bluuebunny/crossref_metadata_2025 huggingface dataset:
Formation of thiol conjugates of shogaols in mice and in humans and modulation of glutathione levels in cancer cells by [6]‐shogaol
Authors: Chen Huadong , Soroka Dominique N. , Sang Shengmin
Published: June 2021

Extensive research has characterized ginger's anti‐inflammatory, antioxidant, and antitumor activities. Shogaols, a series of major constituents in dried ginger with the most abundant being [6]‐, [8]‐, and [10]‐shogaols, show much higher anti‐cancer potencies than gingerols. Previously, we reported the mercapturic acid pathway as a major metabolic route for [6]‐shogaol in mice. However, it is still unclear how the side chain length affects the metabolism of shogaols. In the present study, we first investigate the metabolism of [10]‐shogaol in mouse urine, with special focus on the mercapturic acid pathway, and then investigate the biotransformation of shogaols ([6]‐, [8]‐, and [10]‐shogaols ) in human urine. Our results show that eight major thiol‐conjugated metabolites of [10]‐shogaol were detected in mouse urine, while six major thiol‐conjugated metabolites of [6]‐shogaol, two thiol‐conjugated metabolites of [8]‐shogaol, and two thiol‐conjugated metabolites of [10]‐shogaol were detected in urine collected from human after drinking ginger tea, using liquid chromatography/electrospray ionization tandem mass spectrometry. Our results clearly indicate the mercapturic acid pathway is a major metabolic route for shogaols in mice and in humans. Furthermore, we also investigated the regulation of glutathione (GSH) by [6]‐shogaol in human colon cancer cells HCT‐116. Our results show [6]‐shogaol, after initially depleting glutathione levels, can subsequently restore and increase GSH levels over time. This activation mechanism will also be discussed.

Grant Funding Source: NIH
Accumulation of dietary naringenin and metabolites in mice
Authors: Ke Jia‐Yu , Schwartz Steven J , Riedl Ken M , Yee Lisa D , Kliewer Kara L , Belury Martha A
Published: June 2021

Naringenin is a grapefruit flavonoid that may ameliorate metabolic disturbances and exert anti‐carcinogenic actions in mice. The bioavailability of naringenin and its metabolites in tissues is still unknown. In a pilot study, we used LC‐MS/MS to analyze naringenin metabolites in plasma, inguinal adipose tissue, and liver. Male mice fed a diet with 3wt% naringenin did not show differences in food intake or tissue weights after 12 days of feeding. Plasma contained the highest concentration of naringenin (13.38 ± 4.00 μM). Naringenin monoglucuronides were the major derivatives identified in inguinal adipose tissue, while free naringenin was the dominant form of accumulated naringenin in liver. Currently, we are analyzing tissue samples from ovariectomized female mice fed with high fat diets containing 1% or 3% naringenin to compare the concentration of metabolites in tissues. Evaluation of naringenin metabolites and their concentration in metabolically relevant tissues will improve our understanding of the cellular and molecular mechanism by which diets/foods enriched in naringenin may exert beneficial effects on metabolic disturbances and carcinogenesis.

Grant Funding Source: 1R03CA162551–01A1
The Effect of Whole‐Grape Powder on Body Composition, and Serum Biomarkers of Adipose Tissue and Bone Metabolism in Postmenopausal Women
Authors: Zemenu Elleni , Kelly Owen , DiMarco Nancy
Published: June 2021

The effects of consuming freeze‐dried, whole‐grape powder (GP) or a placebo powder (P) for 12 weeks on body composition, biomarkers of inflammation, adipose tissue, and bone metabolism were determined. A randomized, double‐blind, placebo‐controlled study was performed with 40 post‐menopausal women (mean age 52.4±7.4) from 16 cities in Texas. Body composition and bone mineral densities (BMD) were determined using dual‐energy X‐ray absorptiometry (DXA). Serum concentrations of cross‐linked N‐telopeptide of type I collagen (NTX), alkaline phosphatase (ALP), osteocalcin (OC), insulin‐like growth factor‐1 (IGF‐1), adiponectin, leptin, prostaglandin E2 (PGE2), and leukotriene E4 (LTE4) were assayed using ELISA. Participants BMD were within normal ranges at baseline and at the end of study. NTX (p=0.04) and ALP (p=0.04) were significantly different between groups at baseline. In the placebo group (n=14), OC (p<0.01), adiponectin (p=0.01), and LTE4 (p=0.02) significantly decreased by 60.6%, 45.8%, and 61.9%, respectively. OC (p=0.02) and adiponectin (p=0.02) significantly decreased in GP group (n=11) by 50.9% and 59.7%, respectively. There were no significant differences between groups at 12 weeks. In the short term, freeze‐dried whole‐grape powder may exert no favorable effects on bone or adipose tissue biomarkers in post‐menopausal women.

Grant Funding Source: California Table Grape Commission.
Ursolic Acid Protects Monocytes Against Priming and Dysfunction Induced by Metabolic Stress by Preventing Induction of Nox4
Authors: Ullevig Sarah Lynn , Kim Hong Seok , Hambright William Sealy , Tavakoli Sina , Asmis Reto
Published: June 2021

We showed that mice fed at high‐fat diet (HFD) supplemented with ursolic acid (UA), an anti‐inflammatory phytochemical, were protected against atherosclerosis and monocyte dysfunction. Here we determine the molecular mechanism by which UA prevents metabolic stress‐induced monocyte priming and dysfunction. UA (0.3. 1, 3, 10 μM) protected THP‐1 monocytes against the sensitizing effect of metabolic stress on MCP‐1‐induced chemotaxis. Metabolically stressed monocytes treated with UA showed a decrease in total protein‐ and actin‐S‐glutathionylation, measures of thiol oxidative stress. MAPK phosphatase‐1 protein expression and phosphatase activity, reduced by metabolic stress, were rescued if UA was present during metabolic priming. UA treatment did not change protein and mRNA expression of glutaredoxin‐1, an enzyme that reduces glutathionylated proteins. However, UA blocked metabolic stress induction of Nox4. We therefore propose that UA prevents monocyte priming and dysfunction by suppressing Nox‐4 induction, thereby, preventing Nox4‐dependent dysregulation of redox‐sensitive processes, including actin turnover and MAPK‐signaling. These studies provide a novel mechanism of action for the anti‐inflammatory properties of UA. Funded by NIH RO1 HL070963 & HL115858 (RA).

Grant Funding Source: NIH RO1 HL070963 & HL115858
Effect of Isoflavones from Astragalus Membranaceus on 3T3‐ L1 Adipocyte Differentiation and Insulin Sensitivity
Authors: Choi Youngmin , Noh Jeongsook , Yun Seung‐Won , Kwon Young‐In , Kim Young‐Cheul
Published: June 2021

Bioactive isoflavones found in Astragalus membranaceus have been shown to alleviate hyperglycemia and insulin resistance in animal models of Type 2 diabetes. However, the major constituents and molecular mechanisms underlying the antidiabetic effects of Astragalus remain elusive. Here we examined the effect of formononetin, a major isoflavone found in Astragalus, on differentiation of 3T3‐L1 preadipocyte and regulation of PPARγ, a key transcription factor that mediates insulin‐sensitizing effects of antidiabetic drugs. Formononetin at 1–20μM concentrations dose‐dependently enhanced adipocyte differentiation, consistent with increased protein expression of PPARγ and C/EBPα. Formononetin also markedly induced the expression of PPARγ target genes such as fatty acid binding protein 4 (FABP4) and GLUT4 protein. In addition, formononetin treatment significantly increased glucose uptake as well as expression of insulin receptor substrate‐1 (IRS‐1), a key molecule for insulin signaling and action. Moreover, formononetin‐induced adipocyte differentiation and expression of PPARγ and FABP4 protein were blunted in the presence of PPARγ antagoists. Our results indicate that the antidiabetic effect of Astragalus membranaceus is mediated by formononetin through activation of PPARγ and its downstream target genes and insulin signaling pathway, leading to increased insulin sensitivity.
Lycopene Concentrate Supplementation Decreases Plasma Values for C‐Reactive Protein and Oxidized LDL
Authors: DiSilvestro Robert A , Joseph Elizabeth , DiSilvestro Daniel
Published: June 2021

Various lines of indirect evidence suggest that lycopene can exert anti‐inflammatory effects relevant to problems such as cardiovascular disease (CVD). However, a beneficial influence for lycopene supplementation on measures relevant to inflammation remain largely lacking. In the present study, a lycopene rich extract from tomato (20 mg lycopene/day in Lyc‐O‐Mato® tomato lycopene complex), or placebo, was given to healthy middle aged men for 6 weeks (N = 10 per group). Subjects did not have to follow a low lycopene diet, but a preliminary screening eliminated people with a history of frequent tomato product intake. In the lycopene group, plasma c‐reactive protein concentrations, which rise with CVD‐relevant inflammation, showed a mean decrease of almost 30% (p < 0.05, 2‐tailed paired t‐test). Placebo gave no statistically significant effect. Lycopene also produced a mean decrease of 5.1% in plasma oxidized LDL values, an atherosclerosis‐related measure that can be increased by inflammation (p < 0.05, 2‐tailed paired t‐test). Although this percent decrease was not tremendously large, longer intervention periods and/or combination with other natural products may heighten the effect. Again, placebo was ineffective. Neither of the lycopene effects was boosted by adding supplementation with zinc arginate (15 mg zinc/day) + selenium (200 μg selenium/day) in another group of subjects. These mineral supplements did improve zinc and selenium functional status based on two measures for each mineral. In summary, in a small study, supplementation with a tomato extract‐lycopene by itself produced changes in two cardiovascular disease‐relevant measures related to inflammation.
Astaxanthin mediates inflammatory biomarkers associated with arthritis in human chondrosarcoma cells induced with interleukin‐1β
Authors: Kimble Lindsey , Mathison Bridget , Chew Boon P
Published: June 2021

Arthritis is characterized by chondrocyte dysfunction resulting in inflammation, matrix metalloproteinase (MMP) production and progressive degeneration of articular cartilage. Astaxanthin, a potent antioxidant, may downregulate inflammation‐associated factors involved in arthritis. We studied the uptake and protective effects of astaxanthin against chondrocyte dysfunction using SW‐1353 human chondrosarcoma cells. Cells were pre‐incubated with 0, 0.01, 0.1, or 1.0 μmol/L astaxanthin for 48 h, and oxidative stress induced with 10 ng/mL IL‐1β overnight. Astaxanthin (1.0 μmol/L) accumulated in SW‐1353 cells in a time‐dependent manner. IL‐1β alone suppressed intracellular antioxidant activity, resulting in overproduction of reactive oxygen species (ROS), MMP‐13, inflammatory cytokines and mediators. In contrast, pre‐incubation with astaxanthin increased (P < 0.05) glutathione peroxidase activity and decreased (P < 0.05) ROS, MMP‐13, IL‐6, TNF‐α and inflammatory mediators. Pre‐treatment with astaxanthin also downregulated transcriptional activation of NF‐κB and activator protein‐1, which play critical roles in downstream production of MMP, inflammatory cytokines and mediators. Astaxanthin protects against degenerative factors upregulated by IL‐1β, likely by scavenging ROS required for transcriptional activation. Supported by Washington State University.
Regulation of AMPK‐beta‐catenin pathway via GSK3beta dependently or independently in Hep3B cancer cell growth and apoptosis control with selenium
Authors: Park Song Yi , Lee Yun‐Kyoung , Kim Young‐Min , Park Ock Jin
Published: June 2021

This study approached to address the mechanism of cancer cell suppressive activities of selenium and the consequence of the activation of AMPK on the important signal of cancer cell survival β‐catenin. We have shown that selenium activated AMPK, and the activation of AMPK with selenium suppressed β‐catenin. Selenium induced the translocation of AMPK into nucleus, and prevented the accumulation of β‐catenin, a prime regulatory molecule of many oncogenic genes into nucleus. Upon the inactivation of AMPK either by AMPK siRNA or Compound C, selenium no longer modulated β‐catenin implying that AMPK is an upstream signal for β‐catenin. By applying fluorescent markers of nucleus, AMPK and β‐catenin, it was shown that selenium down‐regulated expressions of β‐catenin and AMPK and β‐catenin were clustered together right outside of nuclear membrane. In the AMPK inactivated states with AMPK siRNA the translocation into nucleus of both these signals was facilitated under selenium treatment. Also, we have found that cancer cell anti‐proliferative effect of selenium was mediated by a GSK3β dependent AMPK/GSK3β/β‐catenin or GSK3β independent AMPK/β‐catenin pathway. These findings imply that selenium has a chemopreventive potential through the regulation of AMPK involving a complex downstream signaling pathways of β‐catenin. [This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (NO. R01–2008‐000–20131‐0)]
Saussurea involucrata extract inhibits metastasis in SK‐Hep1 hepatocarcinoma cells
Authors: Byambaragchaa Munkhzaya , Kim Gi Dae , Hwang Seong Gu
Published: June 2021

The rate of morbidity and mortality of hepatocellular carcinoma has not lessened because of difficulty in treating tumor metastasis. Saussurea involucrata extract (SIE) possesses various anticancer activities, including apoptosis and cell cycle arrest. However, detailed effects and molecular mechanisms of SIE on metastasis are unclear. Thus, this study was undertaken to investigate the antimetastatic effect of Mongolian Saussurea involucrata on HCC cells as well as possible mechanisms. Effects of SIE on the growth, adhesion, migration, aggregation and invasion of the SK‐Hep1 human HCC cell line were investigated. SIE inhibited cell growth of metastatic cells in a dose‐ and time‐dependent manners. Incubation of SK‐Hep1 cells with 200–400μg/mL of SIE significantly inhibited cell adhesion to gelatin‐coated substrate dose‐ and time‐dependently. In the migration (wound healing) and aggregation assay, SIE treated cells showed lower levels of migration and aggregation than untreated cells. Invasion assay revealed that SIE treatment inhibited cell invasion capacities of HCC cells substantially. Testing of mRNA level, quantitative real time PCR showed the inhibitory effects of SIE on MMP‐2 and MMP‐9 and stimulatory effect of SIE on TIMP‐1 which is inhibitor of MMPs. Gelatin zymography analysis for MMPs protein activity showed that SIE treatment may lead reduced activities of MMP‐2 and MMP‐9 in a dose‐dependent manner. The present study not only demonstrated that invasion and motility of cancer cells were inhibited by SIE, but also indicated that such effects were likely associated with the decrease in MMP‐2/‐9 expression of SK‐Hep1 cells. From these results, it was suggested that SIE could be used as potential anti‐tumor agent.
Boric Acid Activated Pathways in Prostate DU‐145 Cells
Authors: Eckhert Curtis , Kobylewski Sarah , Henderson Kimberley , Yamada Kristin
Published: June 2021

Boron intake is associated with reduced risk of prostate cancer and ultrasound determined gland volume. Boric acid (BA) at physiological concentrations is a reversible competitive inhibitor of cyclic ADP ribose (cADPR), depletes luminal endoplasmic reticulum Ca2+ and activates eIF2αP in DU‐145 prostate cells [Henderson and Eckhert 2009; 2006]. Our objective was to characterize the events that follow BA‐cADPR interaction. Treatment [10 μM BA] was followed by a dose dependent expansion of the ER and time dependent formation of TIA‐1 positive stress granules, decrease in global protein synthesis, and activation of the eIF2α/Grp78/ATF4 integrative stress pathway and the ATF6 branch of the unfolded protein response. BA increased expression of ATF4 downstream genes GADD34 and HERP and their respective proteins. CHOP, a pro‐apoptotic protein was decreased by BA treatment a result that is consistent with survival and differentiation. BA increased cytoplasmic ATF6 protein, its movement into the nucleus and downstream proteins Grp78, Grp94, calreticulin and XBP1. Cleavage of XBP1, an indicator of IRE1 activation, was not observed. HERP and EDEM1 mRNA were significantly up‐regulated by BA treatment. Our results extend current knowledge of the BA effect from rapid dose dependent inhibition of cADPR to time dependent activation of pathways known to be expressed in tissues affected by boron status. Funded by payments for teaching.
Changes in Leukocyte Populations among Children Receiving a Follow‐on Cow's Milk‐Based Formula Containing Lactobacillus rhamnosus GG
Authors: Sarvetnick Nora , Murphy Kevin , Pauley‐Hunter Rosemary , Scalabrin Deolinda
Published: June 2021

Background

Lactobacillus rhamnosus GG (LGG) is a probiotic with demonstrated health benefits, and these benefits may be due to LGG‐induced changes in the immune system.

Objective

We examined changes in immune cell populations and activation markers produced by the addition of LGG to a children's follow‐on formula.

Methods

This was a double blinded, randomized, controlled, parallel‐designed, prospective study. Data is reported for participants who completed the study. Participants (1–4 years of age) received either a marketed, follow‐on formula (n=8), formula supplemented with 42 mg of β‐Glucan per serving (n=10), or formula with 6 × 109 colony forming units of LGG per serving (n=9), twice a day for a 3 week period. Flow cytometry was used to immunophenotype peripheral blood leukocytes. Thirty serum cytokines were measured via multiplex analysis. Changes from baseline were analyzed by Kruksal‐Wallis. As this was a pilot study, differences with p‐values < 0.10 were considered of interest.

Results

Participants that received LGG showed reduced levels of IL‐2 and IL‐3. They also had a reduction in the total proportion and general activation of CD4 T cells and a decrease in specific subsets of double negative T cells.

Conclusion

These data suggest that LGG has an anti‐inflammatory effect by reducing available T cell populations that can cause inflammation.
Exploring phospho‐signaling pathways in patients with agerelated macular degeneration (AMD), human pigment epithelial cell line ARPE‐19 and porcine ocular tissue
Authors: Fasler‐Kan Elizaveta , Barteneva Natasha , Meyer Peter
Published: June 2021

The pig has been used extensively in experimental studies of human eye diseases. Pig retina shares many morphological features with the human eye. Retinal pigment epithelial cells constitute an important component of the blood‐retinal barrier and play a pivotal role in the development of AMD. Understanding the underlying molecular mechanisms is a prerequisite for developing therapeutic strategies. We investigated cytokine‐induced changes of the JAK/STAT and NF‐kappa B signaling pathways.

Methods

Porcine ocular cells, human ARPE‐19 cell line, eyes with AMD were used to study the nuclear translocation of STAT and NF‐kB proteins by gel‐shift assay, immunohistochemistry, confocal laser microscopy and flow cytometry.

Results

Histological examination of AMD patients revealed a strong formation of choroidal neovascular membranes in the macular region. Strong activation of STAT3 was observed in these membranes. Strong STAT1 activation was demonstrated upon exposure to interferon‐γ in ARPE‐19 and pig ocular cells in vitro. Human recombinant interferon‐α upregulated STAT1, STAT2 and STAT3, while IL‐6 and IL‐4 activated STAT3 and STAT6, respectively. Confocal microscopy identified the nuclear translocation of the STAT proteins.

Conclusion

Our data demonstrate the upregulation of the members of the JAK‐STAT signaling pathway in AMD patients, porcine ocular cells and ARPE‐19 cell line.
Live kidney slices present a novel method for delineating the mechanisms of calcineurin inhibitor‐mediated nephrotoxicity
Authors: Kelly Mark , Crawford Carol , Loo Ruey‐Leng , Delaney Michael , Farmer Chris , Wildman Scott S.P. , Peppiatt‐Wildman Claire. M.
Published: June 2021

Cyclosporin A (CsA) and Tacrolimus (FK‐506) are potent immunosuppressant drugs widely used to prevent allograft rejection in organ transplantation. They are calcineurin inhibitors (CNIs) and have limiting side effects including nephrotoxicity that impact upon long‐term renal allograft and patient survival in other organ transplantation. The precise mechanisms of CNI‐induced toxicity are unknown, although ischemic injury resulting from impairment of endothelial cell function, reduced production of endogenous renal vasodilators and concomitant release of vasoconstrictors resulting in net vasoconstriction have been described.

We have utilized the live kidney slice model, developed by our group [1], to investigate the mechanisms of CNI‐induced nephrotoxicity in the renal medulla, a region of the kidney that is particularly sensitive to ischemia

Data presented here demonstrates both CsA (600 ng/ml) and FK‐506 (800 ng/ml) cause vasoconstriction (9.9% ± 0.88% and 9.2% ± 0.63%, respectively) of vasa recta via their specific action at contractile pericytes. Tubule diameter was unaffected by bath application of both CsA and FK‐506. Data suggest that CNI‐induced constriction of vasa recta by pericytes may exacerbate renal medullary ischemia contributing to observed nephrotoxicity in solid organ transplantation.

Research supported by the Medical Research Council and University of Kent.
Inhibition of toll‐like receptor signaling by an ITIM‐containing bacterial protein
Authors: Ge Baoxue
Published: June 2021

The translocated intimin receptor (Tir) in enteric bacteria shares sequence similarity with the cellular immunoreceptor tyrosine‐based inhibition motifs (ITIMs). Despite its importance in pedestal formation, the role of the Tir and its ITIMs in the regulation of the host immune response remains unclear. Here, we demonstrate that Tirs from enteropathogenic Escherichia coli (EPEC) interact with the cellular tyrosine phosphatase SHP‐1 in an ITIM phosphorylation‐dependent manner. The association of the Tir with SHP‐1 facilitated the recruitment of SHP‐1 to TRAF6 and inhibited the ubiquitination of TRAF6. Moreover, Tir ITIMs suppressed EPEC‐stimulated expression of pro‐inflammatory cytokines and contributed to intestinal immunity against Citrobacter rodentium infection. These findings identify a previously unknown mechanism by which bacterial ITIM‐containing proteins can inhibit innate immune responses.
Severe Hyperglycemia Down Regulates Toll‐Like Receptors on Neutrophils: Implications for Propensity to Infections in Diabetics
Authors: Jialal Ishwarlal , Polage Christopher , Devaraj Sridevi
Published: June 2021

While diabetics are predisposed to increased infections the exact mechanisms are unclear. This increased propensity to infections in diabetics may be due to impaired innate immune responses. Toll like receptors (TLR) are pattern recognition receptors of the innate immune response and TLR 2 and 4 are increased on monocytes in diabetic patients. Neutrophils play an important role during the early host response to infection by a coordinated series of effector functions that include phagocytosis, generation of reactive oxygen species (respiratory burst) and killing. However, there is a paucity of data on TLR expression on neutrophils in diabetes. Thus, we examined TLR 2 and 4 expression on neutrophils under diabetic conditions. Neutrophils were isolated from healthy human volunteers and incubated with different concentrations of glucose (5.5 mM, 15 mM, 30 mM glucose with Mannitol serving as an osmotic control). Hyperglycemia upregulated TLR 2 and TLR 4 expression at 15 mM compared to 5.5 mM, however, at very high glucose conditions, 30mM, there was a significant decrease in both TLR2 and TLR4 expression (60% and 80% decrease respectively, p<0.001 by ANOVA). Phagocytosis of S. aureus was decreased in PMN incubated with 30mM glucose (50% decrease, p<0.05) and killing of S. aureus was impaired in PMN incubated with the bacteria at 30mM glucose concentrations compared to 5.5 mM glucose. These studies point to impaired TLR2 and 4 expression and activity on neutrophils resulting in decreased phagocytosis and killing of bacteria at very high glucose (30mM glucose) concentrations. Future studies will examine mechanistic pathways for this impaired TLR activity in neutrophils in diabetes, which could explain the increased susceptibility to infection in diabetic patients.
Age changes of immunological reactivity of an organism under the influence of low doses of gamma‐radiation
Authors: Ilderbayeva Gulzhan , Abduldayeva Aigul , Ilderbayev Oralbek , Nurmukanov Dulat
Published: June 2021

We study the influence of low doses of gamma radiation on the immunological reactivity of rats of different ages. The is a conclusion of the ethical committee (protocol N4, 2012, State medical university, Semey). In compare immunological parameters of 12, 18, 24 month rats with increasing age the reduction of W.B.C. and lymphocytes in the blood was identified. Decreased functional activity of the cellular immune system, the reliability of changes in the organism was grown in the organism of old rats. The influence of low doses of gamma radiation in dose 0,2 Grey showed inhibition of functional activity of T‐link cells in 12 month rats, the activation of the metabolism in phagocyte cells and activation of humoral system of immunity. Low doses of gamma‐radiation cause activation humoral immunity and phagocytic reactions. Under influence of low doses of gamma radiation in the 18 month rats leucopenia and lymphopenia was revealed, the activity of MIF in W.B.C, and CD3+, CD4+ decreased, the concentration of CIC increased, the changes in the immune system more expressed than in 12 month rats. The 24 month rats leucopenia and lymphopenia was revealed, the content of CD3+, CD4+, CD8+ cells decreased, inhibition of lymphokine producing function of leukocytes and phagocytes process was observed. In old aging rats adaptation ability on influence of extreme stress factors was decreased, the autoimmune processes were activated.
Altered ZO‐1‐Actin Interaction Induces an Epithelial to Mesenchymal Transition
Authors: Klindworth Kevin O , Lyford Brooks P , Nepomuceno Helene L , King Jonathan M
Published: June 2021

Zonula occludens‐1 (ZO‐1) is a large scaffold protein necessary for tight junction formation and signaling. The ZO‐1 protein includes a unique C‐terminal structure, including an actin‐binding region (ABR) and ZU‐5 terminal domain, which play a relatively unknown role. Mouse ZO‐1 C‐terminal phrGFP‐fusion constructs (ABR and ABR‐ZU5) were prepared and stably transfected into MDCK II cells. Appropriate localization was observed in all stably cell lines except for the highly expressing GFP‐ABR‐ZO‐1 cells. The GFP‐ABR‐ZO‐1 construct markedly altered cell phenotype into spindle‐like cells deviating from the expected epithelial monolayer. GFP‐ABR‐ZO‐1 appears to interact with bundles of F‐actin as examined by confocal microscopy. These studies highlight the ZO‐1‐ABR region as an important component in the tethering of the actin cytoskeleton to the apical junction complex. Proliferation and migration was enhanced in the in GFP‐ABR‐ZO‐1 cells and wound healing was markedly impaired by 5‐fluorouracil, a cell proliferation inhibitor. qRT‐PCR and Western blotting demonstrated that junctional markers (occludin and claudin‐1) were significantly down regulated by ABR overexpression whereas vimentin, a mesenchymal marker, was elevated. The ZO‐1 C‐terminus mediates critical epithelial cell homeostasis events and in addition to epithelial to mesenchymal transition.

Supported by TU‐HHMI program.
Selective tethering of mural cell phosphodiesterase 4D variants allows spatial resolution of cAMP‐mediated events required for adhesion and migration
Authors: Truong Tammy K , Freitag Silja I , Brzezinska Paulina B , Maurice Donald H
Published: June 2021

Mural cells can inhibit vascular lesion progression and stabilize angiogenic vascular tubes during tissue repair or they can act maladaptively and promote atherosclerotic lesion progression and arterial stenosis; effects dependent on binding to selected extracellular matrix (ECM) proteins and on choosing between chemotactic gradients. Although cAMP, acting via protein kinase A (PKA) or exchange protein activated by cAMP (EPAC), impacts mural cell adhesion or migration, a consensus has emerged that individual actions occur within discrete “compartments” populated by cAMP‐signaling complexes (cAMP‐signalosomes). Previously we showed that PKA in leading edge structures in migrating mural cells was in a cAMP‐signalosome also populated by one (PDE4D8) of the five PDE4D variants in these cells. Displacing PDE4D8 from these structures activated PKA, inhibited actin assembly and inhibited migration. Here we report on the subcellular targeting of the five mural cell PDE4D variant, identify their interacting protein partner(s), and assess their impact on mural cell adhesion/migration. Our data show that each PDE4D variant impacts mural cell cAMP‐signaling and selectively control specific cellular functions.
Simvastatin, Atorvastatin, and Pravastatin are Equally Effective in Improving Acetylcholine‐Induced Relaxation in STZ‐Diabetic Rats
Authors: Quidgley Jose , Cruz Nildris , Crespo Maria J
Published: June 2021

Statin therapy improves cardiovascular (CV) status in normocholesterolemic diabetic patients. To further clarify the underlying mechanisms, we studied and compared the effect of atorvastatin (AV), simvastatin (SV), and pravastatin (PV) (10 mg/kg/day) on acetylcholine (Ach)‐induced relaxation in the aortic rings of streptozotocin (STZ)‐induced diabetic rats. EMAX values for the Ach‐induced relaxation were lower in diabetic than in CT rats (53.70 ± 4.07 % vs. 74.61 ± 3.27 %, N=10, P<0.05). Four weeks of statin treatment improved EMAX in diabetic rats (AV 82.13 ± 7.01 %, SV 84.63 ± 6.51 %, and PV 79.40 ± 9.10 %, N=8, P<0.05), but EC50 values remained unchanged (from 0.41 ± 0.098 μM before treatment to an average across the three statins of 0.54 ± 0.252 μM after treatment, P>;0.05). All statins produced a similar improvement in vascular relaxation. Moreover, this positive statin effect was abolished by 1 mM LNAME, suggesting that it is NO‐dependent. The reduction of CV events by statins in diabetic patients may be secondary to improved endothelial function, which by decreasing vascular tone and systemic vascular resistance, will reduce the workload on the heart. Supported by MBRS‐RISE Grant R25‐GM061838.
Subtype specific β‐adrenerigic receptor‐mediated transactivation of epidermal growth factor receptor decreases apoptosis through differential activation of ERK1/2 and Akt
Authors: Grisanti Laurel A , Carter Rhonda L , Yu Justine E , Tilley Douglas G
Published: June 2021

β‐adrenergic receptors (βAR) are critical regulators of cardiac function whose dysregulation during heart failure are associated with diminished cardiac function, however βAR‐mediated EGFR transactivation has been shown to relay cardioprotection via unknown mechanisms. While both β1AR and β2AR are capable of inducing EGFR transactivation the relative impact of each on cardiac function is unknown. We hypothesized that transactivation of EGFR will produce distinct cardiomyocyte signaling responses leading to decreases in apoptosis that are βAR subtype‐specific. To test this hypothesis, rat neonatal cardiomyocytes (RNCM) were treated with dobutamine (β1AR agonist) or salbutamol (β2AR agonist) in the presence or absence of the EGFR antagonist AG1478. Following 10 min stimulation, differences in ERK1/2 and Akt phosphorylation and localization were observed. Apoptosis was decreased which was abrogated with EGFR inhibition. EGFR‐transactivation by βAR decreased apoptosis through ERK1/2 and Akt pathways. These results demonstrate differential impact of β1AR and β2AR‐mediated EGFR transactivation on the subcellular activation of ERK1/2 and Akt and the regulation of cell death in cardiomyocytes.
Cigarette smoke extract causes endothelial nitric oxide synthase dysfunction through stimulation of ubiquitin proteasome system
Authors: El‐Mahdy Mohamed A , Abdelghany Tamer M , Hemann Craig , De Pascali Francesco , Esmat Ahmed , Zweier Jay L
Published: June 2021

Cigarette smoking (CS) is a major risk factor for endothelial dysfunction (ED), a prognostic predictor for the risk of future cardiovascular events. Endothelial nitric oxide synthase (eNOS) dysfunction has been linked to CS‐induced ED; however, the exact mechanism is not fully understood. This study reports the role of the ubiquitin proteasome system (UPS) in CS‐ induced eNOS dysfunction and ED. Exposure of bovine aortic endothelial cells (BAECs) to cigarette smoke extract (CSE) resulted in a concentration‐dependent decrease in nitric oxide (NO) production, accompanied by an increase in superoxide generation and formation of 4‐hydroxy‐2‐nonenal protein adducts. CSE depleted tetrahydrobiopterin (BH4). total biopterin, and decreased the expression of eNOS and guanosine triphosphate cyclohydrolase‐1 (GTPCH‐1). More importantly, exposure of BAECs to CSE led to accumulation of ubiqutinylated protein and increased the 26S proteasomal activity in a concentration‐and time‐dependent manner. Pre‐treatment of BAECs with MG132, an inhibitor of 26S proteasome, restored the levels of eNOS, BH4, GTPCH‐1, and enhanced NO production in BAECs following CS exposure. Our data reveal that CS‐induced stimulation of the UPS resulted in degradation of eNOS and GTPCH‐1 that led to eNOS dysfunction and uncoupling. Thus, controlled interference with the UPS pathway could be a novel modality in the prevention of CS‐induced ED. (NIH # HL38324 to JLZ; Egyptian government)
Time‐dependent mechanisms of ethanol‐evoked acute hypotensive and cardiac depressant effects in conscious female rats
Authors: Ibrahim Badr Mostafa , Fan Ming , Abdel‐Rahman Abdel A
Published: June 2021

The molecular mechanisms for the acute hypotensive and cardiac depressant effects of ethanol in proestrus rats are not known. We tested the hypothesis that exacerbated myocardial oxidative stress contribute to the acute ethanol‐evoked myocardial dysfunction and ultimately the hypotensive response in proestrus female rats. Ethanol (1g/kg) or an equal volume of water (control) was administered via nasogastric tube to 48 proestrous rats (divided to 3 ethanol and 3 water groups; n = 6–8), and hemodynamic measurements continued for 30, 60 or 90 min. To elucidate the role of pro‐oxidant and antioxidant mechanisms in the ethanol‐evoked myocardial dysfunction, the catalytic activities of acetaldehyde (ACA) generating [alcohol dehydrogenase (ADH) and catalase] and eliminating (ALDH2) enzymes were measured in the collected cardiac tissues at the specified time points. Further, we measured the levels of markers of oxidative stress (ROS and Nox activity), myocardial kinases (phosphorylated Akt and ERK1/2), and myocardial levels of: (i) the cardiotoxic aldehyde malondialdehyde (MDA), and (ii) 4‐hydroxy‐2‐nonenal (4‐HNE)‐modified proteins. The ex vivo studies revealed: (i) enhanced (ADH), without a change in ALDH2, activity (indicative of elevated ACA levels), (ii) increases in mediators of oxidative stress such as phosphorylated (p) Akt and pERK1/2, Nox activity enhanced along with ROS, (iii), elevated MDA and 4‐HNE‐modified proteins expression. These molecular responses along with the inhibition of myocardial catalase activity coincided with the drastic reductions in cardiac function and BP at 90 min post‐ethanol. The findings implicate local ethanol/ACA metabolizing enzymes in the myocardial oxidative stress, which along with the accumulation of cardiotoxic aldehydes lead to ethanol‐evoked myocardial dysfunction and hypotension in conscious proestrus female rats.

Supported by NIAAA Grant 2R01 AA14441–06
Endothelin ETA/ETB receptors modulate the hemodynamic interaction of cyclosporine with selective and nonselective nonsteroidal antiinflammatory drugs in rats
Authors: El‐Mas Mahmoud M , Abdel‐Halim Rabab M , Helmy Maged W , El‐Gowelli Hanan M
Published: June 2021

We tested the hypotheses that NSAIDs with different COX‐1/COX‐2 selectivity variably influence the hemodynamic effects of chronic cyclosporine (CSA) and that these interactions are causally related to alterations in endothelin receptors. Tail‐cuff measurements showed that CSA (20 mg/kg/day for 10 days) increased systolic blood pressure (SBP) and heart rate in rats. CSA also caused perivascular fibrosis of the renal arterioles along with opposite changes in the immunohistochemical signal of arteriolar ETA (increases) and ETB (decreases) receptors. In contrast to no effect for the COX‐1/COX‐2 inhibitor indomethacin, celecoxib (a selective COX‐2 inhibitor) abolished the pressor and tachycardic effects of CSA and associated changes in perivascuarr fibrosis and ETA/ETB protein expressions. The blockade of endothelin ETA receptors with atrasentan abolished the pressor, but not tachycardic, response elicited by CSA or its combination with indomethacin. Alternatively, BQ788, ETB receptor blocker, caused celecoxib‐sensitive elevations in SBP and potentiated the pressor response evoked by CSA. Collectively, opposite changes in vascular endothelin receptors (ETA upregulation and ETB downregulation) contribute to the pressor effect of chronic CSA. Moreover, ETB receptors mediate the protection offered by celecoxib against the adverse hemodynamic effects of CSA.
The truncated isoform of the neurokinin‐1 receptor (NK‐1R) is associated with increased cell proliferation accompanied by increased mRNA and protein expression of heat shock protein 70 in HEK293 cells
Authors: Gillespie Earl , Mitra Somdutta , Leeman Susan , Stucchi Arthur F
Published: June 2021

Substance P signals through the NK‐1R, a G‐protein coupled receptor that has two major isoforms: a full‐length form (fl‐NK‐1R) that binds and activates Gq and a truncated form (tr‐NK‐1R) that does not. Our lab showed that the tr‐NK‐1R is increased in colitis‐associated cancer (PNAS 108: 17420–17425, 2011); however, the effects of this increase on carcinogenesis are not understood. To determine the effect of tr‐NK‐1R expression on proliferation, we transfected both NK‐1R isoforms into HEK293 cells, an epithelial cell line that lacks constitutively expressed NK‐1R, and measured proliferation using the MTT Growth Assay. After a 48‐hour incubation in serum, there were 20% more tr‐NK‐1R transfected cells (mean A570 = 0.603, p<0.05, n=8) compared to untransfected cells (mean A570 = 0.494), while there were 40% fewer fl‐NK‐1R transfected cells (mean A570 = 0.290, p<0.05, n=8) compared to untransfected cells (mean A570 = 0.494). The tr‐NK‐1R transfectants also show increased mRNA expression of the heat shock protein (HSP) A1A gene (1740‐fold compared to untransfected cells, p<0.001, n=3) accompanied by a 2.5 fold increase in HSP70 protein (p<0.01, n=6). These data suggest that the tr‐NK‐1R may stimulate proliferation in HEK293 cells, that HSP70 may participate in this effect, and that the same pathways may be pertinent in the transition from inflammation to cancer. Funded by the Robert and Dana Smith Family Foundation.
AKIP1 protects against cardiac injury via enhanced mitochondrial function
Authors: Schilling Jan M , Haushalter Kristofer J , Roth David M , Taylor Susan S , Sastri Mira , Patel Hemal H
Published: June 2021

Cardiovascular disease is a leading cause of morbidity and mortality in the United States but the signaling mechanisms leading to protection are poorly understood. Protein kinase A (PKA) has been implicated in regulating cardiac contractility, gene expression and metabolism. However, PKA has dichotomous roles and is implicated in both, cell survival and cell death. We identified protein kinase A interacting protein (AKIP1) as a modulator that responds to oxidative stress; when up regulated, it shows adaptation to ischemic injury. We hypothesized that AKIP1 plays a critical role in regulating mitochondria to affect protection. AKIP1 gene transferred hearts showed increased mitochondrial localization of AKIP1 and the mitochondria had 1) decreased reactive oxygen species generation, 2) enhanced calcium tolerance, 3) decreased cytochrome C release, and 4) enhanced PKA signaling. Further we show interaction of AKIP1 with the mitochondrial apoptosis inducing factor (AIF); where upon hypoxic and oxidant stress these proteins show increased expression and interaction. Therefore, we infer that AKIP1 is a key molecular scaffold that assembles PKA/AIF complexes at the mitochondria to alter the physiological response of the heart to stress. Understanding the molecular activity and regulation of AKIP1 may provide a novel therapeutic approach for limiting myocardial injury.
Alcohol‐induced depression and associated changes in hippocampal BDNF is attenuated by nicotine
Authors: Kalejaiye Olubukola O , Taylor Robert E , Tizabi Yousef
Published: June 2021

Although various hypotheses for alcohol‐nicotine co‐dependence have been suggested, it is not known whether pharmacological effects of alcohol or nicotine on mood behavior may contribute to such co‐abuse. Chronic exposure to high alcohol levels may lead to neurochemical changes and precipitate depressive‐like behavior, whereas nicotine may exert an antidepressant‐like effect. Here, we sought to determine whether nicotine may mitigate the behavioral and neurochemical effects of alcohol. Adult male Wistar rats were injected (i.p.) with alcohol (1.0g/kg), nicotine (0.3mg/kg) or their combination once daily for 21 days. The behavior of these rats in open field locomotor activity (LMA), the forced swim test (FST), a measure of helplessness and sucrose preference test, a measure of anhedonia were evaluated 18h after the last injection. Chronic alcohol did not affect LMA, but increased immobility in FST and decreased sucrose consumption suggesting a “depressogenic” effect. Nicotine alone did not affect the behavior of the rats, but significantly attenuated alcohol‐induced increase in immobility in FST and decrease in sucrose intake. Parallel to the behavioral changes, alcohol treatment resulted in a significant decrease in hippocampal BDNF which was attenuated by nicotine. These findings suggest that the opposing effects of alcohol and nicotine on depressive‐like behavior may contribute to their co‐abuse.
Subjective and Cardiovascular Responses to Cocaine Differ in Cigarette Smokers versus Nonsmokers
Authors: Brewer Alex Jawann , Arnoudse Nicholas , Mahoney James J , Newton Thomas F , De La Garza Richard
Published: June 2021

In this placebo‐controlled, between‐subjects study, we evaluated the cardiovascular and subjective responses to cocaine (40 mg, IV) administered in the laboratory in cigarette smokers versus non‐smokers. . Participants (N=8/group) were matched by gender, age, years of education, years of drug use, amount of daily drug use, and number of days used in the last month. At present, analyses of the data indicate that cigarette smokers exhibited greater changes in heart rate and blood pressure and several subjective effects than non‐smokers, though these did not reach statistical significance (all p's>;0.05). For example, changes in heart rate were greater at early time points (5 min), but also at later time points (60, 90, 120 min) suggesting sustained effects produced by cocaine in smokers as opposed to non‐smokers. A similar trend was observed for changes in “high”, “good effects”, and “stimulated”. Taken together, the available data support the hypothesis that smokers differ from non‐smokers in their cardiovascular and subjective responses to cocaine. Data acquisition and analyses are continuing.
Intranasal delivery of pGDNF nanoparticles provides neuroprotection in the rat 6‐hydroxydopamine model of Parkinson's disease
Authors: Harmon Brendan Trevor , Aly Amirah , Cooper Mark J , Dines Karla , Padegimas Linas , Waszczak Barbara L
Published: June 2021

Glial cell line‐derived neurotrophic factor (GDNF) has been shown to be neuroprotective toward substantia nigra (SN) dopamine neurons in animal models of Parkinson's disease (PD) and in some clinical trials. However, its delivery to brain has required invasive surgical routes that are untenable for many patients with PD. The goal of these experiments was to test whether intranasal delivery of a GDNF expression plasmid could protect dopamine cells in the rat 6‐hydroxydopamine (6‐OHDA) model of PD. If successful, intranasal delivery of pGDNF could provide a renewable source of GDNF within the brain without need for surgical injections or frequent re‐dosing.

Rats were given intranasal saline, naked pGDNF or pGDNF in a PEGylated polylysine nanoparticle (NP) preparation 7 days prior to receiving a unilateral 6‐OHDA lesion. Three weeks after 6‐ OHDA, rotational behavior to 5 mg/kg d‐amphetamine was assessed, followed by sacrifice. Tyrosine hydroxylase (TH) immunohistochemistry revealed a significant reduction in lesion severity in the SN of rats given intranasal pGDNF NPs versus those given naked pGDNF or saline. In addition, rotational behavior was reduced in rats treated with pGDNF NPs. These results demonstrate the utility of intranasal delivery of pGDNF NPs as a non‐invasive means of gene therapy for PD and possibly other diseases of the brain. Support provided by: 2011–12 Northeastern University Tier 1 Seed Grant and the M.J. Fox Foundation.
Kallikrein 6 signals through PAR1 and PAR2 to promote neurotoxicity and exacerbate glutamate‐mediated excitotoxicity
Authors: Yoon Hyesook , Radulovic Maja , Wu Jianmin , Blaber Sachiko I , Blaber Michael , Fehlings Michael G , Scarisbrick Isobel A
Published: June 2021

Trauma to the CNS generates a proteolytic imbalance that contributes to secondary injury, including axonopathy and neuron degeneration; however the mechanism by which this occurs is not well understood. Kallikrein 6 (KLK6) is a serine protease implicated in neurotoxicity and in these studies we investigate the role of protease activated receptors (PARs) in mediating these effects. We demonstrate KLK6 and the prototypical activator of PAR1, thrombin, as well as PAR1 and ‐2, are each elevated in a murine experimental spinal cord injury at acute or subacute time points. KLK6 and thrombin each promoted cerebellar granule neuron toxicity and potentiated glutamate excitotoxicity in vitro. Importantly, neural injury in each case was reduced or blocked in neurons derived from PAR1 deficient mice. KLK6 was shown to activate ERK in cerebellar neurons in a PI3K and MEK‐dependent fashion. Novel lipopeptide inhibitors specific for PAR1 or PAR2, in addition to neurons derived from PAR1 deficient mice were utilized to demonstrate both receptors play an important role in KLK6‐elicited ERK signaling and in the activation of the proapoptotic protein Bim. Taken together these data point to novel KLK6‐PAR1 and ‐PAR2 mediated signaling cascades in CNS neurons that are positioned to participate in neuron degeneration and glutamate‐mediated excitotoxicity in cases of CNS injury. Supported by NIH R01 NS052741
Quantitative membrane proteomics of esophageal squamous cell carcinoma
Authors: Kashyap Manoj K. , Pawar Harsh , Renuse Santosh , Kim Min‐Sik , Pinto Sneha , Sahasrabuddhe Nandini A , Keerthikumar Shivakumar , Harsha H. C. , Prasad T. S. K. , Mahmood Riaz , Kumar Rekha V. , Rustgi Anil , Pandey Akhilesh
Published: June 2021

Membranous proteins have been the attractive target in different cancers e.g. EGFR, VEGF, and CXCR4. We used ESCC (esophageal squamous cell carcinoma) as a prototype and employed the stable isotope labeling with amino acids in cell culture (SILAC) methodology to compare membrane proteome of cells with different type of differentiation. Normal epithelial (Het‐1A), well (TE1) & poorly differentiated (TE2) cell lines were labeled with light, medium or heavy arginine plus lysine, respectively. The membrane proteome was isolated by means of Na2CO3 treatment. The samples were resolved on SDS‐PAGE, excised into 24 bands. In‐gel trypsin digestion was carried out and samples were analyzed on LTQ‐Orbitrap Velos mass spectrometer. The data was processed and quantified with Proteome Discoverer (Version 1.3) using 1% FDR and unique peptides were considered for quantitation. We identified 1,950 unique peptides from =20,000 peptide spectrum matches (PSMs) were obtained. This analysis led to identification of 625 differentially expressed proteins between normal and ESCC derived cell lines. There was large number of known and novel membranous proteins e.g. EGFR, BAK1, C2orf18 and BCL2L13.