bluuebunny/crossref_metadata_2025 huggingface dataset:
Developmental changes in peptide transport in the small intestine of the Yucatan miniature pig
Authors: Nosworthy Matthew G , Brunton Janet A
Published: June 2021

Di/tripeptides in the small intestine (SI) are transported via the oligopeptide transporter PepT1. Our objectives were to characterize the activity of PepT1 across the length of the SI and to describe ontological changes in PepT1 activity in early life. A laparotomy was conducted in piglets at 1,2,3 and 6 wk of age (n=4 per group). Four 10 cm ligated SI segments were created with the first at 15 cm distal to the ligament of Trietz and at 25, 50, and 75% of SI length. 5 mM glycylsarcosine (gly‐sar) with 3H‐gly‐sar was perfused through each segment for 2 h. The perfusate was sampled every 10 min. Gly‐sar uptake was determined from 3H‐gly‐sar disappearance. Uptake in specific segments and between age groups were compared by ANOVA. In 1 wk piglets, greater dipeptide uptake occurred in the mid gut compared to the initial (p < 0.05) and distal segments (75%) (p < 0.05). In the mid gut, uptake was greater at 1 wk than at 2 and 3 wk (p < 0.05). At 2 and 3 wk, uptake was greatest in the initial segment compared to the other three sites (p <0.001). In 6 wk pigs, gly‐sar uptake was again greater in the mid gut compared to younger pigs (wk 2, p < 0.001). These observations suggest that PepT1‐mediated amino acid uptake in the mid gut may be important during gut stress such as at birth and weaning. Identifying factors that can induce PepT1, and providing amino acids as dipeptides might provide a nutritional advantage when the SI is compromised. (NSERC)
The effects of chromium (VI) and vitamin C on the frequency of micronucleated erythrocytes in Hybro chicken
Authors: Bajraktari Ismet Dervish , Halili Fetah Murtez , Gashi Agim Maliq , Maxhuni Mirjeta , Bislimi Kemajl , Fetahu Shukri
Published: June 2021

Chromium (VI) is a genotoxic metal which is rapidly reduced to chromium III by cellular enzymes. During the reduction to the less‐toxic trivalent form, chromium can react with macromolecules including DNA and cause lesions.

In this investigation, the evaluation of the genotoxic effects of chromium (VI) on the peripheral blood erythrocytes of Hybro chicken has been carried out. The effect of vitamin C on the genotoxicity caused by chromium (VI) has also been investigated. Chickens were divided into 5 groups (I–V) of 7 individuals each and orally administered varying concentrations of Cr (VI) and vitamin C for four months. Our results show that four months ingestion of two different concentrations of chromium (VI) as well as chromium (VI) + vitamin C resulted in a significant increase in micronucleated erythrocytes. No protective effect of vitamin C in the concentrations applied was observed. Based on our data we confirm that Hybro chicken could be used as an alternative test for the evaluation of the genotoxic effects of environmental contaminants.
Prostate specific antigen activation peptide (PSAap) is detectable in breast cell line media
Authors: Voeghtly Laura , Enghild Jan , Latimer Jean , Oury Tim
Published: June 2021

Breast cancer (BrCa) is the most common cancer in women and currently has no universally accepted biomarker for its detection. It has been shown that prostate specific antigen (PSA) is not prostate specific and is secreted in more than 30% of breast tumors. The function of PSA in breast tissue has not been established, but has been shown to be indicative of neoplastic state. We have shown that PSA, a proenzyme, is activated extracellularly allowing its activation peptide (PSAap) to be detected in biological samples. Activated PSA reacts rapidly with protease inhibitors including α1‐antichymotrypsin and α2‐macroglobulin in the blood. These complexes are removed from the circulatory system by hepatocyte‐mediated endocytosis. Previous data from our lab show that this rapid clearance can interfere with serum detection of PSA. Significantly, we have shown that PSAap is cleared through simple renal filtration and detectable in the urine. To determine if PSAap is detectable in biological samples taken from BrCa patients, we first show that PSAap is found in the media of breast tumor lines when stimulated with hormones. These results are similar to our previous studies with prostate cancer (CaP) cell lines which indicate the ability to detect PSAap in the urine of CaP patients. If an increase in PSA production correlates with malignancy we propose that detection of PSAap in urine of BrCa patients may be a useful diagnostic tool.
Proteomic Analysis of the HCV Encoded Peptide NS5 A Cellular Protein Complex
Authors: French Samuel Wheeler , Gonzalez Oscar , Raychaudhuri Santanu , Fontanes Vanessa , Loo Rachel , Loo Joseph , Dasgupta Asim
Published: June 2021

Background: Hepatocellular carcinoma (HCC) is the third most common cause of cancer related death worldwide. One of most common causes of HCC includes infection with hepatitis C virus (HCV) in association with cirrhosis. The HCV protein NS5A is required for viral replication and has been implicated in the development of HCC through alteration of cell signaling. The oncogenic potential of NS5A and its impact on cell signaling renders it a reasonable target for therapeutic intervention.

Aim: To develop a system to determine cellular signaling pathways altered by the HCV protein NS5A through identification of proteins it form complexes with in cellular extracts.

Methods: NS5A protein complexes were purified from stable cell lines expressing NS5AFlag fusion protein, resolved by SDS‐PAGE and silver stained.

Results: Complex purification was optimized and at least two proteins (approximately 40 and 70 kDa) were identified as potential NS5A associated proteins.

Conclusions: We have identified 40 kDa and 70 kDa cellular protein members of the NS5A complex that may play a role in viral replication or augment the NS5A antiviral response mediated through PKR. Pharmacologic inhibition of these interactions may reduce viral replication and ultimately prevent hepatocellular carcinoma.

Supported by: Stein/Oppenheimer Endowment Award, CURE Pilot and Feasibility Study, CURE Named New Investigator Award
Sensitizing Hypermethylator Breast Cancer Cells to Chemotherapeutics using Demethylating Agents
Authors: Sandhu Rupninder , Risher J. Devon , Coleman William B.
Published: June 2021

Basal epithelial breast cancers express a hypermethylation defect characterized by silencing of numerous genes due to DNMT3b overexpression. This feature of basal breast cancers may account for their poor response to chemotherapy and poor long‐term survival. Using an in vitro model, we examined differences in the chemotherapeutic efficacy of doxorubicin hydrochloride (dox) against breast cancer cells depending on their methylation status. Hypermethylator breast cancer cell lines with various levels of DNMT3b (MDA‐MB‐453, BT20, and SUM102) were exposed to demethylating agents before treatment with dox. Demethylating treatment significantly increases the effectiveness of cytotoxic drugs, reducing the dose required for desired therapeutic effect. The dose of dox required to achieve 50% cell kill (IC50) is reduced by 30–35% after 3 days demethylating treatment and 60–65% after 7 days of treatment. These results indicate that: (i) demethylating treatment sensitizes hypermethylator cell lines to effects of chemotherapeutics, (ii) sensitivity is a function of the duration of exposure to demethylating drugs, and (iii) the effectiveness of demethylating treatment depends on the levels of DNMT3b activity in each cell line. These results strongly suggest that the efficacy of breast cancer chemotherapy can be improved by combination treatment with demethylating drugs.
CNS Metastasis Model for HER2 Positive Human Breast Cancers
Authors: Strom David K. , Filippelli Crystal S. , Andres Dale , James Mollie M.
Published: June 2021

Over‐expression of the HER2 oncogene is seen in 20% of all breast cancers. In this population of women, 30% will present with CNS metastasis following the initial diagnosis, and 50% of these women will die of progressive CNS disease. Treatment options are limited and patients’ quality of life is usually compromised.

Our laboratory has developed a model of CNS metastasis of a human HER2 positive breast cancer cell line which can be used to evaluate new therapies. We have found that a left ventricular, percutaneous injection of 1×106 cells into athymic rats causes detectable CNS metastases by 4 weeks in 80–90% of subjects. We are using this model to develop novel strategies for blocking CNS metastasis and treating CNS metastasis once it occurs.
Role of neurokinin‐1 receptor during the transition of inflammation to dysplasia in a colitis‐associated colon cancer model
Authors: Pagan Beatriz , Isidro Angel A , Rivera Edelmarie , Coppola Domenico , Wu Jie , Appleyard Caroline B
Published: June 2021

Long‐standing colitis markedly increases the risk of developing colorectal cancer. Neurokinin‐1 receptors (NK‐1R) have been shown to be involved in chronic colonic inflammation but their role in the transition to dysplasia is unclear.

Aim

To investigate the role of NK‐1R in a novel model of colitis‐associated dysplasia.

Methods

Sprague Dawley rats received trinitrobenzene sulfonic acid (TNBS; 30 mg in 50% ethanol ic), followed six weeks later by reactivation with TNBS (5 mg/kg iv) for three days. To induce colitis‐associated dysplasia the rats then received TNBS (iv) twice a week for ten weeks. One group received the NK‐1R antagonist SR140333 (1 mg/kg ip) twice a week for ten weeks; the rest received vehicle. After sacrifice the colons were removed and analyzed for total macroscopic damage, histological analysis, and real‐time RT‐PCR.

Results

Pathological analysis revealed a decreased incidence of dysplasia in animals treated with the NK‐1R antagonist compared with the vehicle treated group. Animals receiving SR140333 had significantly less macroscopic and microscopic damage, and a down‐regulation of mRNA levels for EGFR and NK‐1R, which correlated with the pathology.

Conclusion

Our results suggest that a selective NK‐1R antagonist can delay the development of damage and dysplasia in a novel model of colitis‐associated dysplasia, offering its potential therapeutic use. 1U56 CA126379‐01 & 1F31 GM078951.
The impact of IL‐1 deficiency in colitis‐mediated colon cancer
Authors: Stillie RoseMarie , Shukkur Farooq M , Stadnyk Andrew W
Published: June 2021

Inflammatory bowel diseases (IBD) predispose individuals to colon cancer. IL‐1, elevated during IBD, is implicated in cancer development in several models. Our objective was to determine the contribution of IL‐1 to carcinogenesis in a mouse model of colitis‐mediated colon cancer. Wildtype (WT), IL‐1R1−/− and caspase‐1−/−mice (n=6–12) were treated with azoxymethane (10 mg/kg) followed by 3 cycles of 3% dextran sulphate sodium. Unlike WT, IL‐1R1−/− and caspase‐1−/− mice had significant mortality (40–50%) after the second and third cycles, although inflammation severity in the mid‐colon did not differ between strains. All strains showed non‐invasive colon carcinoma. Cancer incidence did not differ significantly between strains (WT 60%, IL‐1R1−/− 100% and caspase‐1−/− 100%), however IL‐1R1−/− mice had a higher tumor multiplicity compared to WT and caspase‐1−/− mice (p<0.05). In conclusion, IL‐1R1−/−and caspase‐1−/− mice are more susceptible to chronic inflammation, despite histological inflammation scores that are similar to WT. Although cancer incidence did not differ between strains, the higher tumor number in IL‐1R1−/− mice suggests a protective role for IL‐1 in this model.

Funded by Crohn's and Colitis Foundation of Canada
Effect of EGCG on SGC‐7901 cells migration and metastasis
Authors: Zhang Jingwei , Zhou Yunfeng , Chen Huaqi , Fan Biao , Zhang Yimin , Han Guoge , Wei Lei
Published: June 2021

Objective: The actin binding protein Vasodilator‐stimulated phosphoprotein(VASP) has complex effects on cytoskeletal organization and cell motility, which play a positive role on tumour cells migration and invasion. Our research aim to investigate the effect of epigallocatechin gallate (EGCG), an extractent from green tea, on SGC‐7901 cells migration and invasion. Methods: The expression level of VASP in SGC‐7901 cells was detected by RT‐PCR and Western Blotting. Down regulation of VASP expression was performed with VASP siRNA transfection, while the activation of Rac1 pathway was enhanced by fMLP. Wound‐healing assay (2D assay) and transwell migration assay (3D assay) were employed to analyze invasive migration ability of SGC‐7901 cells. Results: After treated with EGCG, the expression level of VASP in SGC‐7901 cells decreased significantly at mRNA and protein level compared with control group (P<0.05). In 2D assay, the cell migration velocity was reduced by EGCG (P<0.05), accordingly the EGCG group showed lower invasion capacity than the control group in 3D assay (P<0.05).The effect of EGCG on SGC‐7901 cells migration and invasion was enhanced by VASP siRNA transfection, on the contrary decreased by fMLP. Conclusion: EGCG has a inhibitive effect on SGC‐7901 cells migration and invasion by decreasing the expression level of VASP via Rac1 pathway. (This work was supported by National Natural Sciences Foundation of China under Grant No.30770966)
ASK FOR WOMEN'S OPINION FIRST
Authors: Markovic Nenad , Markovic Olivera
Published: June 2021

INTRODUCTION

In 2006, the FDA approved a “new vaccine for prevention of cervical cancer and other diseases in females caused by human papilloma virus.” The news raised hopes for eradication of cervical cancer. Professionals recommended new strategies. Only few voices called for caution against over‐enthusiasm and change of Pap test screening. The authors of this abstract have been involved in the improvement of cervical cancer screening and presented their results on several EB meetings in the past.

MATERIAL & METHODS: One of the objectives within the mission of the Global Academy for Women's Health is to help women making educated decision on issues of their health. In 2006 and 2007, we evaluated most of the published literature on the cervical cancer prevention strategies, and conducted a limited survey among women at risk to learn more what they think about Pap test.

RESULTS: In the US, approximately 1 out of 2,000 women infected with HPV virus developed invasive cervical cancer, but almost all cervical cancers (10,000 /year) were infected with HPV (‘oncogenic’ and other subtypes). These numbers suggest a symbiotic rather than a causal association; HPV infecting the susceptible cancer and promoting its growth thereafter. The survey showed that 99% women were aware of Pap test, but only 70% participated (regularly or irregularly). The reasons for avoiding the test were cited as “uncomfortable” and “waiting for the results is stressful.” However, 93% would participate if an accurate and more convenient home test is available.

CONCLUSION: HPV vaccination is justified but its marketing strategy needs a more realistic approach. A home test added to HPV vaccination could be the future strategy that will embrace all women at risk.
In vitro activation of pro‐α‐defensins by neutrophil serine proteinases of rhesus macaque
Authors: KAMDAR KARISHMA , Maemoto Atsuo , Qu Xiaoqing , Ouellette Andre J
Published: June 2021

α‐Defensins are abundant antimicrobial peptides in the azurophilic granules of phagocytic leukocytes or in the Paneth cells of small intestine. To investigate the mechanisms of myeloid pro‐α‐defensin processing, recombinant pro‐α‐defensin 4 (proRMAD4) was exposed to human neutrophil elastase (HNE), cathepsinG (cG) and proteinase 3 (P3) from azurophil granules and analyzed biochemically and in bacterical peptide assays. ProRMAD4 was converted in vitro to active RMAD4 by these proteases. Only HNE cleaved at the native RMAD4 N‐terminus. cG and P3 activated proRMAD4, but they cleaved at different sites within the proregion. Mutagenesis of acidic proregion amino acids (Asp,Glu) to charge neutral (Asn, Gln) did not significantly alter the bactericidal property of proRMAD4 suggesting H‐bonding interactions also participate in the peptide inhibition. Disulfide variants of proRMAD4 were extensively degraded by HNE, cG and P3 showing that the disulfide array protects the α‐defensin from proteolysis during conversion to the bactericidal form. These data support the hypothesis that azurophil serine proteinases activate macaque myeloid α‐defensins and convert them to active forms. This research was supported by NIH grants DK044632, AI059346, Human Frontiers Science Project
Adiponectin Downregulates C‐Reactive Protein Synthesis and Secretion from Human Aortic Endothelial Cells: Evidence for a Adipose‐Vascular Loop
Authors: Devaraj Sridevi , Torok Natalie , Jialal Ishwarlal
Published: June 2021

C‐reactive protein (CRP), the prototypic marker of infammation, is a cardiovascular risk marker. Also, several lines of evidence point to a pro‐atherogenic role for CRP. We have previously shown that in addition to the liver, human aortic endothelial cells (HAEC) synthesize and secrete CRP. While CRP levels are increased in obesity, metabolic syndrome, diabetes and CAD, levels of the adipocyte‐derived hormone adiponectin are reduced in humans with these conditions. We tested the hypothesis that adiponectin reduces CRP synthesis and secretion in HAECs. Globular adiponectin dose dependently reduced CRP mRNA and protein from HAEC. Adiponectin treatment of HAEC significantly decreased phosphorylation of IKb and decreased NFKb binding activity. There was no effect of adiponectin on STAT or CEBP transcriptional activity. Globular adiponectin also activated AMP kinase resulting in decreased NFKb activity and decreased CRP mRNA and protein. These effects of adiponectin were mimicked by AICAR, an activator of AMPK and reversed by inhibition of AMPK. Globular adiponectin reduces CRP synthesis and secretion from HAEC via upregulation of AMP kinase and downregulation of NFKb. Similar findings were observed in rat primary hepatocytes. Thus, strategies aimed at upregulating adiponectin could attenuate the pro‐inflammatory state by decreasing CRP production.
Extracellular regulatory kinase (ERK1/2) regulates 15‐lipoxygenase Expression in Primary Human Monocytes
Authors: Bhattacharjee Ashish , Pal Srabani , Feldman Gerald M , Cathcart Martha K
Published: June 2021

Interleukin‐13 (IL‐13) induces the expression of the lipid‐oxidizing enzyme 15‐lipoxygenase (15‐LO) in primary human monocytes. 15‐LO catalyzes the oxygenation of certain polyunsaturated fatty acids to their corresponding hydroperoxy derivatives, which play an important role in the pathogenesis of atherosclerosis and inflammation. Previously we identified the functional IL‐13 receptor components, the association of Jaks and the activation of Stats. We have also observed the involvement of a cytosolic signaling complex containing Stat3, p38MAPK and PKCδ, in regulating 15‐LO. Our recent data demonstrate that ERK1/2, another MAP kinase, is involved in regulating IL‐13‐stimulated 15‐LO expression in monocytes. IL‐13 induces the activation of ERK1/2 in a time‐dependent manner. Our results show that the ERK1/2 pharmacological inhibitors block both IL‐13 induction of 15‐LO mRNA as well as protein expression. We further demonstrate that ERK1/2 is present in the signaling complex along with p38MAPK, and PKCδ and that ERK1/2 activity has no role in regulating this complex formation. Further investigations revealed that the PKCδ, p38 MAPK and ERK1/2 pathways are independent, parallel pathways, but are indispensable for 15‐LO expression. Our current studies are focused on exploring the mechanisms by which ERK1/2 regulates the gene expression of 15‐LO, an important regulator of inflammation and apoptosis.

(This work was funded by HL51068)
MMP‐1 and MMP‐3 stimulate macrophage MMP‐9 expression by upregulating components of the PGE2 biosynthetic pathway
Authors: Khan K.M. Faisal , Steenport Michel L. , Du Baoheng , Dannenberg Andrew J. , Falcone Domenick J.
Published: June 2021

Evidence has implicated MMP‐9 expression with the pathogenesis of occlusive and aneurysmal vascular diseases. In previous studies, we characterized a COX‐2→PGE2→EP4 receptor axis that regulates macrophage MMP‐9 expression. In the current studies, we determined whether MMPs in the inflammatory environment regulate expression of COX‐2→PGE2→EP4 receptor axis components. Results demonstrate that active MMP‐1 and MMP‐3 induce macrophage COX‐2 expression, which led to increased secretion of PGE2, PGE2→EP4 and subsequent MMP‐9 expression. MMP‐induced COX‐2 expression was coupled with increased expression of microsomal PGE synthase‐1 (mPGES‐1). Since COX‐2 expression is enhanced by PGE2 via an EP4‐dependent feedback loop, we determined whether PGE2→EP4 regulated levels of mPGES‐1. MMP‐1 and MMP‐3 selectively up‐regulated macrophage expression of PGE2 receptors, EP2 and EP4. Negligible levels of mPGES‐1 were observed in control macrophages and cells incubated with the EP2 agonist butaprost. In contrast, both PGE2 and the EP4 agonist PGE1‐OH stimulated mPGES‐1 expression, and PGE2 failed to induce mPGES‐1 expression in cells incubated with the EP4 antagonist ONO‐AE3‐208. Thus, MMPs regulate macrophage MMP‐9 expression by up‐regulating components of the PGE2 biosynthetic pathway. These studies were supported by HL073375 and the Center for Cancer Prevention Research.
Effects of oral methylphenidate on prefrontal cortex‐associated neurocognitive deficits in the spontaneous hypertensive rat strain
Authors: Kantak Kathleen M , Harvey Roxann C , Dembro Kimberly A , Mutebi Michael M , Singh Teghpal , Kerstetter Kerry A , Dwoskin Linda P , Deschepper Christian F
Published: June 2021

Though several animal models of Attention Deficit Hyperactivity Disorder (ADHD) exist, the spontaneous hypertensive rat (SHR) model is most frequently used. SHR display the same core behavioral symptoms as individuals with ADHD (hyperactivity, inattention, impulsivity), and also show extradimensional set‐shifting and non‐spatial working memory deficits that are unrelated to hypertension. The objective here was to determine if treatment with methylphenidate could prevent these deficits in the SHR. Extradimensional set‐shifting was assessed using the Attentional Set‐Shift Task and non‐spatial working memory was assessed using the Odor Delayed Win‐Shift Task. The SHR and several comparator rat strains were administered either methylphenidate at a clinically relevant dose (1.5 mg/kg), route (oral) and pretreatment time (30 min), vehicle, or were left untreated. Results demonstrated that treating the SHR with low‐dose oral methylphenidate prevented working memory and set‐shifting deficits that were observed in untreated SHR, and eliminated strain differences in both tasks. The findings provide further evidence that the SHR is a valid model for studying neurocognitive deficits associated with ADHD. Moreover, the current behavioral approach is appropriate to assess novel medications that target neurocognitive deficits associated with ADHD. Supported by SBE‐0354378, RO1 DA11716 and P50 DA05312.
Chronic treatment with paliperidone, like lithium and valproate, induces similar changes in expression and phosphorylation at the synaptoneurosomal level in rat prefrontal cortex (PFC)
Authors: McLeod Maria Corena , Oliveros Alfredo , Charlesworth Cristine , Madden Benjamin , Boules Mona , Liang Yian Qi , Williams Katrina , Richelson Elliott
Published: June 2021

Bipolar disorder is treated with mood stabilizers. mRNA data show that these drugs regulate synaptic protein expression. To determine protein levels and post‐translational processing of proteins and to determine if paliperidone (PALI), risperidone active metabolite, regulates synaptic proteins similar to mood stabilizers, we determined protein expression profiles in the PFC with chronic drug treatments. LiCl, valproate (VAL), or PALI was given daily by i.p. injections to 4 male Sprague‐Dawley rats/group for 28 d. Controls = 0.9% saline and 0.3% tartaric acid. Brains were dissected 24 h after final injections. Synaptoneurosomal proteins were extracted and separated by DIGE. Simultaneously, phosphorylation changes were determined (ProQ diamond staining). Reproducibly present data in all biological replicates were analyzed and compared with their respective controls using Interpreter (Ludesi). Proteins with differential expression and phosphorylation were identified by nano‐LC MS. PALI, LiCl, and VAL treatment resulted in similar protein expression profiles and phosphorylation patterns at the synaptoneurosomal level, suggesting that the mode of action for this drug is similar to that of mood stabilizers and supporting its use for this purpose. Interestingly, the expression profile for PALI was more similar to that of LiCl than that of VAL. Supported by a grant from Janssen Pharmaceutica, Inc.
Targeting Gβγ Signaling to inhibit Cellular Migration and Invasion of Breast Cancer Cells
Authors: Kirui Joseph K , Xie Yan , Wolff Dennis W. , Tu Yaping
Published: June 2021

Several GPCR ligands can promote breast cancer metastasis, thus signaling redundancy in the cancer microenvironment theoretically limits the ability of selective GPCR antagonists to prevent metastasis. Disruption of intracellular signaling pathways crucial for metastasis may be a better treatment strategy. We therefore tested the hypothesis that breast cancer cell migration and invasion, key steps of cancer metastasis, could be limited by blockade of the Gβγ subunit mediated responses that occur following GPCR activation. Highly metastatic MDA‐MB‐231 or ‐436 breast cancer cells were placed in transwell inserts without or with a layer of Matrigel to measure migration and invasion, respectively, in response to NIH 3T3 cell conditioned media. The rates of cell migration and invasion were attenuated >60% by expression of a Gβγ (inhibitor peptide derived from the carboxyl terminus of G protein‐coupled receptor kinase 2 (ct‐βARK). M119‐K, a specific Gβγ inhibitor, blocked the migration and invasion of metastatic breast cancer cells >80% (IC50 ≈ 3μM) without affecting cell viability while M119‐H, a structural analog that does not block Gβγ had no effect. Further studies indicated that the inhibition of breast cancer cell migration and invasion by the Gβγ inhibitor peptide or M119‐K is dependent on attenuation of Rac1 activation, a key promoter of breast cancer metastasis. This research was funded by LB595(Y.Tu).
Analysis of direct binding of small molecules to G protein βγ subunits: biophysical analysis and binding site mapping
Authors: Seneviratne Pramodh , Wedekind Joseph , Smrcka Alan V
Published: June 2021

Guanine nucleotide exchange proteins (G proteins) are heterotrimeric proteins composed of three subunits α, β, γ that initiate intracellular signaling cascades. Previously our laboratory identified small molecules that bind to a protein‐protein interaction “hot spot” to achieve selective modulation of downstream effectors. The central hypothesis of the study is that different compounds bind directly to different sub‐surfaces of the Gβγ ‘hot spot’. To assess binding of compounds directly to Gβγ we developed a Surface Plasmon Resonance (SPR) method utilizing immobilized Gβγ subunits. The results indicate that multiple compounds bind directly to Gβγ with affinities in the range of 400 nM to 10 μM. The binding is reversible, but with surprisingly slow on and off rate kinetics. For example: the Kd for binding of gallein to Gβ1γ2 was 420 nM with kon=1 × 103 M−1s−1, koff= 4 × 10−4 s−1. We have separately purified Gβγ subunits with single amino acids within the Gβγ hotspot mutated to alanine, and are assessing small molecule binding by SPR. We also obtained co‐crystals of several small molecules bound to Gβγ that diffract to 2.6 Å and are in the process of solving their three dimensional structures using x‐ray crystallography. Understanding how selective small molecules interact with Gβγ will be important for determining the mechanism of action of these compounds and developing more potent and selective agents.

This work was funded by National Institutes of Health GM 060286
Mice lacking RGS protein activity at Gαi2 exhibit a 5HT1A receptor‐mediated antidepressant‐like phenotype
Authors: Talbot Jeffery N , Clemans Crystal F , Graves Steven M , Huang Xinyan , Neubig Richard R , Traynor John R
Published: June 2021

Serotonin (5HT) acting through 5HT1 receptors plays an integral role in regulating mood. Multiple lines of evidence suggest 5HT1 receptors preferentially couple to Gαi2 relative to the more abundant Gαo. Therefore, transgenic “knock‐in” mice that express RGS‐insensitive Gαi2 (G184S; RGS‐i) were evaluated in the tail suspension test (TST), a classical assay of antidepressant‐related behavior in rodent models. In the TST, RGS‐i animals exhibited basal antidepressant‐like behavior and were 5–10 times more sensitive to the antidepressant‐like effects of the SSRI fluvoxamine and the 5HT1A‐selective agonist 8‐OH‐DPAT. In contrast, the antidepressant potency of the norepinephrine reuptake inhibitor desipramine was unchanged. Yet, 8‐OH‐DPAT‐mediated hypothermia was unaltered in animals of either genotype. Furthermore, the antidepressant‐like phenotype of RGS‐i animals was completely reversed by treatment with the 5HT1A‐selective antagonist WAY‐100635 without altering spontaneous motility. These data point to a critical role for endogenous RGS protein regulation at Gαi2 in mediating the antidepressant‐like effects of 5HT1A receptor activation. By selectively enhancing the antidepressant effects of serotonin, RGS proteins represent a novel therapeutic target for the treatment of depression. Supported by GM39561 and the Bower, Bennet, & Bennet Endowed Chair Research Award.
Modulation of α2C‐adrenergic receptor export trafficking by multiple Rab GTPases
Authors: Filipeanu Catalin M. , Wu Guangyu
Published: June 2021

At 37°C, α2C‐adrenergic receptor (AR) is poorly transported to the plasma membrane. Cold exposure enhances α2C‐AR translocation to the cell surface and this effect is exaggerated in some individuals leading to Raynaud Phenomenon. To identify the factors limiting α2C‐AR transport, the receptor was co‐transfected with well defined Rab GTPase dominant negative mutants in HEK293T cells. Subsequently, the α2C‐AR plasma membrane levels were determined at 37°C and 30°C by [3H]RX‐821002 binding. Inhibition of the endoplasmic reticulum (ER) to Golgi traffic with Rab1N124I equally attenuated α2C‐AR cell surface targeting at both temperatures. Blockade of the retrograde traffic from the Golgi to the ER using Rab2N119I had no effect on α2C‐AR cell surface expression. In contrast, inhibition of the retrograde traffic by Rab6N126I augmented α2C‐AR plasma membrane levels at 30°C, but not at 37°C. Reduction of Golgi to plasma membrane traffic by Rab11N124I did not influenced receptor translocation to the cell surface. However, Rab8T22N decreased α2C‐AR plasma membrane levels with similar extent at 37°C and 30°C. Our data indicate that Rab6‐dependent retrograde traffic is required to prevent exaggerated transport of α2C‐AR to the plasma membrane at low temperature. (R01GM076167, 5P20RR018766)
Morphine and etorphine activate same G protein but exhibit distinct pathways in μ‐opioid receptor‐mediated ERK1/2 activation
Authors: Zheng Hui , Loh Horace H , Law Ping‐yee
Published: June 2021

G protein‐ and β‐arrestin‐dependent pathways are well established mechanisms in agonist‐selective signaling of GPCRs. Classically, GPCR signaled via G protein initially and then switched to β‐arrestin‐dependent signaling. Recently, different GPCR conformations induced by different agonists were suggested to determine the signaling pathway, suggesting ß‐arrestin can mediate signaling without G protein activation. In current study, we could demonstrate agonist‐selective signaling in μ‐opioid receptor (MOR)‐mediated ERK1/2 activation in both cell models and primary hippocampal neurons. Morphine and etorphine activated ERK1/2 via PKC and β‐arrestin respectively. In addition, morphine‐activated ERK1/2 stayed in cytosol while etorphine‐activated ERK1/2 translocated into nucleus. Most importantly, the ERK1/2 phosphorylations induced by both agonists were blocked by PTX and could be restored with the over‐expression of PTX‐insensitive Gi2α mutant. Thus, we concluded that the conformations of agonist‐receptor complexes resulting in the recruitment of different cellular proteins such as PKC or ß‐arrestin will determine the pathway selected for the eventual signals. (Research supported by NIDA grants)
Interaction of the Sigma‐1 Receptor with Endogenous Sphingolipids and Alkyl Amines
Authors: Ramachandran Subramaniam , Chu Uyen B. , Hajipour Abdol R. , Mavlyutov Timur , Jackson Meyer B. , Ruoho Arnold E.
Published: June 2021

The sigma‐1 receptor (S1R) is a mammalian transmembrane protein implicated in anxiety, depression, schizophrenia, analgesia and drug dependence. The S1R displays promiscuous affinity for a range of pharmacological agents, however endogenous ligands are not known. Functions proposed for the S1R include ligand mediated chaperone activity, regulation of Ca2+ release, inhibition of K+ channels and antitumour effects.

We have discovered that members of the class of endogenous sphingolipids (e.g. sphingosine, N, N’ dimethyl sphingosine) bind to the pure or membrane bound S1R with considerable affinity. While sphingosine binds to the S1R its phosphorylated counterpart sphingosine‐1 phosphate does not bind. Specific binding of sphingosine but not its phosphorylated analogue may indicate a mechanism for regulation of S1R function. Furthermore, the characteristics of sphingosine binding to the S1R appear to be different from a competitive interaction suggesting a possible allosteric role of these lipids at the receptor.

In addition to sphingosine a series of aliphatic amines and their N‐substituted phenylpropyl derivatives were also found to interact with the S1R. These compounds modulated the voltage gated K+ channel, Kv1.4 expressed in HEK‐293 cells with EC50's for inhibition correlating strongly with their affinity at the S1R. These studies propose sphingosine as an endogenous modulator of the S1R and indicate a possible allosteric role for these lipids in S1R function and signal transduction.

Supported by NIH R01MH065503
Rapid, non‐genomic vascular actions of genistein suggests a phytoestrogen receptor
Authors: Lin Amanda H.Y. , Leung George P.H. , Leung Susan W.S. , Man Ricky Y.K.
Published: June 2021

Genistein, a phytoestrogen rich in soy beans and soy products, was reported to be a vasorelaxant. This study examined the receptor and related signaling pathways in the rapid vascular actions of genistein. Isometric tension was measured in isolated aortic rings from male spontaneous hypertensive rats (SHR). Acute exposure to genistein at 10 μM, a concentration with no direct relaxation effect, potentiated acetylcholine (ACh)‐induced relaxation and reduced ACh‐induced contraction. Both actions were insensitive to 10 μM actionmycin D (transcription inhibitor) and 10 μM cycloheximide (translation inhibitor). The potentiation of ACh‐induced relaxation by genistein was inhibited by 10 μM NF023 and 10 μM GP antagonist‐2A, the selective Gi/o and Gq α‐subunit antagonists respectively, but not by 10 μM NF449, a selective Gs α‐subunit antagonist. Interestingly, NF023, NF449 and GP antagonist‐2A did not alter the inhibitory effect of genistein on ACh‐induced contraction. These results demonstrate that rapid vascular actions of genistein in modulating ACh‐induced relaxation and contraction responses in SHR are mediated by non‐genomic pathways. Furthermore, involvement of G‐proteins in the enhancement of ACh‐induced relaxation by genistein suggests that genistein exerts its effect through a putative G‐protein coupled phytoestrogen receptor. (Supported by the Research Grant Council of Hong Kong)
High glucose (HG)‐induced angiotensin‐converting enzyme 2 (ACE2) and angiotensin (1–7) [Ang (1–7)] decrease is prevented by captopril, losartan and insulin in rat vascular smooth muscle cells (VSMCs)
Authors: Lavrentyev Eduard N , Malik Kafait U
Published: June 2021

Ang (1–7) prevents the development of diabetic cardiovascular complications. In VSMCs ACE2 is a major source of Ang (1–7). ACE2 expression is PKCβII dependent and it is decreased by exposure to HG via Nox1‐derived superoxides. In diabetic animal models captopril and losartan restore circulating Ang (1–7) levels. This study was conducted to determine if in rat VSMCs these agents and insulin prevent HG‐induced ACE2 and Ang (1–7) decrease. Cells were maintained in normal glucose (NG) or HG (~4.1 mmol/l and ~23.1 mmol/l, accordingly) for up to 72 hours and exposed to captopril, losartan, insulin or angiotensin II (Ang II). The cell lysates were subjected to Western blot, RTQ‐PCR and Ang (1–7) radioimmunoassay. HG‐induced ACE2 and Ang (1–7) decrease was prevented by captopril, losartan and insulin. Ang II alone reduced ACE2 expression in the presence of NG, which was prevented by captopril and losartan, and partially by insulin. HG‐induced Nox1 increase was diminished by captopril, losartan and insulin, whereas Ang II‐induced Nox1 increase was reversed by captopril and losartan but not insulin. HG deceased PKCβII level that was blocked by captopril, losartan and insulin. These data suggest that these agents prevent HG‐induced ACE2 and Ang (1–7) decrease by diminishing HG‐induced Nox‐1 expression and by restoring PKCβII level in VSMCs (Supported by NIH grant 079109‐03 from the HLBI).
Differential regulation of oxidative stress by NO in vascular and nonvascular tissues of endotoxemic rats
Authors: Sahan‐Firat Seyhan , Canacankatan Necmiye , Korkmaz Belma , Yildirim Hatice , Tamer Lulufer , Buharalioglu C Kemal , Sari A Nihal , Tunctan Bahar
Published: June 2021

Endotoxemic shock is a systemic inflammatory response that is associated with increased NO production by iNOS which contributes to the fall in blood pressure (BP), vascular hyporeactivity and multiple organ failure. NO has also been reported to have both stimulatory and inhibitory effects on oxidative stress (OS). We have previously demonstrated that endotoxin (ET)‐induced fall in BP is associated with an increase in nitrite levels in serum, kidney, heart, thoracic aorta (TA) and superior mesenteric artery (SMA), a decrease in MDA levels in the kidney, heart, TA and SMA, and an increase in MPO activity in the heart and TA, but a decrease in the kidney and SMA of rats. We further investigated the effect of NO on the markers of OS in other tissues of the endotoxemic rats. ET‐induced increase in nitrite production was associated with a decrease in GSH levels in the liver, lungs, brain, spleen and femoral artery (FA). MPO activity was increased by ET in the lungs, spleen and FA, but was decreased in the liver and brain. MDA levels were increased by ET in the lungs, brain, spleen and FA, but were decreased in the liver. SOD and catalase activity was decreased in the liver and spleen, but was increased in the lungs, brain and FA. The effects of ET were prevented by an iNOS inhibitor. The data suggest that NO differentially regulates OS depending on the tissue during endotoxemia. This study was supported by Mersin University Research Foundation.
Accessory β3 subunits promote the functional expression of voltage‐gated calcium channels in vascular smooth muscle cells
Authors: Sonkusare Swapnil , Rhee Sung W , Thakali Keshari , Fletcher Terry W , Stimers Joseph R , Telemaque Sabine , Rusch Nancy J
Published: June 2021

Four distinct β subunits (β1–4) promote tissue‐specific expression of the voltage‐dependent Ca2+ channel pore‐forming α1C subunits. Our goal was to identify β subunits promoting the functional expression of α1C in vascular smooth muscle cells (VSMCs). Expression of all four β subunits was detected at the transcript level in rat small renal, mesenteric and femoral arteries. Western blot analysis suggested that β3 and to a lesser extent, β2 subunits were expressed at the protein level in these resistance arteries, which led us to postulate that β3 promotes α1C expression. To evaluate this hypothesis, β3 siRNA was delivered into A10 and A7r5 (embryonic rat thoracic aorta) smooth muscle cell lines by electroporation. RT‐PCR showed nearly 75% reduction in β3 transcript 24 hrs after siRNA delivery, but no change in α1C transcript. At the protein level, β3 subunit expression was reduced 48 hrs after siRNA delivery, and a concurrent loss of α1C expression was observed. Expression levels of GAPDH and α‐actin (internal standards) were unchanged. Patch‐clamp studies showed a reduced voltage‐dependent Ca2+ current density in both cell lines 48 hrs after siRNA delivery (A7r5 cells: −4.4±1.6 pA/pF Vs −11.1±2.4 pA/pF; A10 cells: −2.8±0.4 pA/pF Vs −4.8±0.5 pA/pF, n= 3–4). Thus, β3 subunits may promote the expression of α1C to enhance voltage‐dependent Ca2+ influx at the surface membrane of VSMCs. Supported by R01 HL064806‐07(NJR).
Genetic Ablation of Caveolin‐1 does not Affect Pressure‐Induced Constriction but Alters Endothelin‐1 Pharmacology in Murine Cerebral Arteries
Authors: Jarajapu Yagna , Bishwokarma Bimjhana , Knot Harm , Lisanti Michael , Andersson Karl‐Erik , Christ George
Published: June 2021

Numerous phenotypic changes have been reported in mice with knockout of caveolin‐1 (KO) expression. This study evaluated cerebroarterial reactivity to investigate the role of caveolin‐1 in vascular smooth muscle function. Arterial segments were isolated from KO and wild type (WT) mice of age 6–9 months, pressure‐mounted and diameter was monitored. No difference was observed in constriction to intraluminal pressure up to 110 mmHg, further increase in pressure caused forced dilation. Constrictions to 60 mM KCl, 10 μM phenylephrine and 10 μM serotonin and dilation to 10 μM bradykinin and 16mM KCl were not different in WT and KO arteries. Concentration response curves to endothelin‐1 in WT arteries were biphasic. In KO arteries, constriction to ET1 was significantly decreased and the second low affinity phase was not observed. Decreased ET1 constriction was not restored by 100 μM L‐NAME. Myogenic tone at 70 mmHg was reversed by SKF 96365 and 2APB, known blockers of transient receptor potential (TRP) channels, in WT but only partially decreased in KO arteries. ET1 constriction was equally sensitive to SKF 96365 and 2APB in both groups. Myogenic tone was reversed by Y27632, a rho‐kinase inhibitor in both groups. Decrease in ET1 constriction by Y27632 was significantly lower in KO arteries compared to WT arteries. Constriction to pressure and ET1 may be modulated by caveolin‐1 via activation of TRP channels and rho‐kinase.
Circulating CD34‐positive cells stimulate phosphorylation of endothelial nitric oxide synthase in cerebral arteries
Authors: Santhanam Anantha Vijay Raghavan , Smith Leslie A , Katusic Zvonimir S
Published: June 2021

In animal models, transplantation of CD34+cells has beneficial effect on cerebral ischemia and recovery from stroke, however, the mechanism underlying cerebrovascular effects of CD34+cells is poorly understood. In the present study, CD34+ cells were obtained by magnetic cell sorting of rabbit peripheral blood mononuclear cells. Rabbit basilar arteries were exposed to autologous CD34+ cells (~5×105 cells) in vitro or in vivo. Arteries treated with endothelial growth medium‐2 (EGM‐2, vehicle) served as controls. Twenty four hours after intracisternal delivery of cells, basilar arteries were isolated and studied. In arteries treated with CD34+ cells either in vivo or in vitro, protein expression of phosphorylated Ser‐473‐Akt and phosphorylated Ser‐1177 endothelial nitric oxide synthase (eNOS) were significantly increased (n = 6, P<0.05), while expressions of eNOS, Akt, inducible NOS, cyclooxygenase‐1 (COX‐1) and COX‐2 were not affected (n = 6, P>0.05). In arteries exposed to CD34+ cells in vitro, cGMP content was significantly augmented (n = 4, P<0.01), whereas cAMP levels remained unaffected (n = 4, P>0.05). Our results suggest that in basilar artery, CD34+ cells stimulate phosphorylation of Akt and eNOS thereby increasing endothelial NO production. These findings may help to explain described therapeutic effect of transplanted CD34+ cells in cerebral circulation.
Potent endonthelial‐independent relaxation promoted by a nitrosyl ruthenium complex in rabbit aorta and corpora cavernosa
Authors: Fonteles Manassés Claudino , Cerqueira João Batista Gadelha , Sousa Clauber Mota , de Alencar Cunha Karina Moreira , Lopes Luiz Gonzaga França , de Sousa Moreira Ìcaro , Nascimento Nilberto Robson Falcão
Published: June 2021

Nitric oxide (NO) is a key mediator of both vasodilation and penile erection. Some metal nitrosyl complexes are being tested as NO donors. The current work compared LLNO1 a nitrosyl ruthenium complex to sodium nitroprusside (SNP). The tissues were mounted in organ baths for isometric recordings, precontracted with 1 μM phenylephrine and concentration‐response curves (10−11 a 10−4 M) to LLNO1 were obtained. In order to investigate the mechanisms involved, oxyhemoglobin (10μM), L‐cysteine (1 mM), glibenclamide (100 μM.) or iberiotoxin (1 μM) plus apamin (0,1 μM) were added 30 minutes before LLNO1 curves in separate protocols. LLNO1 completely relaxed phenylephrine‐contracted aortic rings with PD2 of 4.8 [5‐4.5] compared with a Rmax of 100% and PD2 of 5.8[6.3‐5.2] to SNP. This effect was partially blocked by oxyhemoglobin with Rmax reduced to 34.6±3.8% and PD2 [4.3[4.8‐3.9]. The corpora cavernosa strips were also relaxed by LLNO1 with Rmax of 72.6±6.6% and PD2 of 5.9[6.5‐5.3]. Similarly, this relaxation was blocked in 73% by pre‐treating the tissues with oxyhemoglobin. The incubation of tissues during 30 min with L‐cysteine, glibenclamide or iberiotoxin+apamin did not change the effect of LLNO1 in any of the tissues studied. LLNO1 is a potent aortic vasodilator and corpora cavernosa relaxant agent that acts predominatly by releasing NO and with no significant nytroxyl releasing properties.
Macrophages mediate organophosphorus pesticide‐induced airway hyperreactivity in guinea pigs
Authors: Proskocil Becky J , Bruun Donald A , Jacoby David B , Lein Pamela J , Fryer Allison D
Published: June 2021

Organophosphorus pesticide (OP) exposure is implicated in human asthma. At doses that do not inhibit acetycholinesterase, OPs such as parathion (Pth) block inhibitory neuronal M2 receptor function, which increases acetylcholine (ACh) release and potentiates vagally‐induced bronchoconstriction in guinea pigs. We tested whether macrophages mediate Pth‐induced airway hyperreactivity. Guinea pig alveolar macrophages were depleted with liposome‐encapsulated dichloromethylene‐diphosphonate (clodronate; prepared by Nico van Rooijen, Amsterdam) given intranasally 1 and 3 days prior to 1.0 mg/kg Pth sc. 24 hrs after Pth administration, animals were anesthetized and ventilated. In non‐Pth treated animals, bronchoconstriction induced by electrical stimulation of both vagus nerves was not affected by clodronate or PBS liposomes. Pth significantly potentiated bronchoconstriction in animals receiving PBS liposomes but did not potentiate bronchoconstriction in those pre‐treated with clodronate. ACh‐induced bronchoconstriction was not affected by any treatment, indicating that smooth muscle responsiveness was not changed. Macrophages mediate Pth‐induced airway hyperreactivity possibly by the release of interferon‐ã which decreases neuronal M2 RNA expression.

Funding: NIH ES014521, ES014601, HL55543, HL54659, HL071795