bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure‐based ligand discovery for nutrient transporters
Authors: Schlessinger Avner
Published: November 2023

Alterations in cell metabolism support rapid growth and proliferation of cells ‐ key hallmarks of cancer. Solute Carrier (SLC) transporters are membrane proteins that transport solutes such as metabolites and drugs across membranes, and play a major role in mediating nutrient delivery in reprogrammed cancer metabolism networks. Here, we describe a structure‐based discovery approach to identify small molecule modulators for key nutrient SLC transporters. In particular, we use homology modeling, virtual screening, and experimental testing to identify small molecule ligands including drugs, metabolites, and lead‐like molecules. We target a variety of nutrient transporters such as the amino acid transporters LAT‐1 and ASCT2, and the glucose transporter GLUT1, which function cooperatively in cancer metabolism and are highly expressed in the blood‐brain‐barrier (BBB). Our results may explain some of the pharmacological effects (i.e., efficacy and/or side effects) of known drugs via polypharmacology, and rationalize the enhanced brain permeability of drug‐like molecules. Finally, our top hits inhibited proliferation of various cancer cell lines via distinct molecular mechanisms, providing useful chemical tools to characterize reprogrammed metabolic networks, as well as a framework for developing efficacious drugs against these key targets and other SLC transporters.

Support or Funding Information

R01 GM108911
Phorbol Myristate Acetate Suppresses Breast Cancer Cell Growth via Down‐regulation of P‐Rex1 Expression
Authors: Jiang Haihong , Wong Chuu‐Yun A. , Abel Peter W. , Scofield Margaret A. , Xie Yan , Wei Taotao , Tu Yaping
Published: November 2023

Phosphatidylinositol‐3,4,5‐trisphosphate‐dependent Rac exchange factor 1 (P‐Rex1) is highly overexpressed in estrogen receptor‐positive and ErbB2‐overexpressing human luminal breast tumors. The present study was to investigate the role and mechanism underlying phorbol‐12‐myristate‐13‐acetate (PMA), a protein kinase C (PKC) activator, down‐regulation of P‐Rex1 in breast cancer cells. P‐Rex1 mRNA and protein expression were determined by real‐time PCR and western blot analysis, respectively. Cell growth was analyzed by counting cells number and 5‐bromo‐2′‐deoxyuridine (BrdU) cell proliferation assay. The translocation of PKC was examined by immunofluorescence staining. Our results showed that treatment with PMA caused a time‐ and concentration‐dependent decrease in P‐Rex1 protein levels in breast cancer MCF‐7 and BT‐474 cells with a maximum reduction of 87.2 ± 1.1 and 57.0 ± 8.6%, respectively, at a concentration of 10 ng/ml. Interestingly, either PMA treatment or silence of P‐Rex1 by siRNA inhibits MCF‐7 and BT‐474 cell growth by 60–80%, which can be attenuated by restoring P‐Rex1 expression. MCF‐7 and BT‐474 cells express PMA‐sensitive conventional (α and β) and novel (δ, ɛ and η) PKC isoforms. Immunofluorescence staining analysis of breast cancer cells showed translocation of PKCs from cytosol to the plasma membrane and/or nucleus, an indication of their activation upon PMA stimulation. The PKC general inhibitor Gö6983, but not the conventional PKC isoform inhibitor Gö6976, blocked PMA inhibition of P‐Rex1 expression and breast cancer cell growth. Expression of a constitutively active PKCɛ mutant suppressed P‐Rex1 expression and breast cancer cell growth, which was blocked by Gö6983 treatment. In contrast, expression of wild‐type PKCɛ, wild‐type or constitutively active PKCδ and PKCη had no effect. Together, our data suggest that PMA suppresses breast cancer cell growth via P‐Rex1 down‐regulation and PKCɛ activation plays a key role in this regulation. Our study may provide a novel strategy for development of chemotherapeutic agents for P‐Rex1‐overexpressing breast cancer patients that develop resistance to anti‐estrogen and/or anti‐ErbB2 therapies.

Support or Funding Information

Supported by grants from NIH (5P20GM103489 and R01HL116849), Nebraska State LB595 research program and the National Natural Science Foundation of China (31100595 and 31300683).
Differential Specificity of the Human and Mouse Multidrug Resistance Protein 4 (MRP4) Orthologs for Arsenic Metabolites
Authors: Whitlock Brayden D. , Swanlund Diane P. , Le X. Chris , Schuetz John D. , Cole Susan P. C. , Leslie Elaine M.
Published: November 2023

An estimated 160 million people world‐wide are exposed to levels of arsenic in their drinking water higher than the limit of 10 ppb (10 μg/L) set by the World Health Organization. Arsenic is a Group 1 (proven) human carcinogen which causes skin, lung, and bladder tumors, and is associated with numerous other adverse health effects, including neuropathy and cardiovascular dysfunctions. Multidrug resistance proteins (MRPs/ABCCs) mediate the cellular efflux of a chemically diverse array of endogenous and xenobiotic metabolites. Substrates include glutathione (GSH) conjugates of inorganic and methylated arsenic species and dimethylarsinic acid (DMAV). Unlike in humans, arsenic is a poor carcinogen in rodents, requiring at least 100‐fold higher doses for tumour induction. Differences in toxicokinetics between humans and other species are also well documented. We have shown that human MRP4 (hMRP4) reduces the cytotoxicity and cellular accumulation of inorganic and methylated arsenic species, and hMRP4‐enriched membrane vesicle transport studies identified the GSH conjugate of the highly toxic methylated arsenic metabolite monomethylarsonous acid (MMAIII), MMA(GS)2, and the major human urinary metabolite DMAV as the transported forms. The objective of the current study was to determine if mouse Mrp4 (mMrp4) conferred resistance to and/or transported the same arsenic species as hMRP4. HEK293 clonal cell lines stably expressing mMrp4 were established and used in cytotoxicity assays. Our results showed that mMrp4 did not confer resistance to any of the arsenic species tested [arsenite (AsIII), arsenate (AsV), MMAIII, monomethylarsonic acid (MMAV), dimethylarsinous acid (DMAIII) or DMAV]. Studies with mMrp4‐enriched membrane vesicles showed that unlike hMRP4, mMrp4 did not transport MMA(GS)2 or DMAV. Thus, under the conditions tested, arsenicals are not substrates for mMrp4. These results suggest that hMRP4/mMrp4 could contribute to differences in arsenic toxicokinetics between humans and mice. Furthermore, Mrp4/Abcc4(−/−) mice are unlikely to be a relevant model for understanding the in vivo contribution of hMRP4 to arsenic detoxification and elimination.

Support or Funding Information

Canadian Institutes of Health Research, Alberta Innovates Health Solutions, Faculty of Medicine and Dentistry, University of Alberta
Diethyldithiophosphate (DEDTP) Role in Proportion of Macrophages and Natural Killer Cells in Lymph Vessel and 4T1 Murine Breast Tumor Model
Authors: BUELVAS DUNIA MARGARITA MEDINA , Muñiz Elizabeth Estrada , Loyo Libia Vega
Published: November 2023

The diethyldithiophosphate (DEDTP) is a metabolite produced by the degradation of organophosphorus pesticides (POFs), and a dialkylphosphate (DAP) which is also chemically synthesized with a widespread commercial use and so the world population is chronically exposed to this compound. It is suggested that organophosphorus compounds produce deleterious effects on the immune system of the exposed population, since it has been demonstrated that DAPs affect peripheral blood lymphocytes activation and proliferation of T lymphocytes, furthermore, in vivo assays using C57 mice, the exposure to DEDTP enlarges skin tumors of BMK (Baby Mouse Kidney) cells, these results suggest that DEDTP exposure in vivo may modify the immune response and enhance the growth of tumor cells. Therefore, our objective is to determine the effect of DEDTP in the proportion of macrophages and natural killer cells (NKs) in lymph nodes and 4T1 breast cancer tumors in mice. To this end, we used Balb/c female mice of 6–8 weeks old, exposed for 8 days to 0.01 g/kg i.p. of DEDTP and inoculated with 1×105 4T1 cells. Tumors and lymph nodes were excised and measured. The percentage of total (F4/80+) and M2 (CD206+) macrophages and natural killer (NK) cells (CD355+) were assessed by flow cytometry. Exposure to DEDTP for 8 days increased the proportion of M2 macrophages. After 15 days of exposure to DEDTP, NK cells proportion was reduced but increased after 20 days. In mice challenged with 4T1 cells during DEDTP exposure, the number of total macrophages increased while the proportion of CD355+ cells in the lymph node proximal to the tumor decreased after 5 days; 10 days after, the number of M2 macrophages increased. Finally, after 20 days the number of M2 macrophages and NK cells in the proximal lymph node increased. In tumors, the number of infiltrating NK cells and total macrophages were reduced, followed by a reduction of M2 macrophages. Subsequently, after 15 days we observed an increase of infiltrating NK cells and M2 macrophages; after 20 days of tumor, M2 macrophages were increased. None of these changes had a significant impact on the establishment and development of tumors. With these results we conclude that exposure to DEDTP, in the absence of a particular antigen challenge, exerts immunomodulatory effects on individuals, apparently by inducing alternative activation of macrophages, however, this effect had no significant effect in the growth of 4T1 tumor cells. This could probably be because the more aggressive behavior of 4T1 tumor cells than BMK cells (previously tested on C57 mice) and the pronounced Th2 immune response profile of Balb/c mice (contrary to C57 animals). These results suggest that the effect of DEDTP on the immune system is not decisive against an aggressive tumor challenge, but may have relevance against a bacterial or parasitic infectious challenge.

Support or Funding Information

CONACyT grant 153468
Oxidative and Inflammatory Responses of Monocytes or Macrophages to Ambient Particulate Matter
Authors: Bliss Bishop Charles , Solaimani Parissa , Campbell Arezoo
Published: November 2023

Oxidative stress and inflammation are two major responses to ambient particulate matter (PM) and are thought to be responsible for the majority of its adverse health impacts. The hypothesis of this study is that monocytes and macrophages will have different responses to PM and that the oxidative and inflammatory responses will be linked. A human monocytic cell line (THP‐1) was used as a model for monocytes. These cells were also differentiated to macrophages via exposure to phorbol ester (PMA) for 48 hours. These cells were then exposed to three different size fractions of particulate matter: PM10 (coarse), PM2.5 (fine), and PM0.1 (ultrafine) for 24 hours. Oxidative stress was measured using the DCFH‐DA assay. Reactive oxygen species (ROS) increased compared to the control, but the increase was not dependent on PM size. There was no significant difference in ROS levels in monocytes compared to macrophages. Inflammatory cytokines TNF‐α and IL‐1β were measured using ELISA. In both monocytes and macrophages, fine and coarse particles elicited a significant increase. No significance occurred after exposure to ultrafine particles and there was no difference in the response to monocytes compared to macrophages. This study suggests that monocytes and macrophages respond similarly to particulate matter. Also, PM‐induced oxidative and inflammatory responses may not be directly linked. The mechanism by which PM causes these observed responses in immune competent cells is the subject of future studies in the laboratory.

Support or Funding Information

British Petroleum/ Southern California Air Quality Management District (BP/SCAQMD Award #11527) and Western University of Health Sciences
Eating High Fat Chow Enhances Sensitivity of Adolescent Rats to the Behavioral Effects of the Dopamine D2/D3 Receptor Agonist Quinpirole
Authors: Ramos Jeremiah , Hernandez‐Casner Caroline , Serafine Katherine
Published: November 2023

Eating high fat chow increases sensitivity of rats to drugs that act indirectly on dopamine receptors (e.g., cocaine). This effect is greatest in female adolescent rats. Eating high fat chow also enhances sensitivity of adult male rats to the behavioral effects of drugs that act directly on dopamine receptors (e.g., the dopamine D2/D3 receptor agonist quinpirole); however, it is not known if this effect is greater in adolescents. It is also not known if there are sex differences in this diet‐induced enhancement that mirror those previously demonstrated with cocaine. To test the hypothesis that females are more sensitive than males to the diet‐induced effects on quinpirole‐induced yawning, male and female Sprague‐Dawley rats (postnatal day 25) eating either standard laboratory chow (17% kcal from fat) or high fat chow (60% kcal from fat), were tested once per week with quinpirole (0.0032–0.32 mg/kg) for 8 weeks. Eating high fat chow increased sensitivity to the behavioral effects of quinpirole in male rats. Specifically, quinpirole induced more yawning in male rats eating high fat chow, (e.g., an increase in the maximal effect) as compared to standard chow fed controls. In comparison, female rats yawned significantly less than their male counterparts, and eating high fat chow did not increase the frequency of yawning. These data suggest that alternative behavioral assays should be considered to measure sensitivity to the behavioral effects of dopamine receptor agonists in female rats. For example, other directly observable behaviors, such as locomotion or rearing, might provide a way to measure drug sensitivity in female rats. These results further demonstrate the importance of studying drug sensitivity in both male and female subjects.
Inhibitory Effects of Green Coffee Bean Extract on Serotonin Uptake in the Rat Brain Cortex
Authors: Ohia Sunny Edet , Bush Leah , Robinson Jenaye , Njie‐Mbye Ya Fatou , Bagchi Debasis
Published: November 2023

Caffeine/Coffee consumption has been reported to increase the thermic effects of food and fat oxidation (Acheson et al., Am. J. Clin. Nutr. 33: 989, 1980) and can cause weight loss in obese subjects (Westerterp‐Plantenga et al., Physiol. Behav. 89:85, 2006).

Purpose

Since serotonin (5‐hydroxytryptamine, 5‐HT) has been implicated in the regulation of feeding behavior, the aim of the present study was to determine the effect of extract of green coffee bean (GCBE) on the uptake of radiolabeled serotonin ([3H]‐5‐HT) in the rat brain cortex.

Methods

Isolated rat brain cortex slices were incubated in oxygenated Krebs solution maintained at 37°C for 20 minutes and transferred to warm oxygenated buffer solutions containing [3H]‐5‐HT (80 nM) for different time intervals. In some experiments, tissues were exposed to GCBE (300 μM ‐ 3 mM) and the selective serotonin reuptake inhibitors (SSRI) fluoxetine (100 μM) plus clomipramine (10 μM).

Results

A time‐dependent uptake of [3H]‐5‐HT occurred in control brain cortex slices reaching a maximum at 90 minutes. GCBE (300 μM – 3 mM) and the well‐known serotonin reuptake inhibitors fluoxetine plus clomipramine inhibited the time‐dependent uptake of [3H]‐5‐HT. For example, at 30 minutes, GCBE (1 mM) caused a 23 ± 5.4 % (n = 6) decrease in [3H]‐5‐HT uptake, whereas fluoxetine plus clomipramine inhibited [3H]‐5‐HT uptake by 52 ± 3.1 % (n = 6). At 90 minutes, GCBE (1 mM) reduced uptake by 23 ± 4.16% (n = 6), while fluoxetine plus clomipramine inhibited [3H]‐5‐HT uptake by 25 ± 10.2 % (n = 6).

Conclusion

GCBE can inhibit [3H]‐5‐HT uptake and increase 5‐HT availability in rat isolated brain cortex slices in a manner similar to that of the well‐established SSRIs suggesting a potential role of this compound in regulation of feeding behavior.

Support or Funding Information

Cepham Inc. 144 12th Street Piscataway, NJ 08854
An Essential Role of Calmodulin Binding to the Juxtamembranous Segment of the Fourth Submembrane Domain in the Function of the α‐1A Adrenergic Receptor
Authors: Gebert‐Oberle Briana , VerMeer Mark , TRAN QUANG‐KIM
Published: November 2023

The α1 adrenergic receptor type 1A (α1A‐AR) plays crucial roles in the control of adrenergic functions. Calmodulin (CaM) is the ubiquitous transducer of intracellular Ca2+ signals. Here we show a novel physical and functional interaction between CaM and α1A‐AR. α1A‐AR and CaM coimmunoprecipitate in primary vascular smooth muscle cells and cardiac tissue in non‐stimulated conditions. Novel FRET biosensors revealed direct interaction between CaM and the juxtamembranous segment of the fourth submembrane domain of α1A‐AR (SMD4JM), a domain that encompasses the nuclear localization signal (NLS) of α1A‐AR. Characterization of affinity and Ca2+ sensitivity for the interactions with CaM showed that the NLS itself interacts with CaM, but with significantly lower affinity and Ca2+ sensitivity compared to the SMD4JM, suggesting that the entire SMD4JM is required for full interaction with CaM. Conservative mutations in the non‐NLS segment of SMD4JM that reduced CaM binding affinity of the SMD4JM 3‐fold completely converted α1A‐AR from a membrane targeted receptor to an intracellular receptor. These changes are also associated with substantially reduced α1A‐AR‐mediated ERK1/2 phosphorylation. The results demonstrate an essential role for CaM binding to the SMD4JM in the subcellular localization and function of α1A‐AR and indicate a role for CaM in the control of adrenergic functions.

Support or Funding Information

Iowa Osteopathic and Educational Research Funds

National Institutes of Health Grant HL‐112184
Signal transduction of lyso‐type phosphatidylethanolamine lipids on intracellular calcium concentration in dopaminergic cells
Authors: Park Soojin , Im Dongsoon
Published: November 2023

Background

Metabolic products of phosphatidylethanolamines, lyso‐type phosphatidylethanolamine lipids, shortly LPEL, are produced by phospholipase A2. LPEL increased intracellular Ca2+ concentration in mammalian cells. G‐protein‐coupled receptor (GPCR) were supposed to be involved in the response. GPCRs for lysophosphatidic acid (LPA) might be responsible for the action in certain cell types but not in others. In the present study, action mode of LPEL on [Ca2+]i in human dopaminergic cells was studied.

METHODS

Human dopaminergic cells were cultured at 37°C in a 5% CO2 humidified incubator, and maintained in high glucose RPMI1640, containing 5% (v/v) heat‐inactivated horse serum, 100 units/mL penicillin, 50 μg/mL streptomycin, 2 mM glutamine, and 1 mM sodium pyruvate. Intracellular Ca2+ concentration was measured in the cells that were trypsin‐digested, allowed to sediment, resuspended in Hepes‐buffered medium, and then incubated for 40 min with 5 μM of fura 2‐AM. [Ca2+]i levels were estimated by measuring changes in fura 2 fluorescence using a fluorescence spectrophotometer.

RESULTS

LPEL induced increase of [Ca2+]i in the dopaminergic cells. Also, LPA induced similar increase of [Ca2+]i in the cells. LPEL‐ and LPA‐induced responses showed homologous and heterologous desensitization. Structurally different antagonists of LPA1/3, Ki16425 and VPC32183 inhibited LPEL‐induced increase of [Ca2+]i. And a specific inhibitor of LPA1, AM‐095, also inhibited the LPEL‐induced Ca2+ response completely in dopaminergic cells. Expression of LPA1 and LPA6 was found in the cells, but not other LPA receptors. Pertussis toxin, s specific inhibitor of Gi/o proteins completely inhibited LPEL‐induced [Ca2+]i increase. And edelfosine, an inhibitor of phospholipase C, and 2‐APB, an inhibitor of IP3 receptor, also completely inhibited LPEL‐induced Ca2+ responses. HA130, an inhibitor of autotaxin/lysophospholipase D, didn't inhibited LPEL‐induced response.

DISCUSSION

In the present study, LPEL‐induced [Ca2+]i increase was found to be mediated via LPA1 in dopaminergic cells. Heterologous desensitization, complete inhibition by AM‐095, LPA1 antagonist, and Gi/o‐coupling character of LPA1 and the PTX‐sensitivity of LPEL‐induced [Ca2+]i increase strongly supported the hypothesis in dopaminergic cells. Also results indicate that LPEL increases [Ca2+]i sequentially via LPA1 receptors, Gi/o proteins, phospholipase C, IP3, Ca2+ rise, and Ca2+ influx in the dopaminergic cells.
Essential Functions of the N‐terminal Domain of Tim17 in Trypanosoma brucei
Authors: Chaudhuri Evan , Weems Ebony , Singha Ujjal K , Chaudhuri Minu
Published: November 2023

The majority of proteins essential for mitochondrial functions are encoded in the nuclear DNA and subsequently imported from the cytosol by translocases of the mitochondrial outer and inner membranes, termed TOM and TIM, respectively. These translocases are multimeric protein complexes, which are overall conserved from fungi to human. However, mitochondrial protein import machinery in trypanosomatids, a group of ancient and early branching unicellular eukaryotes that causes a fatal human disease African trypanosomiasis, is significantly different. As a part of this machinery, we previously characterized Tim17 from Trypanosoma brucei. TbTim17 is critical for mitochondrial protein import and thus essential for parasite survival. Here we further investigate the function of the divergent N‐terminal domain of this protein. We found that deletion of the first 20 or 30 amino acid residues (Δ20‐ and Δ30‐) of TbTim17 is detrimental for its function. These mutant proteins neither complement the growth defect of TbTim17 RNAi cells nor the import defect of other nuclear encoded proteins into TbTim17 knockdown mitochondria. However, knock‐in of the full‐length protein (FL‐TbTim17) completely restored these functions in RNAi cells. Further analysis showed that both of these mutants of TbTim17 were properly targeted to mitochondria, however, Δ20‐TbTim17 was not membrane integrated and Δ30‐mutant, though integrated into the mitochondrial membrane was sensitive to protease digestion of the isolated mitochondria, indicating that the N‐terminal region is necessary for proper integration of TbTim17 into the mitochondrial inner membrane. Analysis of the protein complexes in the wild type and mutant mitochondria by Blue‐native gel electrophoresis revealed that none of these mutants were assembled into the TbTim17 protein complex. Co‐immunoprecipitation analysis showed that unlike the wild type TbTim17, the mutant proteins were not associated with the endogenous TbTim17 as well as its interacting partner TbTim62, a novel trypanosome specific Tim. Together, these results show that the N‐terminal domain of TbTim17 is critical for its interaction with TbTim62 and required for the formation of a functional TbTim17 protein complex. To elucidate the assembly of the TIM complex in T. brucei, analysis is under progress to identify essential amino acids residues of this region by site‐directed mutagenesis.

Support or Funding Information

NIH Grants 2SC1GM081146‐05, T32HL007727, and G12RR003032
Activation of Human antigen R (HuR) downstream of p38‐MAPK promotes cardiomyocyte hypertrophy through modulation of NFAT activity
Authors: Slone Samuel , Anthony Sarah R , Tranter Michael
Published: November 2023

The RNA binding protein HuR (Human antigen R) interacts with specific AU‐rich domains in target mRNAs and is highly expressed in many cell types, including cardiomyocytes. RNA binding proteins such as HuR are becoming recognized as potential mediators of cardiac physiology and pathology but the role of HuR in cardiac hypertrophy is largely unknown. Activation of Gαq (Gq) proteins downstream of Gq‐coupled GPCRs such as angiotensin receptors (AT1R) or α‐adrenergic receptors (α1‐AR) is a known mediator of pathological hypertrophy, but the responsible signaling pathways downstream of Gq have yet to be fully elucidated.

Our results show that HuR cytoplasmic translocation (a marker of HuR activation) is significantly increased in neonatal rat ventricular myocytes (NRVMs) following treatment with phenylephrine or angiotensin II, agonists of two independent Gq‐coupled GPCRs known to induce hypertrophy. It appears that this Gq‐mediated HuR activation is dependent on p38 MAPK, but not canonical Gq‐PKC signaling. Furthermore, we show that HuR activation is necessary for Gq‐mediated hypertrophic growth of NRVMs as siRNA‐mediated knockdown of HuR inhibits hypertrophy as measured by cell size and expression of ANF (atrial natriuretic factor). To decipher the downstream mechanisms by which HuR translocation promotes cardiomyocyte hypertrophy, we assessed the role of HuR in the transcriptional activity of NFAT (nuclear factor of activated T cells), the activation of which is a hallmark of cardiac hypertrophy. Using an NFAT‐luciferase reporter assay, we show that HuR knockdown or inhibition by the use of siRNA and small molecule inhibitors results in acute inhibition of NFAT.

In conclusion, our results identify HuR as a novel mediator of cardiac hypertrophy downstream of the Gq‐p38 MAPK pathway, and suggest modulation of NFAT activity as a potential mechanism.

Support or Funding Information

This work was partially supported by an AHA Scientist Development Grant to MT (Award #: 16SDG27360004).
RhoA Regulation of Drp1 and Cardiac Mitochondrial Fission
Authors: Brand Cameron S. , Tan‐Sah Valerie P. , Brown Joan Heller , Miyamoto Shigeki
Published: November 2023

G‐protein coupled receptors can signal downstream through various pathways, including through activation of the small G‐protein RhoA. In cardiomyocytes, RhoA signaling is protective against ischemia/reperfusion injury. We have previously shown that this is mediated through downstream activation of Protein Kinase D (PKD), phosphorylation of PKD targets, and diminished translocation of pro‐apoptotic proteins to the mitochondria (Xiang et al, Sci. Signaling 2013). Mitophagy, a process that removes damaged mitochondria and limits mitochondrial death signaling, has also been suggested to be a cardioprotective response to oxidative stress. Mitochondrial fission is considered to be a preliminary step to clearance of damaged mitochondria via mitophagy. We recently observed that adenoviral expression of constitutively active RhoA in neonatal rat ventricular myocytes (NRVMs) increases RhoA levels at the mitochondria. Adenoviral RhoA expression also results in small, fragmented mitochondria as observed by fluorescent confocal microscopy and electron microscopy, indicative of increased mitochondrial fission. The main protein involved in mitochondrial fission, Drp1, translocates from the cytosol to the mitochondria when activated. We used an adenoviral miniSOG‐tagged Drp1 construct to ask whether expression of active RhoA changes Drp1 levels at the mitochondria. Our results show that mitochondrial miniSOG‐Drp1 is increased within 12 hours of adenoviral expression of constitutively active RhoA. In addition adenoviral RhoA expression increases PKD levels at mitochondria. In summary, we show that RhoA associates with mitochondria and can induce mitochondrial Drp1 and PKD accumulation, as well as mitochondrial fission. The relationship between these mitochondrial signaling events is currently under investigation, with the goal of determining whether RhoA‐dependent Drp1 accumulation and mitochondrial fission contribute to cardioprotection.

Support or Funding Information

NIH Grant R37‐HL028143

NIH Training Grant T32‐HL007444
“Characterization of mRNA Transcript Expression of IGF‐I/PI3K Pathway Components in Regenerating Skeletal Muscle”
Authors: Geddis Alyssa V , Mary Abdalla N , Leandry Luis A , Carrigan Christopher T , Matheny Ronald W , Hobbs Stuart S
Published: November 2023

Skeletal muscle regeneration is a complex process involving the coordinated input from multiple intra‐ and extra‐cellular stimuli. Of these processes, actions of the insulin‐like growth factor‐I (IGF‐I) and phosphoinositide 3‐kinase (PI3K) pathways are critical for regeneration to occur. Multiple isoforms of PI3K catalytic and regulatory subunits have been identified. All Class IA PI3K p110 catalytic isoforms (p110alpha, p110beta, and p110delta) are expressed in skeletal muscle, whereas Class IB PI3K catalytic isoform gamma is expressed primarily in immune cells. Given the importance of the IGF‐I/PI3K pathway in skeletal muscle repair, we sought to determine whether gene expression of components of these pathways was sensitive to initial injury intensity. To test this, we injured the left tibialis anterior (TA) muscle of mice by application of a cold (−70° C) probe to induce traumatic injury. The probe was applied for either 5 seconds (5s) or 10 seconds (10s) to induce two different injury intensities, while the uninjured contralateral leg served as an internal control. TA muscles from injured and control legs were harvested three days (3d) or seven days (7d) after the injury for real‐time PCR analysis. Three days following 5s injury, we found increased expression of Igf1 (~4‐fold), Igf1r (~1.3‐fold), Pik3cb (~2‐fold), Pik3cg (~16‐fold), and Pik3r5 (~20.5‐fold) in the injured leg compared to the control leg (P<0.05 for all). Three days following 10s injury, we observed increased expression of Igf2 (~5‐fold), pik3cb (~3‐fold), Pik3cd (~4.5‐fold), pik3cg (~47‐fold), Pik3r1 (~2.6‐fold), Pik3r5 (~52‐fold) (P<0.05 for all). Significant differences between 5s and 10s injuries at 3d were observed for pik3cb, pik3cd, pik3cg, pik3r5 (P<0.05 for all). Seven days after injury, statistical differences were observed only between control leg and 5s injury only for Igf1r (~0.64‐fold; P<0.01); however a number of transcripts were altered following 10s injury [Igf1 (~3.9‐fold); Igf2 (~28‐fold); Pik3cd (~0.71‐fold); Pik3cg (~5.8‐fold); P<0.05 for all)]. These data reveal that during early skeletal muscle regeneration, transcript expressions for some components of the IGF‐I/PI3K pathway are sensitive to initial injury intensity. The increased expression of p110gamma (pik3cg) and its regulatory subunit (Pik3r5) in 3d injured muscle is consistent with injury intensity‐dependent immune cell infiltration.

The views expressed in this abstract are those of the authors and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government.
Interaction of the P‐Glycoprotein‐Inhibitor Tariquidar with Hepatocellullar Organic Anion Transporting Polypeptides (OATPs)
Authors: Stieger Bruno , Häusler Stephanie , Langer Oliver
Published: November 2023

Tariquidar is a third‐generation inhibitor of P‐glycoprotein (also MDR1 or ABCB1) and has been used in clinical trials to enhance the efficacy of anti‐cancer drugs. Ongoing positron emission tomography (PET) studies in human volunteers with [11C]tariquidar revealed high radioactivity uptake in liver. We therefore aimed to test the role of hepatocellular OATPs in the uptake of tariquidar into human liver. Uptake experiments were performed using CHO cells stably transfected with OATP1B1, OATP1B3 or OATP2B1 with standard substrates in the absence and presence of tariquidar as inhibitor. Using 1 mM estrone‐3‐sulfate or 5 mM taurocholate and up to 100 mM tariquidar, no inhibition of OATP1B1 transport activity was observed suggesting that tariquidar is not interacting with OATP1B1. Using the same experimental approach, no inhibition of OATP1B3 by tariquidar was observed. In contrast, tariquidar inhibited in a concentration dependent manner OATP2B1‐mediated transport of 1 mM estrone‐3‐sulfate. Finally, we used 1 mM tariquidar as substrate for all three OATPs and found that OATP2B1 and OATP1B3 mediates uptake of tariquidar. These data demonstrate that OATPs may be involved in hepatocellular uptake of tariquidar and confirm that interaction of substrates and inhibitors with OATPs are complex processes. Interaction of tariquidar with OATPs could potentially lead to changes in pharmacokinetics of concomitantly administered (anti‐cancer) drugs.
The roles of peripheral blood natural regulatory T cells and the expression of TNF‐α in aortas and cardiac tissues during the progression of atherosclerosis
Authors: HAO CHUNYAN , DUAN HUBIN , LUO FEITONG , LI SHUZHEN
Published: November 2023

Objects

To explore the immune regulation effect of peripheral blood natural regulatory T cells and the expression of TNF‐α in aortas and cardiac tissues during the progression of atherosclerosis.

Methods

36 male SD rats (body weight 100±10g) were randomly divided into three groups, healthy control group (A group), early stage atherosclerosis group (B1 group), late stage atherosclerosis group (B2 group), each group have 12 rats. Rat atherosclerosis model can be established by treating rats with celiac injection of vitamin D3 accompanied by high sucrose‐high fat diet in this experiment. The frequency of CD4+CD25+ Foxp3+ regulatory T cell in peripheral blood was measured by fluorescence‐activated cell sorter (FACS) in each group. Aortas and cardiac tissues were observed with HE staining for pathological changes. The expression of TNF‐αin the rat aortic roots and cardiac tissues was detected by immunohistochemical assay

Results

There is no plaque in the rat aortic roots in Group A. There is fibrous plague in the rat aortic roots in Group B1. There is atheromatous plague in the rat aortic roots in Group B2. The expression of TNF‐αprotein in Group B1 and B2 was higher than those in Group A (P<0.05). Peripheral frequency of CD4+CD25+ Foxp3+ regulatory T cell was decreased in Group B1 compared with those in Group A and B2 (P<0.05).

Conclusion

In the early stage of atherosclerosis, peripheral frequency of nTreg was decreased, where as the expression of TNF‐αprotein in aortas and cardiac tissues were increased, suggesting an increasing inflammatory response. In the late stage of atherosclerosis, peripheral frequency of nTreg was increased, suggesting an important negative feedback mechanism that results in the attenuation of prolonged or excessive immune responses, which otherwise may cause severe collateral damage.

Support or Funding Information

This study was supported by the Scientific research project of Shanxi provincial health department (No. 201301068), Doctor Start‐up fund of Shanxi Medical University and The first hospital of Shanxi Medical University, Science and technology innovation projects of Colleges and universities of Shanxi Province
Sex and tissue‐dependent expression of transporters and enzymes involved in GHB disposition
Authors: Felmlee Melanie A
Published: November 2023

Purpose

To investigate tissue‐dependent sex differences in expression of monocarboxylate transporters (MCTs and SMCTs), aldo‐keto reductases (AKRs) and alcohol dehydrogenase iron containing 1 (Adhfe1), key determinants of the disposition of gamma‐hydroxybutyric acid (GHB).

Methods

mRNA expression of MCTs (1/4), SMCTs (1/2), AKR1A1, AKR7A2, and Adhfe1 were evaluated by qPCR in male and female Sprague‐Dawley rats (9 weeks of age; N = 3 – 4 per gender) in kidney, liver and intestine.

Results

Gender differences were observed for both enzymes and transporters involved in GHB disposition. Hepatic expression of MCT1, MCT4, AKR7A2 and Adhfe1 were significantly lower in females (−1.4 to 2.5 fold). Expression of SMCT1 (+3.5 fold) in the kidney and SMCT1 (+5 fold), SMCT2 (+11 fold) and AKR1A1 (+2 fold) in the jejunum were significantly higher in females. Differences were also observed in expression among tissues. MCT1 had significantly higher expression in the liver (4.5 fold compared to intestine), while MCT4 had significantly higher expression in the intestine (20 – 30 fold compared to liver). SMCT1/2 expression was highest in the kidney and ileum, with SMCT1 expression significantly lower in the jejunum. AKR1A1 and AKR7A2 were expressed in all tissues with AKR7A2 having significantly higher expression in the kidney. Adhfe1 was expressed in liver and kidney, but not in the intestine.

Conclusions

The tissue‐specific sex differences in expression of monocarboxylate transporters, AKRs and Adhfe1 are consistent with the higher plasma concentrations and decreased renal clearance observed in females following supratherapeutic GHB doses. Future studies are needed to determine the mechanisms underlying the observed sex differences in transporter and enzyme abundance. Altered transporter and enzyme expression leading to higher systemic GHB concentrations has the potential for increased risk of GHB‐related toxicity and death in females.

Support or Funding Information

University of the Pacific
The Generation Rx Laboratory: An Innovative Pharmacology Education Research Hub
Authors: Kwiek Nicole Cartwright , Rooney Katherine , Downing Molly
Published: November 2023

The Generation Rx Lab is a multi‐faceted drug education research lab that aims to improve science learning in grades K‐12, educate the public about medication safety principles, and provide a unique teaching opportunity for college students to practice communicating science. In this lab at the nationally renowned Center of Science and Industry (COSI), museum visitors of all ages conduct hands‐on experiments related to the science of drug action. Because drug topics spark young learners’ interest, we hypothesize that teaching science through a pharmacology context provides an effective platform for the general public to learn science, engage in the scientific process, and explore science professions. To test this hypothesis, we created and implemented a variety of educational programs. We share preliminary results from two types of educational programs: hands‐on science experiments and clinical simulations using a human patient simulator. Regardless of program type, our preliminary findings demonstrate that teaching science in the context of drug action increases the general public's understanding of both science and medication safety principles, as well as builds interest in science and health‐related careers. In addition, results from assessing the experiential impact on student lab staff demonstrate that the Generation Rx Lab teaching experience provides our students with a unique opportunity to improve their science communication skills while positively impacting their desire to include a teaching component in their future profession.

Support or Funding Information

Funding is provided by the Cardinal Health Foundation and the Ohio State University Office of Research
Novel Gut Barrier Modulatory Effect of Vildagliptin in Amelioration of Bacterial Translocation in Cholestatic Rats
Authors: Zaky Doaa Abd‐Allah , Al‐Shorbagy Muhammad Yousef , Nassar Noha Nagah , Abdallah Dalaal Moustafa
Published: November 2023

Intestinal barrier dysfunction is frequently implicated in the etiology of septic complications following obstructive jaundice. The present study was conducted to investigate the potential gut barrier modulating effect of vildagliptin (VLD), a dipeptidyl peptidase‐IV (DPP‐IV) inhibitor, on common bile duct ligation (CBDL) in rats. VLD (10 mg/kg/d; p.o) was administered for 10 successive days after CBDL in male Sprague‐Dawley rats. VLD reduced portal endotoxemia induced by CBDL and suppressed rectal temperature elevation, body weight loss and colon and spleen indices to different extends. Moreover, it reversed villous blunting, low villous density, mucosal thinning and inflammatory cell infiltration noticed microscopically in the ileal segments of the CBDL rats. These events were accompanied by the reduction of intestinal myeloperoxidase activity and lipid peroxidation, the elevation of the major anti‐oxidant molecule, glutathione, as well as the normalization of ileal nitric oxide level. Furthermore, ileal DPP‐IV was inhibited in association with the rise in ileal glucagon‐like peptide‐1 (GLP‐1) and insulin‐like growth factor‐1 (IGF‐1) levels in VLD treated animals. The current findings suggest that VLD anti‐oxidant, anti‐inflammatory, as well as enhancements of incretin and IGF‐1 account for the hardwearing of the intestinal barrier that functions competently in the absence of the supporting bile elements.
20‐HETE Increases Large Artery Stiffness and Systolic Blood Pressure in Metabolic Syndrome
Authors: Soler Amanda , Hutcheson Brenda , Hunter Ian , Zhang Frank , Gotlinger Katie , Schwartzman Michal L , Falck John , Rocic Petra
Published: November 2023

Hypertension is a risk factor that is associated with the metabolic syndrome. Large artery stiffness, which is in part a result of elastin degradation, plays a causal role in the development of isolated systolic hypertension, which is especially common in the elderly. Levels of 20‐hydroxyeicosatetraeonic acid (20‐HETE), a cytochrome (CYP)‐derived arachidonic acid metabolite, are greatly elevated in hypertensive animal models and loosely associated with obesity in humans, but 20‐HETE's role in the regulation of arterial stiffness in the metabolic syndrome has not been investigated. We hypothesized that elevated 20‐HETE in metabolic syndrome increases matrix metalloproteinase 12 (MMP12) activation leading to increased elastin degradation, increased large artery stiffness and increased systolic blood pressure. Our study shows that elastin degradation was increased ~4 fold in large arteries of metabolic syndrome rats (JCR:LA‐cp, JCR) vs. Sprague‐Dawley (SD) control rats. This correlated with increased large artery stiffness (75%±2% JCR vs. SD). 20‐SOLA (2,5,8,11,14,17‐hexaoxanonadecan‐19‐yl 20‐hydroxyicosa‐6(Z),15(Z)‐dienoate) a 20‐HETE antagonist, blocked elastin degradation in JCR rats, concomitant with decreasing MMP12 activation. Importantly, both 20‐SOLA and MMP12 inhibition (pharmacological and MMP12‐shRNA‐Lnv) decreased large artery stiffness in JCR rats (3%±2% JCR+20‐SOLA vs. SD, 18%±4.3% JCR+MMP12 inhibition vs. SD). 20‐SOLA and MMP12 inhibition also decreased systolic but not diastolic blood pressure in JCR rats (113±6 mmHg SD, 159±4 mmHg JCR, 130±4 mmHg JCR+20‐SOLA, 139±5 JCR+MMP12 mmHg inhibition). These data suggest that a portion of the systolic but not diastolic component of hypertension in the metabolic syndrome JCR rats is 20‐HETE‐sensitive and dependent on large artery stiffness/compliance. The source(s) of 20‐HETE and MMP12 responsible for increased elastin degradation and large artery stiffness are likewise unknown. Intra‐abdominal lipectomy (removal of visceral fat=5% body weight) in JCR rats decreased MMP12 activation, elastin degradation and large artery stiffness to levels achieved with 20‐SOLA and observed in SD control rats, suggesting: 1) that this 20‐HETE is largely derived from visceral (intra‐abdominal) adipose tissue, and 2) that MMP12 involved in the regulation of vascular stiffness is also derived primarily from visceral adipose tissue. Future implications of these findings may be important for systolic hypertension management through 20‐HETE and/or MMP12 inhibition.

Support or Funding Information

Supported by NIH R01HL093052.
MicroRNA Mediation of Endothelial Inflammatory Response to Smooth Muscle Cells and Its Inhibition by Atheroprotective Shear Stress
Authors: Chiu Jeng‐Jiann , Chen Li‐Jing
Published: November 2023

In atherosclerotic lesions, synthetic smooth muscle cells (sSMCs) induce aberrant microRNA (miR) profiles in endothelial cells (ECs) under flow stagnation. Increase in shear stress induces favorable miR modulation to mitigate sSMC‐induced inflammation. We addressed the role of miRs in sSMC‐induced EC inflammation and its inhibition by shear stress. Coculturing ECs with sSMCs under static condition causes initial increases of 4 anti‐inflammatory miRs (146a/708/451/98) in ECs followed by decreases below basal levels at 7 days; the increases for miR‐146a/708 peaked at 24 hours and those for miR‐451/98 lasted for only 6 to 12 hours. Shear stress (12 dynes/cm(2)) to cocultured ECs for 24 hours augments these 4 miR expressions. In vivo, these 4 miRs are highly expressed in neointimal ECs in injured arteries under physiological levels of flow, but not expressed under flow stagnation. MiR‐146a, miR‐708, miR‐451, and miR‐98 target interleukin‐1 receptor‐associated kinase, inhibitor of nuclear factor‐κB kinase subunit‐γ, interleukin‐6 receptor, and conserved helix‐loop‐helix ubiquitous kinase, respectively, to inhibit nuclear factor‐κB signaling, which exerts negative feedback control on the biogenesis of these miRs. Nuclear factor‐E2‐related factor (Nrf)‐2 is critical for shear‐induction of miR‐146a in cocultured ECs. Silencing either Nrf‐2 or miR‐146a led to increased neointima formation of injured rat carotid artery under physiological levels of flow. Overexpressing miR‐146a inhibits neointima formation of rat or mouse carotid artery induced by injury or flow cessation. Our findings indicate that Nrf‐2‐mediated miR‐146a expression is augmented by atheroprotective shear stress in ECs adjacent to sSMCs to inhibit neointima formation of injured arteries.

Support or Funding Information

This work was supported by MOST‐103‐2321‐B‐400‐001 and MOST‐103‐2325‐B‐016‐003 (to J.‐J.C.) in Taiwan.
Effects of dietary supplementation of Angelica gigas Nakai root powder on heat shock gene expression of peripheral blood mononuclear cells (PBMC) isolated from broiler chickens grown under normal and heat stressed environment
Authors: Lumbera Wenchie Marie Lara , Vergara Emil Joseph Sanvictores , Yang Seung‐Hak , Hwang Seong Gu
Published: November 2023

The study was undertaken to assess the difference in the heat response of Ross broiler chickens subjected to heat stress with varying amounts of Angelica gigas Nakai (AGN) root powder supplemented in the diet. A total of one hundred ninety‐two, day old broiler chicks were randomly divided into two groups: control (29°C) and heat stressed (36°C for 5 hours). The two groups were divided into four subgroups having different treatments composed of the control (no AGN feed supplementation), 0.1%, 0.3% and 0.5% AGN root powder supplementation. After 4 weeks, animals were sacrificed and samples were collected for analysis. PBMC was isolated using Ficoll‐Paque Plus density gradient centrifugation of blood samples collected from the brachial vein. PBMC cultures were stimulated with LPS then subjected to cell viability assay, RT‐PCR and Western blot analyses (Hsf1, Hsp70, Hsp90, IL‐1β, IL‐2, IL‐6, IL‐8, TGF‐β, iNOS and IFNγ). Cell viability assay showed that AGN root powder promoted cell proliferation on PBMC (with or without LPS stimulation) of broiler chickens under normal temperature conditions. More importantly, the plant also alleviates heat shock through both cytotoxicity prevention and dose dependent cell proliferation on PBMCs (with or without LPS stimulation) of heat stressed broilers. Both RT‐PCR and Western blot analysis showed enhanced expression of heat shock genes and proteins Hsf1, Hsp90 and Hsp70. Immunomodulatory activity of AGN was also demonstrated through upregulated mRNA and protein expressions of cytokines IL‐1β, IL‐6, IL‐8 and IFNγ and down regulation of IL‐2, TGF‐β and iNOS. These findings suggest the potential of AGN root powder as a feed supplement in broiler chickens to enhance cellular tolerance against heat shock through the enhancement of heat shock genes and protein expressions in addition to its immunomodulatory activity on avian PBMC.