bluuebunny/crossref_metadata_2025 huggingface dataset:
PECAM‐1 and Metastatic Tumor Cell Proliferation
Authors: Abraham Valsamma , Paramabath Andrew , Debs Robert , DeLisser Horace
Published: June 2021

PECAM‐1 mediates homophilic and heterophilic ligand binding as well as intracellular signaling. Although endothelial PECAM‐1 has been implicated in metastatic tumor cell proliferation, the mechanism of this involvement is unknown. Mutants of PECAM‐1 in which PECAM‐1‐dependent homophilic binding, heterophilic binding or intracellular signaling had been disabled by targeted mutations were generated, and then expressed in REN cells (an EC surrogate). The resulting cells were then studied in tumor co‐culture assays using B16 melanoma, LOX human melanoma and murine 4T1 breast cancer. For all three tumors, tumor cell proliferation was simulated 2‐4 fold in the presence of cells expressing PECAM‐1, an activity that was lost if homophilic but not heterophilic or intracellular signaling was disabled. Consistent with the presence of a soluble factor, conditioned media (CM) from the co‐culture of REN cells expressing wild type PECAM‐1 stimulated tumor cell proliferation. An antibody array analysis of CM from murine EC/tumor cell co‐cultures identified TIMP1 (a simulator of tumor cell proliferation) as a possible factor whose expression was significantly inhibited by anti‐PECAM‐1 antibody. Consistent with this, we found that TIMP1 was significantly increased in the presence of cells expressing mouse PECAM‐1. This stimulation of TIMP1 expression was lost if homophilic binding was disrupted but was preserved if heterophilic binding or intracellular signaling were disabled. These data suggest that in vivo, endothelial PECAM‐1 regulates metastatic tumor cell proliferation through processes that involve PECAM‐1‐dependent homophilic ligand interactions and the release of endothelial‐derived TIMP1.
Trends in Breast Lesions in Lagos, Nigeria: Changing or Static?
Authors: Obadofin Omobolade , Daramola Adetola
Published: June 2021

Breast lesions are one of the commonest lesions seen in many surgical pathology laboratories and most of these are benign. However about 1 in 4 are malignant. The age standardized incidence rate of breast cancer in Nigeria is relatively low compared to breast cancer rates in North America, but the morbidity and mortality is much higher.

Objective

To analyze the trend in frequency of breast lesions over a 15 year period and determine if there are any significant changes in Lagos, SW Nigeria.

Method

Records of all breast specimens received from January 1999 – December 2013 were retrieved and analyzed. The 15 years were divided into 3 time periods: 1999‐2003, 2004‐2008 and 2009‐2013.

Results

34,084 biopsies were received over the 15 years of which 5,599 were breast specimens which accounted for 14.9, 18.8 and 16.0% of total biopsies in the 3 time periods respectively. Benign breast lesions (BBL) constituted 77.6, 76.3 and 64.6%, while malignant breast lesions (MBL) accounted for 22.4, 23.7 and 35.4% respectively. This shows a progressive decrease in the ratio of BBL to MBL. The decrease is more significant in the last 5 years (2009‐2013).
The peak age for BBL was consistently in the 3rd decade while that for MBL was the 4th decade in the 1999‐2003 time period then changed to the 5th decade for the latter 2 time periods.
2.7% of the breast biopsies were males'. Of these, 75% were BBL while 25% were MBL. Over the 3 time periods, MBL constituted 35.6, 25.5 and 16% of male breast lesions and 30, 23.6 and 35.9% of female breast lesions.

Conclusion

Over the last 15 years, the proportion of MBL to BBL has increased; the peak age for BBL has remained the 3rd decade while that for MBL has increased from the 4th to the 5th decade. However, the proportion of MBL to BBL in males has decreased whereas this proportion has increased in females.
Polycystin‐1 and Gα12 regulate epithelial‐mesenchymal transition in renal epithelial cells
Authors: Xu Jen , Racioppi Ryan , Kong Zhanglin , Wu Yong , Li Suyan , Lu Tzong‐Shi , Kong Tianqing
Published: June 2021

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common hereditary diseases. Mutations in polycystin‐1 (PC1) and polycystin‐2 (PC2) account for the etiology of 85% and 15% of cases, respectively. Its pathology of cystogenesis and deterioration of kidney function includes focal inflammation, fibrosis, and disruption of cell polarity, cell‐cell, and cell‐matrix adhesions. Epithelial‐mesenchymal transition (EMT) is also a tubulointerstitial pathology in ADPKD. However, the specific mechanism for EMT in the kidneys of ADPKD remains poorly understood. In Madin‐Darby canine kidney (MDCK) cells, active Gα12 caused the differential shedding of E‐cadherin and N‐cadherin. Further study showed that deletion of Pkd1 led to activation of Gα12, increased the shedding of E‐cadherin in mouse kidney. In addition, we found that a disintegrin and metalloproteinase domain‐containing protein 10 (ADAM10) was the key sheddase. This differential cleavage of E‐cadherin and N‐cadherin results in the switch from E‐cadherin to N‐cadherin that was observed in kidney tissue from ADPKD mice and patients. Gα12 activation or Pkd1 deletion led to the transition of β‐catenin from cell membrane to nucleus and increased levels of EMT markers (α‐SMA, vimentin). The specific ADAM10 inhibitor GI254023X blocked the E‐cadherin to N‐cadherin switch and reduced the EMT markers. Our results demonstrate a novel mechanism of EMT in ADPKD. Inhibition of this pathway, especially of ADAM10 activity, could be a novel therapeutic regimen for ADPKD.
Protective Role of Rho Guanosine Diphosphate (GDP)‐Dissociation Inhibitor Ly‐GDI in Acute Pulmonary Alveolitis
Authors: gao hongwei , yan chunguang , Abdulnour Raja‐Elie , Wu Min , Wang Ximo
Published: June 2021

Ly‐GDI is an inhibitory protein of Rho GTPases. Growing evidences indicate that Ly‐GDI plays an essential role in regulating actin cytoskeletal alteration which is indispensible for the process such as phagocytosis. However, the role of Ly‐GDI in inflammation remains largely unknown. In the current study, we found that Ly‐GDI expression was significantly decreased in the IgG immune complex‐injured lungs. To determine if Ly‐GDI might regulate the lung inflammatory response, we constructed adenovirus vectors that could mediate ectopic expression of Ly‐GDI (Adeno‐Ly‐GDI). In vivo mouse lung expression of Ly‐GDI resulted in a significant attenuation of IgG immune complex‐induced lung injury, which was due to the decreased pulmonary permeability and lung inflammatory cells, especially neutrophil accumulation. Upon IgG immune complex deposition, mice with Ly‐GDI over‐expression in the lungs produced significant less inflammatory mediators (TNF‐α, IL‐6, MCP‐1, and MIP‐1α) in bronchoalveolar lavage fluid when compared control mice receiving airway injection of Adeno‐GFP. Mechanically, IgG immune complex‐induced NF‐κB activity was markedly suppressed by Ly‐GDI in both alveolar macrophages and lungs as measured by luciferase assay and electrophoretic mobility shift assay. These findings suggest that Ly‐GDI is a critical regulator of inflammatory injury after deposition of IgG immune complexes and that it negatively regulates the lung NF‐kB activity.
Developing and Evaluating Communication Skills among Students In an Undergraduate Stem Research Program
Authors: Bender Carol , Cubeta Jennifer
Published: June 2021

Public interest in science is waning at a time when it is more important than ever that Americans understand how science impacts their lives. Recognizing that it is imperative that young scientists develop communication skills that allow them to engage not only with other scientists, but also with the public, the Undergraduate Biology Research Program (UBRP) at the University of Arizona offers a variety of activities to insure that students develop these skills. These activities include regular small group meetings where students learn to communicate about their research to each other and develop an “elevator speech” for members of the public; an annual poster conference that requires them to write an abstract, construct a poster, and present the poster to an audience that includes scientists and the general public; an outreach program that enables them to explain science to economically disadvantaged sixth graders; and a weekly community radio program (“Thursday Thesis”) that allows them to talk about their research to the listening audience. Collectively these activities heighten students' sensitivity to the importance of communicating effectively with different audiences and provides them with valuable practice. Such training allows scientists to develop the communication skills needed to cultivate a more informed public – one that cares about science and sees it as relevant. This presentation will describe the development, implementation, and assessment of the UBRP program's communication scheme which can be adapted for use with other training programs.
More than a Replacement Therapy: Amphetamine Treatment Reverses the Behavioral and Neurochemical Consequences of Cocaine Self‐ Administration
Authors: Siciliano Cody , Calipari Erin , Jones Sara
Published: June 2021

Cocaine abuse results in disruption of dopamine (DA) system function, and it has been postulated that this hypodopaminergic state underlies maladaptive behaviors in addiction. As such, an effort has been made to identify compounds that normalize the DA system deficits in order to improve treatment outcomes for cocaine addiction. Although amphetamine (AMPH) has been examined as a potential treatment, with promising behavioral results in rodents, monkeys and humans, the mechanism for AMPH‐induced decreases in cocaine reinforcement has yet to be elucidated. Cocaine SA has been shown to result in escalation of cocaine intake, and increased reinforcing efficacy of cocaine, effects that are thought to model the transition from recreational use to addiction. We found that continuous AMPH treatment (5 mg/kg/day) during cocaine SA attenuated both escalation of intake, and increases in cocaine reinforcement, suggesting that AMPH treatment has a protective effect against the behavioral consequences of repeated cocaine use. Using ex vivo voltammetry we found that AMPH treatment blocked cocaine SA‐induced reductions in basal phasic DA signaling and prevented the development of cocaine tolerance at the DA transporter. In addition, AMPH, when administered during abstinence, completely reversed cocaine tolerance and alterations in phasic signaling. Together, these data demonstrate that some effects of AMPH on cocaine intake are independent of agonist replacement therapy. While agonist therapies for cocaine addiction are controversial and AMPH has a high abuse potential, these data may be to drive the design of more targeted pharmacotherapies with limited abuse liability.
Effects of Ethanol on Cocaine Self‐administration in Monkeys under a Fixed‐interval Schedule or Food‐Drug Choice Procedure
Authors: Czoty Paul , Coller Michael , Nader Michael
Published: June 2021

As many as 90% of cocaine abusers also drink ethanol (EtOH), suggesting that EtOH enhances sensitivity to the reinforcing effects of cocaine. However, little is known about the interaction of these drugs. In the present studies we determined the effects of acute i.v. administration of EtOH (0.5‐2.0 g/kg over 10 minutes) on cocaine self‐adminstraiton using two paradigms in male nonhuman primates. In one set of studies, rhesus monkeys (n=4) self‐administered cocaine (0.003‐0.3 mg/kg/injection) under a 300‐sec fixed‐interval schedule. The second set of studies used a food‐cocaine choice procedure in socailly housed cynomolgus monkeys (n=8) in which a complete dose‐effect curve for cocaine (0.0, 0.003, 0.01, 0.03, 0.1 mg/kg/injection) was generated daily. Use of socially housed monkeys permitted an assessment of the effects of environmental enrichment (in dominant‐ranked monkeys) and chronic social stress (in subordinate‐ranked monkeys) on EtOH/cocaine interactions. In rhesus monkeys, 1.0 g/kg EtOH decreased self‐administration of low cocaine doses but was without effect on self‐administration of higher cocaine doses. In dominant monkeys, 1.5‐2.0 g/kg EtOH increased cocaine choice but no EtOH dose significantly affected cocaine choice in subordinate monkeys. Taken together, the results suggest that acute administraiton of EtOH can potentiate the relative reinforcing effects of low cocaine doses under a food‐cocaine choice procedure, but higher doses are necessary to alter self‐administration under a simple schedule of reinforcement. Surprisingly, conditions modeling social stress (subordination) did not lead to greater sensitivity to EtOH's effects on cocaine reinforcement. Support: DA10584, DA21658.
Buspirone Does Not Alter the Reinforcing, Subjective or Physiological Effects of Intranasal Cocaine
Authors: Stoops William , Bolin B , Lile Joshua , Rush Craig
Published: June 2021

Despite extensive research efforts, no effective medications have been identified for managing cocaine use disorder. The purpose of this study was to evaluate buspirone, a dopamine D3/ D4 receptor antagonist and serotonin 5‐HT1A receptor partial agonist, as a candidate cocaine pharmacotherapy using human laboratory methods. To this end, nine current cocaine users completed a repeated measures, inpatient protocol in which they were maintained on placebo and buspirone (30 mg/day) in counterbalanced order for 3 days before completing experimental sessions. The reinforcing, subjective and physiological effects of placebo and intranasal cocaine (15 and 45 mg) were assessed during those sessions. Cocaine functioned as a reinforcer and produced other prototypic effects (i.e., increased ratings of good effects and heart rate in a dose‐ and time‐related manner). Although cocaine was well tolerated during buspirone maintenance, there were no systematic differences observed in these outcome measures as a function of buspirone condition. These data indicate that this dosing regimen of buspirone did not change the pharmacodynamic effects of cocaine, consistent with clinical trial data suggesting that buspirone does not reduce cocaine use. However, longer dosing periods and higher buspirone doses should be tested to more fully determine the potential utility of buspirone for managing cocaine use disorder.

Supported by R21DA034095.
Interactions of Modafinil or Methylphenidate with the Reinforcing and Neurochemical Effects of Cocaine
Authors: Tanda Gianluigi , Mereu Maddalena , Hiranita Takato , Chun Lauren , Lopez Jessica , Coggiano Mark , Quarterman Juliana , Newman Amy , Katz Jonathan
Published: June 2021

Modafinil (MOD) and methylphenidate (MPH) are FDA‐approved wakefulness‐promoting agents, and MPH is also approved as a medication to treat attention deficit disorders. College students, who misuse MOD and MPH as “smart drugs” to increase cognitive performance, are also likely a population at high risk for abuse of illicit stimulants, such as cocaine (COC). Since MOD and MPH like COC target the dopamine (DA) transporter, and their interactions have not yet been fully described, it is critical to determine the effects of MOD on the reinforcing effects of COC. In the present study MOD (0.1‐10 mg/kg iv) failed to maintain self‐administration (SA) behavior above vehicle levels in rats trained with COC (0.1‐1 mg/kg iv), whereas MPH SA was maintained above vehicle levels at doses comparable to those for COC SA (0.1‐1 mg/kg iv). However, both MOD (10‐32 mg/kg ip) and MPH (1‐10 mg/kg ip) pre‐treatments dose‐dependently potentiated COC SA, shifting the dose‐response curve to the left. COC, at doses that maintain SA, also produced dose‐related stimulation of DA levels in the nucleus accumbens shell, a brain area involved in drug reinforcement. While MPH enhanced this cocaine‐dependent increase in DA, MOD had no significant effects. In summary, MOD had an unusual stimulant profile compared to COC and MPH. The results suggest MOD may enhance reinforcing effects when combined with sub‐threshold doses of COC. Further the results indicate a basis for clinical use of MOD as an agonist substitution therapy for stimulant abuse, as it shares many pharmacological effects of COC, but appears to have low abuse liability of its own.

Support: NIDA/IRP
Behavioral and biochemical analyses of the fast acting antidepressant‐like potential of dextromethorphan
Authors: Nguyen Linda , Logsdon Aric , Lucke‐Wold Brandon , Matsumoto Rae
Published: June 2021

Ketamine at sub‐anesthetic doses exerts robust, rapid antidepressant effects in depressed patients, but widespread use is limited by abuse liability and adverse events. In this preclinical study, the over‐the‐counter antitussive dextromethorphan (DM) was investigated as a potential alternative to ketamine due to overlapping pharmacology. Male, Swiss Webster mice were acutely injected with DM, and behavioral (antidepressant‐like effects in the forced swim test, FST, and tail suspension test, TST) and biochemical (pro‐ and mature brain‐derived neurotrophic factor, or BDNF, expression using western blot) tests were performed. Also, the role of AMPA and σ1 receptors in the antidepressant‐like effects of DM was examined because mounting evidence suggests these mechanisms contribute to a fast onset of antidepressant efficacy and are relevant to ketamine's effects. Our results revealed that DM produces antidepressant‐like actions in both the FST and TST, similar to ketamine. In the FST, pretreatment with NBQX (AMPA antagonist) or BD1063 (σ1 antagonist) blocked the antidepressant‐like effects of DM, indicating that AMPA and σ1 receptors play pivotal roles in mediating its antidepressant‐like behaviors. DM however did not alter hippocampal pro‐BDNF or BDNF levels, while ketamine increased pro‐BDNF. Together, the data demonstrate that DM shares some, but not all, antidepressant‐like effects with ketamine. In light of a recent clinical report of DM's efficacy for bipolar depression and our preclinical findings, further studies are needed to examine DM's potential as a safe, effective fast acting antidepressant.
CNS effects of a novel Phosphodiesterase 4B selective inhibitor A‐33
Authors: Zhang Chong , Xu Ying , O'Donnell James
Published: June 2021

Phosphodiesterase 4 (PDE4) inhibitors such as Rolipram, have been studied extensively for their roles in alleviating mood and cognitive abnormalities, such as depression, anxiety and learning and memory deficits in rodents. However, one major hurdle in this field is the dose‐limiting side effects, i.e. nausea and vomiting. Targeting specific subtypes of PDE4 might produce similar therapeutic effects while reducing the unwanted side effects. This study aims to characterize the CNS effects of the first PDE4B selective inhibitor A‐33. In vitro studies using the mouse hippocampal cell line HT‐22 indicate that A‐33 dose‐dependently increased cAMP levels and phosphorylation of CREB (cAMP response element binding protein), which peaked at 12 h after treatment. In mice, A‐33 significantly reduced the immobility time of in the forced swim and tail suspension tests, 2 classical rodent tests for depressive behaviors. A‐33 also significantly increased expression of phosphorylated CREB in the mouse hippocampus 2h after i.p. administration of the compound, to a level that is comparable to those induced by rolipram. However, A‐33 did not exhibit any anxiolytic‐like or memory enhancing effects in the study. Moreover, A‐33 was also tested for its potential to induce emesis in a rodent surrogate test, ketamine/xylazine induced anesthesia duration. It did not cause a significant reduction of duration of anesthesia, which is consistent with a previous report stating its non‐emetogenic property in ferrets. In summary, the current study shows that A‐33 exhibits antidepressant‐like effects that is comparable to rolipram without causing any significant adverse effects.
RGS Regulation of Gαi2 in the Mouse Hippocampus Modulates 5‐HT1A Receptor Mediated Antidepressant‐Like Activity
Authors: Senese Nick , Jutkiewicz Emily , Traynor John
Published: June 2021

Regulators of G‐protein signaling (RGS) are negative regulators of heterotrimeric G protein coupled receptor signaling. A single point mutation (G184S) in Gαi2 abolishes the RGS negative regulatory effects. Mice expressing two knock‐in copies of this RGS insensitive (RGSi) Gαi2 protein display a robust antidepressant‐like phenotype due to increased 5‐HT1A receptor (5‐HT1AR) signaling. The objective of this study was to test the hypothesis that the 5‐HT1ARs in the hippocampus are critical determinants of the phenotype.

Mice (C57BL6) expressing RGSi‐Gαi2 and their wild‐type littermates were implanted with intra‐hippocampal drug infusion cannulae. Antidepressant‐like activity was evaluated by measuring time spent immobile in the tail suspension test (TST). The RGSi‐Gαi2 mice showed decreased immobility in the TST and this was reversed by infusion of the selective 5‐HT1AR antagonist WAY‐100635 (WAY) directly into the hippocampus. Conversely, direct hippocampal infusion of the 5‐HT1AR agonist 8‐OH‐DPAT produced decreased immobility in wild type mice that was reversed by the peripheral administration of WAY. Peripheral, but not hippocampal, administration of 8‐OH‐DPAT to wild‐type mice caused a 5‐HT1AR‐mediated hypothermia. The mutant mice showed no differences in 5‐HT1AR or Gα protein expression in the hippocampus compared with their wild‐type littermates.

These results are consistent with the hypothesis that the antidepressant‐like phenotype in the RGSi‐Gαi2 knock‐in mice is due to altered 5‐HT1AR signaling in the hippocampus. These findings identify hippocampal 5‐HT1AR specifically coupled to Gαi2, and the RGS proteins that regulate them, as potential targets for antidepressant medications. Supported by MH097012 and GM007767.
The Use of Thalidomide in Treating PTSD and Co‐morbid Pain in an Animal Model of PTSD
Authors: Dib Patrick , Standifer Kelly
Published: June 2021

Single‐prolonged stress (SPS) is an established animal model for post‐traumatic stress disorder (PTSD). We have previously reported that SPS induces increases in serum TNF‐α levels as well as long‐lasting mechanical allodynia and thermal hyperalgesia at day 3 post SPS. The aim of this study was to determine the effects of TNF‐α blockade on development of PTSD and co‐morbid pain symptoms. To achieve this, male Sprague‐Dawley rats were subjected to SPS; half of the SPS and control group rats were immediately presented with 50 mg/kg thalidomide (THL), a potent TNF‐α blocker, in 10% DMSO vehicle, daily, i.p for 5 days; non‐SPS rats received vehicle injections. THL almost completely prevented mechanical allodynia and reduces thermal hyperalgesia associated with SPS (p<0.05) that is normally noted as early as day 3 post SPS, without altering baseline sensitivity itself. Using a 2‐way ANOVA, a significant interaction was observed between THL and SPS (p<0.0001). Additionally, utilizing the elevated plus maze test, THL treatment reduces anxiety like behaviors, specifically mobility time, immobility time, open entries and time spent in open arms (p<0.05). Also, using a 2‐way ANOVA, a significant interaction was observed between THL and SPS (p<0.05) This suggests that immunomodulatory drugs such as THL, may offer a novel therapeutic method to prevent or reduce symptoms of PTSD and co‐morbid pain if administered shortly after the traumatic event.

Support

Department of the Army DMRDP W81XWH‐11‐2‐0077
The Impact of Phosphatidylinositol‐4,5‐bisophosphate (PIP2) on Serotonin Transport Function
Authors: Hofmaier Tina , Kudlacek Oliver , Salzer Isabella , Stockner Thomas , Böhm Stefan , Sitte Harald
Published: June 2021

The serotonin transporter (SERT) plays a key function in the termination of serotonergic neurotransmission. SERT is the main pharmacological target in treating depressive disorders and also a target for various drugs of abuse. Drugs like amphetamine‐type stimulants (ATS) reverse the direction of transport which finally leads to an increased serotonin concentration in the synaptic cleft. Transmembrane proteins get in close contact with the lipid environment and are partitioned in specific lipid microdomains. Our previous studies already implicated that the phosphatidylinositide phosphatidylinositol‐4,5‐bisophosphate (PIP2), an important signaling molecule, influences amphetamine effects at SERT. Furthermore, we elucidated a binding site on the intracellular face of SERT where PIP2 specifically attaches. However, upon mutation of the binding site, PIP2 interaction with SERT was largely reduced, i.e. more than 50%. The objective of this study was to further investigate the interaction between SERT and PIP2 and to explore other positively charged SERT areas. This was achieved by using a computational approach. We identified a putative second binding site which is close to the inner leaflet of the plasma membrane. Introduction of single point mutations and by pharmacological manipulation of cellular PIP2 levels led to a decrease of amphetamine‐induced substrate efflux. These results give rise to a better understanding of the PIP2 –SERT interactions and open up new possibilities to target SERT dependent amphetamine‐induced actions.
Mechanisms Contributing to Lack of Antidepressant Efficacy in Juveniles and Adolescents
Authors: Mitchell Nathan , Fraser Rheaclare , Owens W.Anthony , Horton Rebecca , Vitela Melissa , Gould Georgianna , Koek Wouter , Daws Lynette
Published: June 2021

Depression is a major health problem where most patients are not effectively treated. This problem is compounded in children where only two antidepressants are clinically approved. Both are selective serotonin (5‐HT) reuptake inhibitors (SSRIs), which are often less efficacious in young populations compared to adults. We found the antidepressant‐like effects of SSRIs in juvenile mice (21 days post‐partum) was reduced relative to adult mice; however, there was no difference in expression of hippocampal 5‐HT transporter (SERT), the target protein of SSRIs, to account for the reduced SSRI efficacy. Increased extracellular 5‐HT after SSRI treatment is thought to trigger downstream events for therapeutic response. Our data suggests transporters capable of 5‐HT uptake other than SERT, such as organic cation transporters (OCTs) and the plasma membrane monoamine transporter (PMAT), may be preventing extracellular 5‐HT from climbing to therapeutically relevant levels after SSRI treatment. The binding site density of [3H]decynium‐22 (D22), an OCTs/PMAT blocker, was greater in juvenile mice than adults. Western blots revealed the greater [3H]D22 binding was likely driven by greater PMAT protein expression in juvenile mice. In our preliminary studies, D22 (0.01mg/kg) produced antidepressant‐like effects in juvenile but not adult mice. Using in vivo chronoamperometry, a technique which measures region specific 5‐HT clearance in brain, studies are underway to determine whether the antidepressant‐like effects of D22 in juvenile and adolescent mice are related to its ability to inhibit 5‐HT clearance.

Support: NIH MH093320 / MH064489
Structural Basis for Allosteric Enhancement of Agonist Affinity by G Proteins
Authors: Mahoney Jacob , DeVree Brian , Velez‐Ruiz Gisselle , Rasmussen Soren , Kuszak Adam , Pardon Els , Steyaert Jan , Kobilka Brian , Sunahara Roger
Published: June 2021

G protein‐coupled receptors (GPCRs) are allosteric machines that translate extracellular ligand binding into intracellular signaling events. Many GPCRs display higher affinity for agonists in the presence of G proteins, but the mechanism for this allosteric enhancement of agonist binding is unclear. Based on recent structural data, we hypothesized that in an active receptor conformation, the orthosteric binding site constricts around its ligand, thus slowing dissociation and enhancing affinity. Using purified receptors, we characterized the effects of G protein and G protein‐mimetic nanobodies on the ligand‐binding properties of multiple GPCRs. When β2‐adrenergic receptor (β2AR) was bound to nucleotide‐free Gs heterotrimer, we observed slowed association of the antagonist [3H]DHAP. A Gs‐mimetic nanobody (Nb80) slowed both [3H]DHAP association and dissociation. However, this effect was not antagonist‐specific, as Nb80 also inhibited association of radiolabeled agonists. Together, these data suggest that transit into or out of the orthosteric site in the active β2AR conformation (bound either to Gs or Nb80) is restricted due to closure of the orthosteric site. The M2 muscarinic acetylcholine receptor and mu opioid receptor behaved similarly to β2AR, suggesting that G protein‐mediated closing of the orthosteric site may extend to GPCRs beyond β2AR and the amine receptor family.
The Effects of Siwu Mixture on Chemotherapy‐induced Rat's Ovary
Authors: Zhang Yu‐li , Xie Wei , Xue Xiao‐ou
Published: June 2021

Objective

To observe the influence of Siwu Mixture on structure changes of ovary and ovarian granulose cell apoptosis in rat model.

Method

Thirty female SD rats were randomly divided into siwu , model and normal group.The former 2 groups were respectively given Siwu Mixture and purified water intragastrically after the intraperitoneal injection of Cisplatin, and the normal group was given purified water intragastrically after the injection of saline solution. The vaginal secretion smears were taken everyday. On the 8th experimental morning, serum hormones were assayed and bilateral ovaries were taken. We observed the ultra‐structure and microstructure changes of ovary,imaged micro measurement the particles cell layer maximum thickness at Image Analyzer and use TUNEL method to detecte particles cell apoptosis.

Result

1. Compared with normal group,the FSH was elevated statistically (7.06±1.88 vs 9.19±0.55 U·L‐1, P<0.05) in model group rats while there was no statistical differences in serum hormones between normal and siwu group,the apoptosis index was elevated statistically (0.03±0.01 vs 0.37±0.09 %, P<0.05) in model group rats, while the particles cell layer maximum thickness was decreased statistically(16.16±5.35 vs 10.87±1.45 μm, P<0.05) in model group rats.Compared with model group, the apoptosis index was lower statistically in siwu group rats(0.37±0.09 vs 0.23±0.1%, P<0.05)while there was no statistical difference in the particles cell layer maximum thickness between the two groups(10.87±1.45 vs 16.16±5.35 μm, P>0.05),and the microstructure and ultra‐structure changes of siwu group's ovary demonstrated the lower degree of injury .

Conclusion

Siwu Mixture could promote the recovery of ovarian function ,but cannot afford complete fix.
Ferulic Acid Methylester from Tamarix aucheriana, Shows Anticancer Activity against Human Colorectal Cancer Cells: Underlying Molecular Mechanisms
Authors: Abaza Mohamed , Afzal Mohammad , Al‐Attiyah Rajaa , Guleri Radhika
Published: June 2021

Natural products have long been used to treat many diseases, including cancer and therefore, these are good candidates for drug development.The present study focuses onthe isolation and identification of potential chemo‐therapeutic/preventive constituents from Tamarix aucheriana using bioactivity guided fractionation. Ferrulic acid methylester (FAME) was identified as an active agent. The potential of FAME to control the growth of human colorectal cancer cells, enhance their sensitivity to conventional chemotherapeutic drugs and the potential biological targets and molecular mechanisms of FAME action were investigated. FAME showed time‐ and dose‐dependent anti‐mitogenic effect on colorectal cancer cells with little cytotoxicity for normal human fibroblasts. FAME inhibited the colony formation, induced morphological changes, induced apoptosis, and inhibited DNA‐binding activity of NFkB, the various proteolytic activities of proteasome and invasion in colorectal cancer cells. FAME up‐regulated p15, p18, p19, p21, p27, p53, and p57, pro‐apoptotic gene expression and down‐regulated Cdk1, Cdk2, Cdk4, and Cdk6, anti‐apoptotic gene expression. FAME showed differential potential to enhance the sensitivity of colorectal cancer cells to conventional chemotherapeutic drugs. These results indicate the chemo‐therapeutic potential of FAME.

Acknowledgment: This study was supported by Kuwait University, Research Grant # SL05‐04
Renal Effects of Glucose Transporter 4 in Nω‐nitro‐L‐arginine /High Salt Induced‐Hypertensive Rats
Authors: igbe ighodaro , Omogbai Eric , Oyekan Adebayo
Published: June 2021

The aim of study was to determine the renal effects of glucose transporter 4 (GLUT4) in a hypertensive nephropathy rat model. GLUT4 has been implicated in insulin resistance and hypertension in several animal models, however its role in hypertensive nephropathy still remain unclear. Hypertensive nephropathy was induced by Nω‐nitro‐L‐arginine (L‐NNA), a nitric oxide (NO) synthase inhibitor, 100 mg/ml in drinking water and high salt (HS) diet (4 % NaCl), for 15 days in the presence of insulin, a GLUT 4 agonist (1 U/day) and indinavir, a GLUT4 inhibitor (80 mg/kg/day). Decreased basal renal medullary and cortical blood flow was enhanced in LNNA/HS/indinavir group (p<0.01) but attenuated (p<0.05) by insulin. Proteinuria was increased (p<0.01) in LNNA/HS/indinavir group but attenuated (p<0.01) by insulin. Insulin‐treated rats decreased urine NO (p<0.01) and urine Na2+ (p<0.01) compared to other treated animals. In indinavir‐treated animals, urine Na2+ was increased by benzamil, an epithelial sodium channel (ENaC) inhibitor (p<0.01) and hydrochlorothiazide, a sodium/chloride co‐transporter (NCC) inhibitor (p<0.05). GLUT4 exerts a renoprotective role which may be related to increase NO production. The antinatriuretic effects of GLUT4 appear to be due to enhancement of ion transport activity of ENaC and NCC at the renal tubules.

This study was supported by National Institutes of Health grant HL03674.
Pregnancy‐Related Hormones Modulate Protein Expression of Hepatic DMEs and Transporters in HepaRG Cells
Authors: Farooq Muhammad , Vrana Marc , Parsad Bhagwat , Unadkat Jashvant
Published: June 2021

Pregnancy modulates the activity of a variety of drug metabolising enzymes (DMEs) and some transporters (TRs) resulting in altered clearance of their respective substrate drugs. Previously, we have demonstrated that the in vivo induction of CYP3A activity could be replicated in HepaRG cells exposed to pregnancy‐related hormones (PRHs). However, data on the effect of PRHs on the protein expression of other DMEs and TRs is lacking. The aim of the current study was to fill this gap in knowledge.

Terminally differentiated HepaRG cells were treated for 72h (n=3) with cortisol (C), estradiol (E2), placental growth hormone and pituitary growth hormone (GHs) individually or in combination (PRHs) at their 10X plasma concentrations observed during the third trimester. Then, the cells were lysed, membrane fraction isolated, and target proteins were quantified using surrogate peptide based LC‐MS/MS methods.

C caused 蠅100% increase in CYP3A4, CYP2C9, OATP2B1 and OCT1 protein expression and 蠅 50% decrease in UGT2B7, UGT2B15, MRP2 and P‐gp protein expression. Identical trend was observed with the combination of hormones (PRHs).

Our findings predict that the clearance of some drugs (e.g. by CYP3A4) will be increased during pregnancy while that of others (e.g. by UGT2B7) will be decreased. While additional studies with PRHs at their plasma concentrations (1X and 10X) observed during all three trimesters are needed, HepaRG cells appear to be an excellent in vitro model to elucidate the mechanism by which the expression (and therefore activity) of various DMEs and TRs is modulated during pregnancy.

Supported by NIH grant P01DA032507
Multidrug and toxin extrusion transporters play a key role in natriuresis as a dopamine transporter
Authors: Kajiwara Moto , Ban Tsuyoshi , Hanada Yuki , Matsubara Kazuo , Masuda Satohiro
Published: June 2021

Background and aim

The intrarenal dopaminergic system is likely responsible for regulating over 50% of net renal salt and water excretion when salt intake increases. Although dopamine synthesis is localized at proximal tubular cells, the molecular mechanism underlying dopamine secretion into urine remains unknown. Multidrug and toxin extrusion (MATE, SLC47A) is expressed in high levels in the brush border membrane of proximal tubular cells mediating the efflux of organic cations, such as metformin and cisplatin, from epithelial cells into urine. Therefore, we hypothesized that MATE mediates secretion of dopamine, a cationic catecholamine, into tubule lumen and consequently regulates natriuresis.

Methods

Urinary Na+ and dopamine concentration were respectively measured with an ion‐selective electrode (cobas6000, Roche) and a liquid chromatography‐tandem mass spectrometry.

Results

Uptake of [3H]dopamine by HEK293 cells expressing human (h) MATE1, hMATE2‐K, and mouse MATE1 exhibited saturable kinetics showed that dopamine is a substrate of MATE transporters. After acute saline‐loading experiments, urinary dopamine was detected in wild‐type mice (WT), but was hardly present in MATE1‐knockout mice (KO). Another experiment showed that the amount of urinary Na+ excretion in WT increased to 12.3‐fold than that of the control group, although the amount in MATE1‐KO increased to only 1.5‐fold than that of the control group.

Conclusion

In conclusion, our results indicated a critical role of MATE in dopamine secretion into tubular lumen, along with MATE's regulation of Na+ excretion into urine.
Re‐visiting Busulfan Disposition: Significance and Clearance Mechanisms
Authors: Myers Alan , Zhang Yan‐Ping , Mallick Pankajini , Abdallah Inas , Hassan Hazem , Kramer Mark , Andersson Borje , Ghose Romi
Published: June 2021

Busulfan (Bu) is a chemotherapeutic with narrow therapeutic range and extensive inter‐individual pharmacokinetic (PK) variability. Bu disposition occurs in part by glutathionylation, but newer pharmacogenomic studies point towards, but not proven, other important metabolism pathways. The objectives of this study were to determine the dangers of impaired Bu metabolism in hematopoietic stem cell transplantation patients, and to explore alternative routes of Bu clearance in vitro. In a retrospective analysis of MDACC patients (n=752), the mean Bu clearance was 101 mL/min/m2 that ranged from 52‐150. Among these patients, about 20% had a low Bu clearance 蠄 80. There was a statistically significant correlation between low Bu clearance and poor survival (p<0.01). Furthermore, low Bu clearance was associated with a greater risk of liver toxicity (p=0.0021) and graft‐vs‐host disease(p=0.0014). Bu stimulated P‐gp ATPase activity only at high conc (100 µM), with a 3.6‐fold increase over basal activity. Therefore, it is unlikely that P‐gp will affect the PK of Bu in clinical practice. A LC‐MS/MS assay (LLOQ=0.2µM) was developed to measure the extent of Bu depletion in HLM. Compared to control, the %Bu remaining at 2 hr was 77% and 74% for Bu 3 µM and 90 μM, respectively. No significant depletion of Bu was observed in HLM pre‐treated with CYP inhibitor 1‐ABT. We have shown that CYP enzymes likely have a role in Bu metabolism, and are a focal point for further studying the troubling cross‐patient variabilities in Bu PK, treatment efficacy and toxicity.
Dobutamine Stress Testing as an Early Diagnostic for Radiation‐Induced Heart Disease
Authors: Lieblong Benjamin , Bhatti Sabha , Mulkey Robin , Fletcher Terry , Rusch Nancy , Boerma Marjan
Published: June 2021

Radiation‐induced heart disease (RIHD) is a potentially severe side effect caused by irradiation of all or part of the heart in radiotherapy for intrathoracic and chest wall tumors. Currently, there is no recommended approach for early detection of the disease. However, dobutamine stress testing (DST) is used clinically to unmask other forms of heart disease. Our study sought to determine whether DST may represent an early diagnostic strategy to detect RIHD. Male Sprague‐Dawley rats received X‐irradiation locally to the heart via 5 fractions of 0 (sham), 6, or 9 Gy, and then animals were subjected to DST after 2, 3 and 6 months. Dobutamine was infused in increasing doses (10 ‐ 60 µg/kg/min i.v.) while echocardiography and electrocardiography were performed. At 3 months, radiation caused a dose‐dependent increase in myocardial collagen deposition and alterations in left ventricular function, both of which progressively worsened by 6 months. However, as early as 2 months after irradiation, DST dose‐dependently caused ST‐segment elevation on ECG in irradiated rats, while functional alterations were not present at this time point. The ST‐segment elevations may be an indication of myocardial ischemia. Our results suggest that DST may induce ST segment abnormalities in irradiated hearts prior to detection of functional or histological abnormalities, and thereby may represent a strategy to diagnose and treat myocardial ischemia during early RIHD to prevent later cardiac events.

Supported by CA1487679 to MB (NIH/NCI)
β2‐Adrenergic Receptor Expression on Hematopoietic Cells is Critical for Survival Following Myocardial Infarction
Authors: Grisanti Laurel , Repas Ashley , Gao Erhe , Gumpert Anna , Carter Rhonda , Koch Walter , Tilley Douglas
Published: June 2021

β‐adrenergic receptors (βAR) are critical regulators of cardiac function normally and during heart failure (HF). The importance of βAR on cardiomyocyte contractility and survival is well defined however, following myocardial infarction (MI), inflammatory responses occur, which are critical for healing and scar formation. Catecholamines acting through βAR, particularly the β2AR subtype, are known to modulate immune responses, however, the influence of β2AR in regulating the inflammatory response following MI is unknown. To investigate the contribution of β2AR on immune cells following MI, wild‐type (WT) mice were irradiated and then received β2AR knockout (KO) or WT control bone marrow (BM) transplants to create immune cell specific KO animals. Following BM reconstitution, mice were subjected to MI and cardiac function and survival were monitored. Cardiac function, as assessed by echocardiography, did not differ between WT and KO chimeric mice. However, mice lacking β2AR in their BM resulted in 100% mortality from cardiac rupture within two weeks of receiving MI in contrast to their WT counterparts that had ~20% death. Masson trichrome staining demonstrated infarct expansion in KO chimeric mice was more rapid than their WT counterparts. While there was no change in total cell infiltration into the heart following MI, a retention of granulocytes occurred in the spleen of KO chimeric mice resulting in reductions in infiltrating leukocyte populations. Alterations CCR2 on KO BM resulted in decreased cellular migration which could be rescued using a β2AR lentivirus to re‐express the β2AR. These results demonstrate the critical role of β2AR in mounting an immune response and promoting healing following MI.
Impaired Function in Rats Following Delayed Reversal of Volume Overload Heart Failure is Rescued by Levosimendan
Authors: Wilson Kristin , Guggilam Anuradha , Galantowicz Maarten , West T. , Cismowski Mary , Lucchesi Pamela
Published: June 2021

Objectives

This study investigated if delayed surgical intervention in rats with aortocaval fistula (ACF)‐induced volume overload HF adversely impacts LV functional recovery and if pre or post‐surgical treatment with levosimendan (Levo, myofilament Ca2+ sensitizer) alters LV recovery.

Methods and Results

ACF or Sham surgery was performed in male Sprague‐Dawley rats (200‐240 g) 4 or 8 weeks prior to reversing (REV) the shunt in a subset of rats. REV during established HF at 8 wks resulted in impaired LV systolic (%FS, Ees, PRSW: 31.7%, 0.428 mmHg/µL, 57.4 mmHg, respectively; p<0.05 vs Sham) and diastolic function (tau, dP/dtmin; 12.2 msec, ‐6294 mmHg/sec, respectively; p<0.05 vs Sham), while REV during pre‐HF at 4 wks resulted in delayed, but improved LV systolic (%FS, Ees, PRSW: 39.7%; 0.813 mmHg/µL; 87.4 mmHg, p<0.05 vs ACF) and diastolic function (tau, dP/dtmin; 10.8 msec; ‐7128 mmHg/sec, p<0.5). A subset of ACF rats reversed at 8 wks received Levo (1 mg/kg) for 4 wks prior to REV (Levo‐P) or for 11 wks following REV (Levo‐R). Levo‐P improved LV function prior to and after REV (%FS = 42% and 31.8% at Wks 8 & 19, respectively), while Levo‐R improved LV function through the end of the study (%FS, Ees, PRSW, tau, dP/dtmin: 42.4%, 0.859 mmHg/µL, 98.7 mmHg, 8.7 msec, ‐9583 mmHg/sec, respectively; p<0.05 vs Sham). Improved LV function in Levo‐R correlated with normalized α‐to‐β‐myosin heavy chain and increased cTnI Ser‐23/24 phosphorylation (2‐fold vs REV).

Conclusion

These results demonstrate that LV dysfunction persisted when volume overload was reversed during established HF but was rescued if Levo was given continuously post‐REV.
Targeting mitochondrial oxidants in a pediatric model of sepsis‐induced cardiomyopathy
Authors: Sims Clark , Singh Preeti , Singh Sharda , Mayeux Philip
Published: June 2021

Multi‐organ failure is a frequent complication of infant sepsis. In fact, septic cardiomyopathy represents one of the major predictors of morbidity and mortality. Children have limited cardiac reserve as compared to adults, which could explain why 50% of children with septic shock present with low cardiac output and elevated systemic vascular resistance, referred to as “cold shock.” There are no effective therapies to treat sepsis and physicians must rely on supportive care. Reports suggest that sepsis can cause mitochondrial dysfunction in the heart, which may lead to cardiomyopathy. We developed a model of pediatric sepsis with “cold shock” using a clinically relevant cecal ligation and puncture (CLP) model in 17‐19 day‐old rat pups (developmentally comparable to a 5 to 6‐month‐old human infant). The goals of our study were to characterize the effects of sepsis on cardiac function using echocardiography and then evaluate the therapeutic potential of the mitochondria‐targeted antioxidant, MitoTEMPO. At 18 hours, stroke volume, cardiac output, ejection fraction, and fractional shortening were decreased by 36%, 37%, 20% and 30% respectively, in CLP (n = 5) versus Sham (n = 7; P < 0.05). When given at the time of CLP, MitoTEMPO (10 mg/kg, ip, n = 7) prevented the decrease in both ejection fraction and fractional shortening but not stroke volume or cardiac output. Still, survival at 18 hours was increased from 51% in CLP to 78% with MitoTEMPO, perhaps by reducing mitochondrial injury thereby preserving cardiac contractility. The data also suggest that MitoTEMPO should be evaluated in combination with agents that reduce systemic vascular resistance to improve cardiac output.
The Role of Piezo1 in Regulating Cytosolic Calcium Level in Aortic Smooth Muscle Cells in Rats
Authors: Zhang Jinglin , Tjong Jeff , Tang Nelson Leung‐sang , Yao Xiaoqiang
Published: June 2021

Introduction

Piezo1 proteins are large integral proteins with 24‐39 transmembrane domains. Piezos are expressed in several tissues. Piezo ion channel family is evolutionarily conserved in mechanotransduction. We examined the expression level of Piezo1 in rat aortic smooth muscle cells (ASMCs) and determined stretch induced cytosolic calcium rise in these cells.

Method

mRNA and protein expressions of piezo 1 and 2 proteins in rat tissues were detected by RT‐PCR and western blot study, respectively. Stretch‐induced cytosolic calcium elevation in RASMC was determined by confocal microscopy.

Results

In rats, Piezo1 mRNA and protein was highly expressed in aorta, aortic smooth muscle cells and mesenteric artery, but low in cerebral artery, nodose ganglion and petrosal ganglion. Stretch‐induced [Ca2+]i elevation was found to be increased in Piezo1 over‐expressing rat ASMCs compared with native rat ASMCs.

Conclusion

The results indicated that Piezo1 may play a functional role in stretch induced [Ca2+]i elevation in rat arterial smooth muscle cell.