin treatment significantly inhibited the orthotopic xenograft tumor growth and tumor invasion with no systemic toxicity and body weight loss. In conclusions, our data showed low dose deguelin inhibited the invasion/migration of oral cancer in vitro and in‐situ by Akt‐independent downregulation of NF‐κB signaling. These findings may have clinical implications in utilizing deguelin to treat metastatic oral cancers.
Butyrate uptake and effect on proliferation, differentiation, viability and apoptosis of Caco‐2 cells: influence of polyphenols
Authors: Gonçalves Pedro , Araújo João JR , Martel Fátima
Published: June 2021

The short‐chain fatty acid butyrate (BT) is one of the main end‐products of anaerobic bacterial fermentation of dietary fibre within the human colon. Among its recognized effects, BT is known to inhibit colon carcinogenesis. Polyphenols have several properties, including anti‐oxidant, anti‐carcinogenic and anti‐inflammatory activity. The aim of this study was to investigate the influence of some polyphenols upon the apical uptake of BT into human colonic adenocarcinoma Caco‐2 cells, and to evaluate their influence upon the effect of BT on proliferation, differentiation, viability and apoptosis.

In the first part of the study, we demonstrated that MCT1‐mediated apical uptake of 14C‐BT in Caco‐2 cells is modulated by either acute or chronic exposure to some polyphenols, and that resveratrol acts as a competitive inhibitor of 14C‐BT uptake. In the second part of the study, we evaluated the effect of those polyphenols that chronically increased the apical uptake of 14C‐BT, alone or together with BT, on cell viability, cell proliferation, differentiation and apoptosis. In general, combination of polyphenols with BT did not significantly modify the changes in proliferation, differentiation, viability and apoptosis induced by BT alone. In conclusion, changes in uptake of BT induced by polyphenols do not correlate with changes on cell viability, cell proliferation, differentiation and apoptosis.
NADH can induce necrosis of C6 glioma cells and Neuro2a cells
Authors: Ma Yingxin , Zhao Cuiping , Xia Weiliang , Ying Weihai
Published: June 2021

NADH is a fundamental molecule in energy metabolism and mitochondrial functions. Our previous studies have shown that NADH can decrease genotoxic agent‐induced death of primary cultures of astrocyte. The aim of this study is to determine the effects of NADH on the survival of cancer cells. Our LDH assay showed that 0.1 to 1 mM NADH induced 15–65% death of C6 glioma cells at either 24 or 48 hrs after NADH treatment (p < 0.01), which was confirmed by Trypan blue assays. We also found that NADH produced similar cancer cell‐killing effects compared with NAD+. Our study has excluded the possibility that NADH produced its effects through its degradation products. NADH was further shown to significantly decrease the survival of Neuro2a cells. Our study has suggested that NADH is a novel cancer cell‐killing agent, which may be used to treat cancer (Supported by the Key Research Grant of Shanghai Municipal Education Committee, Pujiang Scholar Program Award and a 973 Grant).
Novel role of STAT1 in the recovery from doxorubicin induced immuno‐suppression
Authors: Parajuli Nirmala , Datta Sebak , Pohle Susanne , Villunger Andreas , Koller Johann Benedikt , Hannesdóttir Lára , Labi Verena , Doppler Wolfgang
Published: June 2021

Signal Transducer and Activator of Transcription 1 (STAT1) is known to play a role in sensitizing cells to chemotherapy induced apoptosis. Doxorubicin (Dox), a commonly used chemotherapeutic drug is known to exert serious side effects, most remarkably cardiotoxicity and myelosuppression. In this study we analyzed the role of STAT1 in Dox induced toxicity and recovery using STAT1 knock out (KO) mice. Dox injected STAT1 wild type (WT) mice showed an early onset of acute toxicity whereas toxicity in KOmice was later but prolonged. A strong effect of Dox on the organs related to immune function such as shrinkage of spleen and thymus were only observed in KO mice on day 16 after Dox injection. Flow‐cytometry analysis of peripheral blood cells from KO mice showed an increase in the granulocyte population but a decrease in B lymphocytes, while no change was noted in WT mice. A severe depletion of bone marrow cellularity was observed on day 3 in both WT and KO mice. However, after 16 days WT mice were able to partially restore the depleted bone marrow, while KO mice were recovering more slowly. On day 30, WT mice showed significant recovery on the population of bone marrow progenitors and B lymphocytes compared to a little recovery in the KO mice. In summary, STAT1 KO mice are differentially sensitive to Dox induced lympho‐toxicity, thus STAT1 has an important role in the recovery from Dox induced immuno‐suppression. Supported by IFTZ grant (WD).
Ramaline inhibits LPS‐induced NO release in macrophages by regulating MAPK and NF‐κB activities.
Authors: Park Hye‐Jin , Yim Joung Han , Pyo Suhkneung
Published: June 2021

Lipopolysaccharide (LPS) induces innate immune responses through TLR4 signaling and activated macrophages produce various inflammatory mediators including Nitric oxide (NO). NO produced by inducible NO synthase (iNOS) mediates a number of pathophysiological processes. Therefore, inhibiting NO production in macrophages is a potential therapeutic target in inflammatory diseases. Herein, we demonstrate that ramalin, a newly discovered antioxidant that is extracted from polar lichen, inhibits production of inflammatory mediators in macrophage‐like Raw 264.7 cell line. RT‐PCR and Western blot analysis demonstrated that ramalin suppressed the level of NO release through down‐regulating both mRNA and protein levels of iNOS. Western blot analysis also revealed that ramalin abrogated LPS‐induced phosphorylation of ERK, p38, and JNK as well as the expression of p65. Attenuated activity of NF‐κB by ramalin was also confirmed by Luciferase promoter assay. In addition, not only the surface level of LPS‐induced TLR4 but also its mRNA and protein levels were decreased by ramalin. Together, these results suggest that ramalin reduces NO release in macrophage through down‐regulation of TLR4 signaling which in turn suppresses MAPK and NF‐κB activities, and it would be a possible therapeutic approach to the treatment of inflammatory diseases.
An optimized method for Paroxysmal Nocturnal Hemoglobinuria screening flow cytometric diagnosis
Authors: Peiro Ana M , Bastande Nuria , Fernández M Luz , Lucas J , López‐Abadía E , Tarín Fabián
Published: June 2021

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare clonal item cell disorder results from an absence of the glycosylphosphatidylinositol (GPI) anchor. Flow cytometry (FC) is considered the gold standard for detecting, traditionally CD55 and CD59. However, there has been no consensus on monoclonal antibodies or immunophenotypic criteria. We describe a technique for PNH diagnosis by FC. A hospital‐based, case–control immunophenotyped study was done of 84 patients with positive >1 PNH symptoms. GPI‐linked antigens and antibodies CD14 (FITC), CD4 (PE) and CD45 (APC) were used to assess the PNH+ monocytes and CD16 (PE), CD24 (FITC) and CD45 (APC) to establish the proportion of GPI‐deficient granulocytes and CD33 (PE), CD55 (APC) and CD59 (FHC). Screening was positive in 11% cases. Patients associated 38% thrombosis, 11–22 % autoimmune haemolytic anemia, aplastic anemia (AA) or leukopenia, 6–7% haemoglobinuria or myelodysplastic syndromes (MDS). Among the markers CD45, CD16, CD24, CD55, and CD59 all were effective in the detection of PNH+ granulocytes, although the combination of CD16 and 24 was most sensitive in monocytes. Conversely, CD55 and CD59 showed limited usefulness. Patients with clones >30% have evidenced of thrombosis and classic PNH and <10% AA and MDS. This simple FC inmunophenotyping screening panel might constitute a rapid and cost‐effective indicator for further confirmatory testing of PNH.
Tecarfarin: A Novel Oral Anticoagulant and Characterization of it Pharmacokinetics, Tissue Distribution, and Mass Balance in Sprague‐Dawley Rats
Authors: Rao Satish , Shao Lei , Irwin Ian , Palme Monica , Druzgala Pascal
Published: June 2021

Tecarfarin is a novel vitamin K epoxide reductase (VKOR) inhibitor being developed for oral anticoagulation therapy for the prophylaxis and treatment of thromboembolic complications associated with atrial fibrillation and other coagulation disorders. It is metabolized to a single inactive metabolite, ATI‐5900, by human microsomal esterases, a CYP450‐independent pathway. We characterized the pharmacokinetics, tissue distribution, and mass balance of tecarfarin after IV and oral administration to Sprague‐Dawley rats. IV administration of tecarfarin was associated with a moderately rapid clearance and a low volume of distribution (Vss). Hydrolysis to ATI‐5900 was moderately fast, with Tmax of the metabolite occurring within 1 hour after that of the parent. Plasma exposure to the metabolite (AUClast) was approximately 30–50% that of the parent. Elimination half‐life (t1/2) of the parent ranged from 2.98 – 6.26 hr. Tecarfarin was well absorbed after oral administration, with oral bioavailability ranging from 80 – 90%. Following oral administration of 14C‐tecarfarin, most of the drug‐derived radioactivity was eliminated within 48 hrs, with 57% being eliminated in the feces, and 33% in the urine. In addition, tissue accumulation was low as most tissues had lower concentration of radioactivity than plasma. These results suggest that the metabolism of tecarfarin is dependent on various esterases in vivo.
Human UDP‐glucuronosyltransferases (UGTs) are involved in the metabolism of the natural resveratrol analogues arachidin‐1, arachidin‐3 and piceatannol
Authors: Radominska‐Pandya Anna , Nair Vipin , Bratton Stacie M. , Gallus‐Zawada Anna E. , Prather Paul L. , Bolivar Fabricio Medina
Published: June 2021

Resveratrol is known to have protective effects against cardiovascular disease, ageing, and cancer. Two natural resveratrol analogs, trans‐Arachidin‐1 and ‐3 (tA1, tA3) were extracted from peanut hairy roots and their glucuronidation was compared to that of trans‐Piceatannol (tPice), a hydroxylated analog of resveratrol that has been shown to be anti‐inflammatoryand anti‐proliferative. The glucuronidation activities of 9 human recombinant UGTs towards these compounds were evaluated using HPLC and LCMS/MS. Single glucuronide products were seen for both tA1 and tA3; while multiple tPice glucuronides were produced. Extrahepatic UGT1A7 and ‐1A10 were found to be the most active and were used for further characterization. UGT1A7 showed Michaelis–Menten kinetics with Km values ranging from 27–56 μM. UGT1A10 demonstrated atypical kinetics toward all three compounds suggesting the presence of multiple binding sites. LCMS/MS assignment of regioselectivity is underway. Our results indicate, for the first time, that lung‐expressed UGT1A7 and intestinal UGT1A10 may contribute significantly to the first pass metabolism of these polyphenols, which can be possible alternatives to resveratrol therapy due to their potential increased bioavailability, a major obstacle to the pharmaceutical use of resveratrol. (NIH‐GM075893 to AR‐P; NSF‐EPSCoR EPS‐0701890 to FM‐B)
Crystal Structure of a Cytochrome P450 2B6 Genetic Variant in Complex with the Inhibitor 4‐(4‐Chlorophenyl)imidazole at 2.0 Å Resolution
Authors: Gay Sean C. , Shah Manish B. , Talakad Jyothi C. , Maekawa Keiko , Roberts Arthur G. , Wilderman P. Ross , Sun Ling , Yang Jane Y. , Huelga Stephanie C. , Zhang Qinghai , Stout C. David , Halpert James R.
Published: June 2021

The structure of the K262R genetic variant of human cytochrome P450 2B6 in complex with the inhibitor 4‐(4‐chlorophenyl)imidazole has been determined using x‐ray crystallography to 2.0 Å resolution. Production of diffraction quality crystals was enabled through a combination of protein engineering, chaperone coexpression, modifications to the purification protocol, and the use of unique facial amphiphiles during crystallization. The 2B6‐4‐CPI complex is virtually identical to the rabbit 2B4 structure bound to the same inhibitor with respect to the arrangement of secondary structural elements and the placement of active site residues. Based on the new P450 2B6 crystal structure and previous 2B4 structures, substitutions at residue 262 affect a hydrogen‐bonding network connecting the G and H helices, where subtle differences could be transduced to the active site. Docking experiments indicate that the closed protein conformation allows smaller ligands such as ticlopidine and clopidogrel to bind to the 2B6 active site in the expected orientation. However, the inability to dock the larger ligands raloxifene and itraconazole in favorable conformations suggests that 2B6 undergoes some degree of structural reorganization to accommodate bulkier molecules, as previously inferred from multiple P450 2B4 crystal structures.

NIH grants ES003619, ES006676
NMR‐based Structural Model of Rabbit Cytochrome P450 2B4 with the Analgesic Amidopyrine, the Intermediate Desmethylamidopyrine, and its Product Aminoantipyrine: Implications for the Structural Basis of Sequential Metabolism
Authors: Roberts Arthur George , Vu Kathy , Halpert James R
Published: June 2021

Rabbit cytochrome P450 2B4 (CYP2B4) and the analgesic amidopyrine were used as prototypes of sequential drug metabolism by mammalian cytochromes P450 (CYPs). CYP2B4 metabolizes amidopyrine by two N‐demethylation steps to form the product didesmethylamidopyrine. Despite numerous studies of amidopyrine with this enzyme, little is known about the structural basis of sequential metabolism. In order to narrow our gaps in understanding, NMR studies were undertaken to build a structural model of binding of amidopyrine, the singly demethylated intermediate, and its product to CYP2B4. Heme‐induced relaxation of ligand protons was used to position the ligands in the active site of CYP2B4. Saturation transfer and NOE experiments were used to probe the protein and aqueous environments of the ligands, respectively. This information was combined and then used as constraints for a molecular dynamics simulation of ligands bound to CYP2B4. Implications of the model will be discussed in the context of sequential metabolism by CYPs as a whole and how this technology can be applied to other CYPs. (Supported by NIH grant ES003619).
Suppression of cytochrome P450 reductase expression in hepatoma cells replicates the hepatic lipidosis observed in hepatic reductase‐null mice
Authors: Porter Todd D , Banerjee Subhashis , Stolarczyk Elzbieta I
Published: June 2021

Cytochrome P450 reductase (POR) is a microsomal electron transport protein that is essential to P450‐mediated drug metabolism and sterol and bile acid synthesis. The conditional deletion of hepatic POR gene expression in mice results in a marked decrease in plasma cholesterol levels counterbalanced by the accumulation of triglycerides in lipid droplets in hepatocytes. It is unclear why lipids accumulate when POR expression is extinguished, as POR is not known to be involved in lipid processing in cells. Moreover, it has not been determined if this hepatic lipidosis is dependent on the transport of lipids from peripheral tissues to the liver. To address these questions, we have developed a stable, siRNA‐mediated cell culture model for the suppression of POR. McARH7777 rat hepatoma cells were transfected with a POR siRNA‐producing plasmid and grown in standard media under continuous neomycin selection for up to 20 days. Real‐time PCR analysis revealed less than 20% of normal POR mRNA levels by day 5, and less than 2% by day 20. Immunoblot analyses demonstrated a greater than 50% reduction in POR protein expression by day 10, and nearly complete loss by day 20. Lipid accumulation was evident by Oil Red O staining by day 15. Immunofluorescence studies demonstrated a marked decrease in POR expression in treated cells at day 20, and a marked accumulation of lipid droplets by Nile Red staining. These results demonstrate that the suppression of POR expression in hepatoma cells results in the accumulation of lipid droplets similar to that seen in the livers of mice in which POR expression is extinguished. This cell culture model should facilitate investigation of the mechanism by which POR contributes to lipid processing in hepatocytes.
Comparative protective effect of N‐acetyl cysteine and tetramethylpyrazine in rats with gentamicin nephrotoxicity
Authors: Ali Badreldin H. , Al Salam S. , Alhusseni I. , Nemmar A
Published: June 2021

Gentamicin (GM) is used against serious and life‐threatening infections, but its use is limited by the occurrence of nephrotoxicity, which involves the generation of free radicals. In this work we tested the effect of a compound with antioxidant properties, tertamethylpyrazine (TMP), on GM‐induced nephrotoxicity, and compared it with an established anti‐oxidant compound N‐acetyl cysteine (NAC). Six groups of rats were studied: (1) control, treated orally (p.o.) and intraperitoneally (i.p.) with saline; (2) treated i.p. with GM (80 mg kg‐1 per day for 6 days); (3) TMP, given p.o. (100 mg kg‐1 per day for 10 days) + GM (same dose as above during the last 6 days); (4) NAC, given i.p. (500 mg kg‐1 per day for 10 days) + GM as above; (5) TMP (100 mg kg‐1 per day for 10 days) + saline; (6) NAC (500 mg kg‐1 per day for 10 days) + saline. GM nephrotoxicity was characterized by reduced creatinine clearance, increased creatinine and urea concentrations in plasma. These functional and structural alterations were prevented or ameliorated by NAC treatment, while TMP had only a slight mitigating effect. The concentration of GM in the renal cortex of the rats given GM + NAC (but not TMP) was lower than that found in rats treated with GM alone. The mechanism by which NAC and, to a lesser extent TMP, protected against GM‐induced nephrotoxicity may be related, at least in part, to the decrease in oxidative stress in renal cortex.
Effects of Portulaca polysaccharide on the dysbacteria in intestinal tract of mice
Authors: Yang Jingyun , Hu Xinjun , Meng Xiangcai , Kang Yu‐Ming
Published: June 2021

We found the effects of Portulaca polysaccharide (PP) in normal flora in intestinal tract of mice. The decontaminated (DC) model was induced by oral Lincomycin. The mice were divided into normal, DC, PP treated and Bacillus bifidus praeparatum treated groups. After treatment for 7 days, the mice were sacrificed, and the intestinal flora was measured. Lincomycin decreased Bifidobacterium and Lactobacillus in intestinal tract of mice. Plasma endotoxin was increased in the DC model. After treatment for 7 days, the number of Bifidobacterium and Bacteroid in normal, PP treated and Bacillus bifidus praeparatum treated groups were higher than that of DC group. The plasma endotoxin in normal, PP treated and Bacillus bifidus praeparatum treated groups were lower than that of DC group. These results indicate that the treatment with PP or Bacillus bifidus praeparatum prevented the dysbacteria in intestinal tract of the DC mice. The present study demonstrates that the PP is an important microecological modulator.
Octreotide Ameliorates Gastric Lesions In Chronically Mild Stressed Rats: Possible Involvement of Antioxidant, Anti‐inflammatory and Antiapoptotic Mediators.
Authors: Nassar Noha N , Abdallah Dalaal M. , Zaki Hala F , Schlaan Mona F.
Published: June 2021

Chronic mild stress (CMS) is linked to different pathophysiological changes that have not been thoroughly investigated. Hence, the present study aimed to evaluate the effect of random exposure to 9 different unpredictable stressors for 21 days on the emergence of hemorrhagic peptic ulcer spots in rats. The possible modulatory effect of a somatostatin‐analogue, octreotide, at two dose levels (50 and 90 μg/kg) was studied in this model of CMS. In addition, both doses of octreotide were given daily for 21 days to normal rats. Exposure to CMS produced anhedonia, increased plasma cortisol level, and reduced body weight, effects which were accompanied by typical gastric lesions confirmed histopathologyically. This insult was also able to elevate levels of gastric mucosal lipid peroxides, PGE2 and the inflammatory cytokine TNF‐α, as well as caspase‐3 activity. On the other hand, CMS mitigated the levels of the anti‐inflammatory cytokine IL‐10 and total antioxidant capacity (TAC). Octreotide administered to normal rats only suppressed gastric mucosal PGE2 level and elevated that of IL‐10. Furthermore, when administered during exposure to CMS, octreotide prevented anhedonia and maintained normal body weight partially at the low dose level and completely with the higher one. These beneficial effects were associated by a remarkable suppression of gastric lesions formation and reverted all derangements in cortisol level and the gastric mucosa, effects that were reflected microscopically. The present study shows that exposure to CMS induces gastric ulceration and that octreotide possesses antiulcer effects via antioxidant, anti‐inflammatory and anti‐apoptotic actions.
Propenone derivatives inhibit TNF‐α‐ and TNBS‐induced intestinal inflammation in vitro and in vivo
Authors: Park Su‐Young , Thapa Dinesh , Kang Youra , Park Min‐A , Cho Mi‐Yeon , Lee Jong‐Suk , Heo Se‐Woong , Lee Eung‐Seok , Kim Jung‐Ae
Published: June 2021

In inflammatory bowel disease (IBD), colon epithelial cells increased the expression of a variety of inflammatory mediators, cell adhesion molecules and chemokines. In this study, we examined the effect of novel propenone derivatives, 1,3‐diphenyl‐propenone (DPhP), 3‐phenyl‐1‐thiophen‐2‐yl‐propenone (PhT2P), 3‐phenyl‐1‐thiophen‐3‐yl‐propenone (PhT3P) and 1‐furan‐2‐yl‐3‐phenyl‐propenone (FPhP), on inflammatory cytokine‐induced responses in HT‐29 human colon epithelial cells. We found that DPhP most effectively blocked TNF‐α‐induced adhesion of U937 monocytes to HT‐29 cells, which is an initial step of colon inflammation. DPhP inhibited the expression of IL‐8, MCP‐1 and ICAM‐1 in a concentration‐dependent manner. When HT‐29 cells were transfected with NF‐κB‐Luc plasmid, DPhP significantly inhibited TNF‐α‐induced luciferase activity. In trinitrobenzene sulfonic acid (TNBS)‐induced rat colitis model, DPhP dose‐dependently protected from the TNBS‐induced weight loss, myeloperoxidase activity increase. At the same time, DPhP suppressed the TNBS‐induced mRNA expression of IL‐8, IL‐6 and MCP‐1. Taken together, the results suggest that DPhP may be a potential candidate to suppress the colon inflammation via inhibition of major pro‐inflammatory cytokines involved in intestinal inflammation.
Chocolate Milk Consumption During Recovery from Endurance Exercise Affects Intracellular Events of Skeletal Muscle Protein Synthesis and Proteolysis
Authors: Lunn William R , Colletto Megan R , Karfonta Kirstin E , Anderson Jeffrey M , Carbone John W , Rodriguez Nancy R
Published: June 2021

PURPOSE

The aim was to determine effects of fat‐free chocolate milk consumption during recovery from endurance exercise on intracellular proteins (ICP) influencing skeletal muscle synthesis and proteolysis.

METHODS

8 active males (23.7 ± 1.6 y, 76.0 ± 3.8 kg, BMI = 24±1, 14.4 ± 2.0% body fat, VO2peak = 53.1 ± 1.6) participated in a randomized, crossover‐design study during which they consumed a eucaloric diet for 2wks. Protein intake was 1.5 g·kg−1. After each week, participants consumed 16 oz. of either fat‐free chocolate milk (MILK) or a non‐nitrogenous, isocaloric control beverage (CON) after a 45‐min run at 65% of VO2peak. Protein expression of PI3K and relative phosphorylation of eEF2, mTOR, eIF4E‐BP1, and rpS6and relative phosphorylation of Akt and FOXO3a, total protein expression of ubiquitin and PSMA2, and activity of caspase‐3 and the 26S proteasome were quantified for protein synthesis and proteolysis, respectively, from muscle biopsied during 3 h of recovery.

RESULTS

Significant results for 0 to 3 h recovery (p<0.05): FOXO3a (Ser253) decreased (1.39 ± 0.14 to 0.98 ± 0.10 AU) and proteasome core activity increased (1.79 ± 0.22 to 2.30 ± 0.19 RFU) for CON vs MILK. Caspase‐3 activity decreased for MILK (3.71 ± 0.61 to 1.68 ± 0.37 AU). Akt (Ser473) was reduced for MILK and CON (0.76 ± 0.13 to 0.27 ± 0.08 AU and 0.72 ± 0.21 to 0.29 ± 0.12 AU, respectively). eIF4E‐BP1 (Thr37/46) increased for MILK (0.63 ± 0.03 to 0.88 ± 0.08 AU).

CONCLUSION

Findings suggest that drinking fat‐free chocolate milk after endurance exercise attenuates ICP activity specific to proteolysis and enhances that of protein synthesis during recovery.
Amino Acid Transporter Expression is Increased in Human Skeletal Muscle Following Essential Amino Acid Ingestion
Authors: Drummond Micah J , Glynn Erin L , Fry Christopher S , Volpi Elena , Rasmussen Blake B
Published: June 2021

Essential amino acids (EAA) stimulate skeletal muscle mTORC1 signaling and protein synthesis in humans. It has recently been reported that amino acid transporters may play a role in the regulation of mTORC1 signaling and translation initiation. The purpose of this study was to profile the expression of several amino acid transporters in human skeletal muscle before and after the ingestion of EAA. Muscle biopsies were obtained from the vastus lateralis of 7 young adult subjects (3 male; 4 female) before and 1–3h after ingesting 10 grams of EAA. Blood and muscle samples were analyzed for leucine kinetics using stable isotopic techniques and qRT‐PCR was used to determine mRNA expression for L‐type (LAT1/SLC7A5, CD98/SLC34A2, LAT3/SLC7A7), sodium‐coupled neutral (SNAT2/SLC38A2 and SNAT4/SLC38A4), proton‐assisted (PAT1/SLC36A1 and PAT2/SLC36A2), cationic (CAT2/SLC7A2), and excitatory (EAAT1/SLC1A3) amino acid transporters. EAA ingestion increased blood leucine concentration, delivery to muscle, transport into muscle, muscle concentration and muscle protein synthesis in association with an increase in LAT1, CD98, SNAT2, and PAT1 expression during the first hour post‐ingestion (P<0.05). We conclude that an increase in EAA availability upregulates human skeletal muscle amino acid transporter expression which likely plays a key role in stimulating anabolic signaling and muscle protein synthesis.

Supported by NIA P30 AG024832 (MJD) & NIAMS R01 AR049877 (BBR)
Age‐dependent capacity to accelerate protein synthesis dictates the extent of compensatory growth in skeletal muscle following undernutrition
Authors: Fiorotto Marta L , Davis Teresa A. , Sosa Horacio A , Estrada Irma J , Watson Keri L , Karhson Debra
Published: June 2021

In both humans and animals, impaired growth during early life compromises adult lean body mass and muscle strength despite skeletal muscle's large regenerative capacity. To identify the significance of developmental age on skeletal muscle's capacity for catch‐up growth following an episode of undernutrition, mice were suckled on control dams fed a 20% protein diet or from birth to 11 (early malnutrition, EM) or 11 to 22 (late malnutrition, LM) days of age on dams fed a low‐protein (8%) diet. They were refed by cross‐fostering (EM) to control dams or weaning (LM) to a control (20% protein) diet. Hindlimb muscle mass and fractional protein synthesis rates (FSR) were measured on days 0, 2, 7 and 21 of refeeding (n=7–9 per age per treatment). On day 0, EM and LM muscles weighed 76% and 84% of age‐matched control values, respectively (P<0.001). After 21 d of refeeding, muscles weighed 94% (EM; NS) and 88% (LM; P<0.001) of control values. FSR were significantly higher in EM than control muscles 2 and 7 d after refeeding (P<0.01), whereas in LM pups, values were similar to controls at all ages. The increase in FSR in EM pups upon refeeding was attributable to increased ribosomal abundance. The data suggest that skeletal muscle's ability to effect catch‐up growth is dictated by a developmental stage‐dependent capacity for ribosome biogenesis. Funded by NIH AR46308 and USDA/ARS/CSREES 6250‐51000‐033
Investigating the hemodynamic parameters involved in microvasculature O2 regulation in skeletal muscle of Zucker Diabetic Fatty rat exposed to surface hypoxia
Authors: Dias Gemma Marie , Milkovich Stephanie , Goldman Daniel , Ellis Christopher G
Published: June 2021

Pre‐diabetes is marked with elevated insulin levels that attenuate ATP release from red blood cells (RBCs) in response to hypoxia. In our lab using 7 week old Zucker Diabetic Fatty (ZDF) obese rats we have observed a significant reduction in RBC velocity (Vel), supply rate (SR) and O2 saturation in skeletal muscle resulting in a decreased capillary O2 supply compared to control animals (ZDF lean). To test whether pre‐diabetic animals can support the tissue metabolic demand, a custom O2 exchange gas chamber was used to create a hypoxic (HX) challenge by altering pO2 at the surface of exposed skeletal muscle from 55 to 15 mmHg for 2 minutes and back 55. Hemodynamic data for each capillary segment for each pO2 was quantified with intravital video microscopy and a functional image analysis system. Previous analysis indicates that mean SR for each pO2 significantly differed within the lean group (p=0.0011), but not within the obese group. The data was reanalyzed to determine whether Vel or hematocrit (Hct) contribute to the SR change. Hct but not Vel, significantly increased in response to the hypoxic challenge in the lean group (p=0.0003). In the obese group neither Vel or Hct responded to the hypoxia. This suggests that Hct contributes to O2 regulation and that in pre‐diabetes the O2 regulatory mechanisms of the microvasculature are impaired and unable to support a hypoxic challenge. (NIH R33 HL089094).