bluuebunny/crossref_metadata_2025 huggingface dataset:
Endotoxin neutralization activity (ENA) of bovine plasma and bovine Immunoglobulin (IgG)‐rich fractions as compared to human plasma
Authors: Weaver Eric M , Klein Gerald L , DeVries Bethany K , Maas Kenneth , Shaw Audrey L
Published: June 2021

Translocation of endotoxin and other bacterial components from the gut is an important source of inflammation. A novel source of proteins with potential ENA is edible serum bovine immunoglobulin (SBI). SBI is >;50% IgG and has GRAS (generally recognized as safe) status, permitting its use in clinical nutrition. This study compared the ENA of 4 test materials: human plasma (n=4); bovine plasma (n=4); purified human IgG (n=2); and SBI (n=7). Endotoxin B55:05 at 400 EU/ml was added to serial dilutions of the test materials and incubated. Post incubation samples and controls were assayed for LPS recovery. Response curves were generated and endotoxin neutralization capacities (ENC50) were calculated for each test material. A simple t‐test compared test materials ENA and ENC50. The expected curvilinear response for ENA of human plasma, bovine plasma and SBI samples was observed. Human IgG did not effectively neutralize endotoxin in this assay. The ENA of bovine plasma and SBI was not significantly different from human plasma (P< 0.10). Calculated ENC50 (mg) of human plasma, bovine plasma and SBI was similar at 0.107±0.035, 0.089±0.053, and 0.129±0.044, respectively. The LAL assay can be used to assess ENA and ENC of both human and bovine plasma. SBI is a potential source of ENA with a similar level of ENC50 to human plasma. The use of SBI in clinical nutrition to preserve ENC merits research. This study was funded by Entera Health.
Expression of transcription factors involved in lipid metabolism in mice fed algae, yeast, or fish oil
Authors: Clevenger Loren Taylor , Barnes Kimberly , Ketz John , Rodavich Mary
Published: June 2021

Fish oil (FO) contains high levels of the omega‐3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), unlike algae oil (AO) which contains DHA but not EPA, and yeast oil (YO) which contains EPA but not DHA. AO, fed at equal DHA levels, and YO fed at equal EPA levels, were not as effective as FO at reducing body fat or serum lipids. Our objective was to identify transcription factors with altered mRNA expression in the liver of mice fed 12% lipid diets containing either soy oil (SO; no DHA or EPA), FO, AO, YO, or AO+YO for either two or four weeks. The RNA was extracted, reverse transcribed, and amplified with primers for SREBP‐1c, PPAR‐γ, and PPAR‐α. The expression of a long‐chain PUFA receptor, GPR‐120, was also measured. The only difference detected was a trend (P = 0.10) for FO‐fed mice to have increased GPR‐120 expression following 4 weeks of diet consumption. Therefore, the GPR‐120 receptor may be involved in the lipid‐lowering effects of FO.
Exercise Training on Left Ventricular Interstitial Collagen in Spontaneously Hypertensive Rats
Authors: Pagan Luana Urbano , Damatto Ricardo , Cezar Marcelo , Lima Aline , Bonomo Camila , Guizoni Daniele , Martinez Paula , Gimenes Rodrigo , Okoshi Marina , Okoshi Katashi
Published: June 2021

Systemic hypertension can cause myocardial interstitial collagen matrix remodeling and exercise may be beneficial in this process. The aim of this study was to evaluate the influence of exercise training on left ventricular interstitial collagen matrix in spontaneously hypertensive rats (SHR) before clinical evidence of heart failure.

Methods

Sixteen month old SHR were divided into two groups: sedentary (SED, n=8) and exercised (EX, n=8). Physical training was performed 5 times per week for 16 weeks in a treadmill. Myocardial total and soluble collagen and hydroxyproline concentrations were determined by spectrophotometry. Collagen I and III and lysyl oxidase gene expression were analyzed by real‐time PCR.

Statistics

Student's t test (p<0.05).

Results

Myocardial total and soluble collagen concentration did not differ between groups. Hydroxyproline concentration was lower in EX (0.22±0.04 vs. 0.32±0.08 μm/mg, p<0.05). Gene expression was not significant different between groups.

Conclusion

Exercise training decreases myocardial hydroxyproline concentration in spontaneously hypertensive rats.
Muscle Ring Finger 1 (MuRF1) and MuRF2 Regulate Gene Expression Mediated by the E2F Transcription Factors and are Necessary but Functionally Redundant During Developmental Cardiac Growth In Vivo
Authors: Willis Monte S , Wadosky Kristine M. , Patterson Cam
Published: June 2021

We recently reported MuRF1 and MuRF2 are functionally redundant during cardiac development. Specifically, mice missing all four MuRF1 and MuRF2 alleles [MuRF1/MuRF2 double null (DN)] were born with a massive spontaneous hypertrophic cardiomyopathy and heart failure; mice that were null for one of the genes, but heterozygous for the other (i.e. MuRF1−/− //MuRF2+/− or MuRF1+/−//MuRF2−/−) were phenotypically identical to wild type mice. Microarray analysis of genes differentially expressed between MuRF1/MuRF2 DN, mice missing 3 of the four alleles, and wild type mice revealed a significant enrichment of genes regulated by the E2F transcription factor family. Over 85% of the differentially expressed genes had E2F promoter regions (E2f:DP; p<0.001). Western analysis of E2F revealed no differences between MuRF1/MuRF2 DN hearts and wild type hearts; however, chromatin IP studies revealed that MuRF1/MuRF2 DN hearts had significantly less binding of E2F1 in the promoter regions of genes previously defined to be regulated by E2F1 (p21, Brip1, and PDK4, p<0.01). These studies suggest that MuRF1 and MuRF2 play a redundant role in regulating developmental physiologic hypertrophy, by regulating E2F transcription factors essential for normal cardiac development by supporting E2F localization to the nucleus, but not through a process that degrades the transcription factor.
Physical rehabilitation based on the phenomenon of ischemic preconditioning in patients with ischemic heart disease
Authors: Lyamina Nadezhda P , Kotelnikova Elena V , Karpova Ella S , Senchikhin Valery N , Lyamina Svetlana V , Downey H Fred , Manukhina Eugenia B
Published: June 2021

Ischemic preconditioning (IP) triggers mechanisms that are cardioprotective during non‐fatal ischemia. Exercise programs are frequently prescribed for patients with ischemic heart disease (IHD). Here we report a strategy for physical rehabilitation of IHD patients based on presumed IP. Patients with stable IHD (age 38–65 y; ejection fraction >;40%), incomplete revascularization after transcutaneous coronary angioplasty, and positive results of a diagnostic exercise stress test were studied. The IP group (53.9±6.2 y; n=11) performed treadmill exercise for 10 days at 70–80% of diagnostic intensity, with duration determined by the time to ST depression and/or appearance of anginal symptoms. The control (C) group (56.1±4.8 y, n=10) performed standard exercise training for 10 days at 50–60% of diagnostic intensity for 30 min. Monitored safety indices: ECG, BP, heart rate, and myocardial injury markers. Post‐training, for the IP group compared to the C group, diagnostic exercise duration increased by 17.5±3.6% vs 4.1±1.2% (p <0.05); MET by 9.3±2.6% vs 2.4±1.1% (p <0.05); double product by 6.5±2.9% vs 2.1±1.0% (p=0.021); maximum ST depression decreased by 46.3±6.8% in the IP group vs 9.8±1.6% in the C group (p <0.001). Safety indices were within reference ranges in both groups. Thus, exercise‐induced IP effectively improves exercise tolerance in IHD patients.
Deficiency of Apoptosis Inducing Factor (AIF) decreases complex I activity and increases the ROS generation in isolated cardiac mitochondria
Authors: Szczepanek Karol , Hu Ying , Lesnefsky Edward , Chen Qun
Published: June 2021

Objective

Genomic downregulation of mitochondrial AIF content in Harlequin (Hq) mice increases cardiac injury during ischemia‐reperfusion (IR). We investigated if the AIF deficiency in cardiac mitochondria affects the rate of oxidative phosphorylation (OXPHOS) and the electron transport chain (ETC)‐mediated generation of reactive oxygen species (ROS).

Methods

The rate of OXPHOS, complex I activity, and the net release of H2O2 were determined in isolated mitochondria from Hq or wild type (WT) mouse hearts.

Results

Downregulation of AIF expression inhibited the rate of OXPHOS using glutamate and malate (Glu+mal) as complex I substrates (Table). The complex I activity was also decreased in Hq mouse heart mitochondria vs. WT. The net release of H2O2 was increased in Hq mitochondria vs. WT, using both complex I and complex II substrates (Succinate + rotenone, Succ+ROT).

Conclusion

Mitochondrial AIF deficiency leads to a defect at the proximal portion of ETC and increased generation of ROS from the ETC. This setting likely contributes to increased cardiac injury in Hq mice during IR.

This work was supported by the American Heart Association (QC, 11SDG5120011) and the Department of Veterans Affairs (EJL).
Chronic hypoestrogenism promotes apoptotic death in cardiac myocytes of the left ventricular myocardium
Authors: Liskova Yuliya Vladimirovna , Kovalevskaya Elena A , Salikova Svetlana P , Stadnikov Alexander A , Blinova Elena V
Published: June 2021

This study characterizes the remodeling of the left ventricular (LV) myocardium in rats with surgically‐induced hypoestrogenism (hypo‐E). The research was done on 15 adult outbred female rats: 10 rats underwent bilateral ovariectomy, while 5 rats served as a control. The LV myocardium was examined with light microscopy, immunocytochemistry (Ñas‐3 and Bcl‐2 protein expression) and morphometry on 2, 6 and 12 months (m) after the surgery. We found that the volume fraction of myocardial interstitial tissue was significantly increased in all hypo‐E rats (38.9±5.4% at 2m, 48.8±6.4% at 6m, 35.3±5.3% at 12m vs. 15.6±2.6% in control, đ<0.05). At the same time, after an initial increase in cardiac myocyte (CM) diameter at 6m of hypo‐E (9.96±1.57μm vs. 8.68±1.64 μm in control, đ<0.05), the average CM diameter was reduced to 7.6±1.7 μm at 12m, suggestive of CM atrophy. The latter observation was further confirmed by the marked decrease in CM volume fraction in these rats (64.6±4.8% vs. 84.2±2.51% in control). Furthermore, the progressive reduction of Bcl‐2+CM in hypo‐E rats (0.21±0.04% at 6m and 0.17±0.01%, at 12m vs. 0.6±0.03% at 2m, p<0.05) in concert with the increase of Cas‐3+ cells (0.4±0.01% at 6m and 0.38±0.05%, at 12m vs. 0.22±0.03% at 2m, p<0.05) has suggested the gradual increase in CM apoptosis. Thus, our data demonstrated that chronic hypo‐E in rats was associated with a significant increase in apoptotic death of LV myocytes.
High MUC2 production in goblet cells caused ER stress and susceptibility to apoptosis
Authors: Tawiah Adelaide Ama , Moreau France , Chadee Kris
Published: June 2021

MUC2 produced by goblet cells is the gel forming mucin in mucus that protects the intestinal epithelium against harmful substances and the development of tumors. In this study, we investigated the role of MUC2 in colonic epithelial cell apoptosis, a process essential in preventing tumor formation. A high MUC2‐producing colonic goblet cell line (HT29‐H) and a clone silenced for MUC2 (HT29‐L) by lentivirus‐shRNA were used. Cells were exposed to apoptosis‐inducing agents and apoptosis quantified by caspase 3 and Poly (ADP‐ribose) polymerase cleavage, DNA fragmentation and lactate dehydrogenase (LDH) release. Expression of endoplasmic reticulum (ER) stress markers was evaluated to monitor ER activities as well as expression of genes involved in MUC2 biosynthesis. HT29‐H was found to be highly susceptible to apoptosis as compared to HT29‐L cells. Over expression of MUC2 in HT29‐H cells caused increased expression of ER stress markers and decreased MUC2 folding genes as compared to HT29‐L or parental wild‐type cells. Our findings indicate that MUC2 induces apoptosis in an ER stress‐dependent manner and strongly suggests that it may be a useful therapeutic agent in colorectal carcinomas. Supported by CIHR.
Role of bile acids in autophagy and alcohol‐induced liver injury
Authors: Manley Sharon , Ni Hongmin , Yang Hua , Guo Grace , Apte Udayan , Ding Wen‐Xing
Published: June 2021

Autophagy is a lysosomal degradation process and cellular protective mechanism. Bile acids display hepatotoxicity by inducing cell death and has been shown to modulate alcohol‐induced liver injury but the mechanism is not clear. We found that Farnesoid X Receptor deficient (FXR‐/−) mice which have increased hepatic bile acid levels had impaired autophagy. This is supported by the finding that the levels of LC3‐II and p62, two proteins which are normally degraded by autophagy, are significantly higher in FXR−/− mouse livers compare to that of wild type. Because inhibition of autophagy exacerbates alcohol‐induced liver injury, we hypothesized that FXR−/− mice may have exacerbated liver injury after alcohol treatment. Indeed, alcohol treated‐FXR−/− mice had increased serum alanine aminotransferase (ALT) and hepatic triglyceride levels compared to wild type mice. Alcohol‐treatment increased the expression of autophagy genes in wild type mice, but not in FXR−/− mice, suggesting the expression of autophagy genes might require FXR. In conclusion, the impaired autophagy in FXR−/− mice may contribute to the increased alcohol‐induced liver injury.

This work was supported by NIH R01 AA020518–01 (WXD) and P20 RR021940 (COBRE).
An Interleukin‐15 Fusion Protein Enhances the Oncolytic Effects of Myxoma Virus in a Murine Melanoma Model
Authors: MacNeill Amy L , Tosic Vesna , Doty Rosalinda A , Liu Jia , McFadden Grant , Roy Edward J
Published: June 2021

Myxoma virus (MYXV) has potential as an oncolytic virus, but is not fully effective as a single‐agent therapy. We hypothesized that intratumoral (IT) expression of interleukin‐15 (IL‐15) by MYXV would recruit cytotoxic effector cells and improve treatment efficacy. To test this, MYXV expressing a fluorescent protein (MYXV‐Tred) and MYXV‐Tred encoding murine IL‐15 (MYXV‐IL15) were evaluated in a murine melanoma model. C57/BL6 mice that received weekly IT injections of virus lived significantly longer than controls. Unexpectedly, the median survival time of mice treated with MYXV‐IL15 was similar to MYXV‐Tred. In MYXV‐IL15‐treated tumors, virus titer, IL‐15 concentration, lymphocyte grade, and CD3+ cell counts were elevated at 4 days post‐inoculation (dpi), but decreased at 7 dpi. In subsequent experiments, recombinant MYXV was injected IT every 3 days. Additionally, to further enhance IL‐15 functions, MYXV‐Tred was modified to express IL‐15 fused with IL‐15 receptor alpha (MYXV‐RLI). The median survival time and number of IT Ly‐49+ cells were significantly increased in RAG mice bearing melanomas treated with MYXV‐RLI compared with MYXV‐IL15. These findings suggest that IT expression of an IL‐15/IL‐15Rα fusion protein enhances the oncolytic effects of MYXV.
Development of experimental protocol for photodynamic therapy on colorectal tumors: determination of irradiation by fluorescence spectroscopy
Authors: Duarte Janaina , de Oliveira Ferreira Alexandre , Junior Landulfo Silveira , Plapler Helio
Published: June 2021

In the last decades, there was a significant improvement in traditional techniques such as surgery, chemotherapy and radiotherapy used to treat different types of neoplasias. Such therapies have intense side effects because they are not selective to the tumor tissue and these limitations and disadvantages have led to the search and development of new therapeutic alternatives to treat cancer, including photodynamic therapy (PDT) which, according to several studies conducted in the last two decades, have proved effective in malignant and pre‐malignant conditions, such as head and neck tumors, lung cancer, prostate cancer, brain tumors, duodenal and colorectal tumors. However, there is difficulty in determining the optimal protocol of PDT considering the main variables involved such as laser and photosensitive drugs. Kinects by fluorescence spectroscopy in tumors can be a useful tool to verify the ideal period of irradiation after photosensitive administration. This work developed a new protocol of kinects by fluorescence spectroscopy in colorectal tumor in Wistar rats to support the photodynamic therapy. For this protocol it was used chloroaluminum phthalocyanine tetrasulfonate and aluminum phthalocyanine chloride incorporated into liposomes as photosensitive drugs. The kinetics of both drugs was performed by means of fluorescence spectroscopy during 48h monitoring of chemically induced colorectal tumors in Wistar rats. The best irradiation period indicated by kinetics for both drugs was between 22 and 26 hours and the fluorescence spectroscopy has been showed useful to determine the PDT irradiation.
Aptamer‐Drug Conjugates for Targeted Therapy of CD30‐expressing Lymphomas
Authors: Parekh Parag A , Kamble Sanchit R , Zihua Zeng , Zu Youli
Published: June 2021

CD30 is a biomarker for diagnosis and targeted therapy of Anaplastic Large cell lymphoma (ALCL) and Classical Hodgkin Lymphoma (CHL). Our previous studies have demonstrated specific binding of RNA aptamer to CD30 positive tumor cells. However, for in vivo targeted therapy there is a concern about biostability of the RNA aptamers.

To overcome this, an ssDNA aptamer was developed via a hybrid SELEX approach using CD30‐expresing lymphoma cells and purified CD30 protein to yield the aptamer C2NP which specifically bound to CD30 expressing ALCL and CHL cells at concentrations ≤10 nm and did not react to control tumor cells. Importantly, C2NP is highly stable and more than 50% of them remained after incubation in human serum at 37°C for 24 hours, indicating the suitability for in vivo use.

For targeted therapy study, C2NP was conjugated with a chemotherapeutic agent and a fluorescent reporter to form an aptamer‐drug conjugate (ADC). The specificity of ADC was confirmed by flow cytometry and intracellular delivery of chemo‐drug was observed by microscopy. Chemical conjugation of drug had no effect on aptamer binding specificity and affinity to lymphoma cells. In addition, potential therapeutic effects of the aptamer‐drug conjugate are under investigation by using cultured ALCL and CHL cell‐lines currently. This is the first study to use an aptamer‐drug conjugate for targeted therapy of CD30‐exprssing lymphomas.
H2S‐induced cardiac protection is caused by activation of mTORC2/Akt/Bim pathway in an isolated rat heart ischemia / reperfusion model
Authors: Zhou Yue , Wang Daying , Gao Xiufang , Lew Karsheng , Richards Arthur , Wang Peipei
Published: June 2021

It is documented that H2S protects hearts against ischemia / reperfusion (I/R) injury through Akt activation. However, the mechanism of Akt phosphorylation is unknown. The purpose of this study was to investigate the role of AKT kinases PDK1, mTORC2 and phosphatases PTEN, PP2A and PHLPP2 in H2S‐induced Akt activation. Hearts from adult Sprague‐Dawley rats were isolated and subject to I/R (20 min zero flow ischemia followed by 60 min reperfusion), divided into 4 groups: Control and NaHS treatment with or without PP242, an mTOR inhibitor. Cardiac function and cell injury were as assessed by rate‐pressure product (RPP) and LDH release respectively. Hearts were freeze clamped at the end of reperfusion for Western blot analysis. Our data demonstrated that NaHS treatment improves RPP (50±1.2, vs. Cont 31±4 % Baseline, p<0.05); decreases LDH release (19±10 vs. Cont 170±87 mM, p<0.05). NaHS increases p‐Akt by 3.0 fold (p<0.05 vs. Cont). There is no change of PDK1, PTEN, PP2A and PHLPP2. mTOR and p‐Bim are up‐regulated by 1.5 and 1.9 fold. The protective effect and Akt activation are blocked by PP242. These results suggest that H2S‐induced cardiac protection is due to activation of mTOR/Akt/Bim pathway.
PAR‐2 RECEPTORS MODULATE NERVE‐EVOKED CONTRACTION OF RESISTANCE ARTERIES
Authors: Kerr Paul , Zwozdesky Myron , Chen Michael , Wei Ran , Plane Frances
Published: June 2021

The sympathetic nervous system regulates arterial diameter and thus blood flow and blood pressure. Protease‐activated receptors (PARs) are a novel class of G‐protein coupled receptors activated by protealytic cleavage of the extracellular domain to reveal a tethered ligand. Four sub‐types of PARs have been identified, of which PAR‐1 and PAR‐2 are expressed in the vasculature. We have investigated whether activation of PAR‐2 can modulate nerve‐mediated vasoconstriction in rat isolated mesenteric resistance arteries denuded of endothelium. Stimulation of peri‐vascular nerves elicited frequency‐dependent increases in tone which were abolished by the α1‐adrenoceptor antagonist prazosin. Activation of PAR‐2 by either trypsin, the tethered ligand mimetic peptide FLIGRL or AC264613, a synthetic PAR‐2 ligand, significantly attenuated nerve‐mediated responses. Increases in tone to bath applied noradrenaline were attenuated but to a significantly lesser extent. The actions of trypsin were prevented by the trypsin inhibitor aprotinin. Thrombin, which activates PAR‐1 receptors, did not attenuate nerve‐mediated or noradrenaline‐evoked increases in tone. These data indicate that PAR‐2 receptors can modulate nerve‐mediated vasoconstriction of resistance arteries and so may be novel regulators of sympathetic vascular tone.
Reversal of P‐glycoprotein mediated multidrug resistance by Cryptotanshinone and Dihydrotanshinone
Authors: HU Tao , Yeung John H.K. , Wang Lin , Zhou Xuelin , Or Penelope M.Y. , To Kenneth K.W. , Cho Chi Hin
Published: June 2021

Multidrug resistance of cancer cells is an obstacle to successful cancer chemotherapy. Overexpression of P‐glycoprotein (P‐gp), an ATP‐binding cassette (ABC) membrane transporter, can mediate the efflux of drugs out of cancer cells, leading to multidrug resistance and chemotherapy failure. Therefore, development of safe and effective P‐gp inhibitors plays an important role in chemotherapy.

This study investigated the reversal of P‐gp mediated multidrug resistance by several tanshinones including Tanshinone I, Tanshinone IIA, Cryptotanshinone and Dihydrotanshinone isolated from Danshen (Salvia miltiorrhiza), a traditional Chinese medicine. Bi‐directional transport assay in Caco‐2 cells showed that Cryptotanshinone and Dihydrotanshinone could decrease digoxin efflux ratio in a concentration‐dependent manner, indicating inhibitory effects on P‐gp function. Whereas, Tanshinone I and Tanshinone IIA had no effects. Moreover, Cryptotanshinone and Dihydrotanshinone could increase the intracellular accumulation of doxorubicin in P‐gp overexpressing SW620 Ad300 cancer cells. To this end using MTT assay Cryptotanshinone and Dihydrotanshinone could potentiate the cytotoxicity of doxorubicin and irinotecan in SW620 Ad300 cells as well. These findings suggest that Cryptotanshinone and Dihydrotanshinone could be used as an adjuvant therapy together with anticancer drugs in chemotherapy to improve their therapeutic effects for colon cancer.
Gastroprotective, ulcer healing and antioxidant activity of protoberberine alkaloid isolated from partially purified fraction of crude dichloromethane stem extract of coscinium fenestratum
Authors: Okechukwu Patrick Nwabueze , Marunga Joan , Chiam Nyet Chiang , Godspower Pamela Rufus , Akowuah Gabriel A.
Published: June 2021

Protoberberine alkaloids such as berberine, jatrarrhizine, and palmatine have been reported to be a major constituent in C. fenestratum. Formulations of these are alkaloids are widely used in traditional systems of medicine including, Ayurveda and Traditional Chinese Medicine and has demonstrated wide range of pharmacological activities including; antihypertensive, anti‐inflammatory, antioxidant, antidepressant, anticancer, antidiarrhoeal, antidiabetic cholagouge, hepatoprotective and anti‐microbial. Our previous study showed that crude and partially purified fraction (PPF) obtained from dichloromethane (DCM) stem extract of C.fenestratum has a very significant antiulcer activity. Therefore in this study we evaluated the ulcer healing and antioxidant effects of PPF of DCM stem extract Coscinium fenestratum. The ability of the PPF to heal gastric mucosa against injuries caused by necrotizing agent's 0.3M HCl/60% EtOH, indomethacin and stress were investigated. Determination of DPPH, hydroxyl, nitric oxide and superoxide radical‐scavenging activity (antioxidant), was performed using simple biochemical reactions and LC‐MS profiling of the bioactive compound was also determined. PPF demonstrated a significant (p< 0.001) ulcer healing effect, in HCl/EtOH‐induced gastric ulcer, it reduced ulcerative lesion index by 76.2%, stress 93.5%, indomethacin was 74.60%, acetic acid 75% compared to negative control group. DPPH radical scavenging activity showed IC50 value of 0.194mg/ml and superoxide radical scavenging activity IC50 value of 1.54 mg/ml. LC‐MS profiling indicated the presence of Palmitine.
Activator of G protein Signaling‐3 (AGS3) regulates CXCR4 and CCR7 signaling in murine lymphocytes and bone marrow‐derived dendritic cells
Authors: Branham‐O'Connor Melissa , Robichaux William G. , Maher Ellen M. , Zhang Xian , Blumer Joe B.
Published: June 2021

Activator of G‐protein Signaling 3 (AGS3), an accessory protein for G‐protein signaling, has functional roles in the kidney and CNS. Here we show that AGS3 is expressed in spleen, thymus and bone marrow‐derived dendritic cells (BMDCs), and is upregulated upon leukocyte activation. Using a recently developed bioluminescent resonance energy transfer (BRET) platform, we observed that AGS3 forms a chemokine regulated, Gαi‐dependent complex with CXCR4 and CCR7, suggesting that AGS3 may integrate signals from chemokine receptors. Indeed, Gαi2‐null mice exhibit altered lymphocyte differentiation, secretion and motility. We thus explored the role of AGS3 in immune cell function by characterizing chemokine receptor signaling in mice lacking AGS3. Although there were no differences in lymphocyte subsets, we observed defects in chemotaxis of AGS3‐null BMDCs, B‐ and T‐cells. This defect was not due to alterations in cell surface receptor expression, but may be explained by observed reductions in chemokine‐stimulated calcium release and altered Erk and Akt activation in AGS3‐null cells. These studies indicate a role for AGS3 in the regulation of G‐protein signaling in the immune system, providing unexpected venues for the potential development of therapeutic agents that modulate immune function by targeting these regulatory mechanisms.
Neuroblastoma cell proliferation involves prostaglandin E2 and subsequent β‐catenin stabilization
Authors: Jansen Sepp , Van Oppen Jessica , Hedemann Ilja , Holman Rian , Faraj Sayf , Kok Jan Willem , Diks Sander , Timens Wim , Gosens Reinoud , De Bont Eveline , Schmidt Martina
Published: June 2021

Neuroblastoma (NBL) is a neural crest derived tumor which accounts for ±15% of pediatric cancer deaths. Despite recent efforts to elucidate the genetic background of NBL, the molecular etiology remains unclear. Recent evidence points to the cyclic‐ AMP (cAMP) elevating ligand prostaglandin E2 (PGE2) and the oncoprotein β‐catenin as two novel players in NBL. Here, we aimed to define a potential role and interplay of PGE2 and β‐catenin in survival of NBL.

β‐Catenin expression was analyzed by immunohistochemistry in 41 human NBL biopsies. Further, we studied survival of 8 human NBL cell lines in response to PGE2 and cAMP elevation by the adenylyl cyclase activator forskolin.

Our findings show that β‐catenin expression inversely correlates with NBL patient survival. In addition, we show that expression of a degradation‐resistant β‐catenin mutant (S33Y) increased NBL cell proliferation. Similarly, both PGE2 and forskolin enhanced proliferation, promoted GSK‐3 phosphorylation, β‐catenin stabilization and β‐catenin dependent gene transcription. In line with our hypothesis, reduction of endogenous PGE2 by a cyclooxygenase‐2 inhibitor attenuated NBL cell survival, a process being partially reversed by exogenous PGE2. We conclude that PGE2‐induced cAMP stabilizes β‐catenin and promotes NBL tumor cell survival via enhanced β‐catenin target gene transcription.

This study is supported by the UMCG.
Effects of anandamide and other CB1 ligands on cognitive function
Authors: Kangas Brian D. , Bergman Jack
Published: June 2021

The primary psychoactive ingredient of marijuana, Δ9‐ tetrahydrocannabinol (THC), has medicinal effects that promote continued development of CB1 agonists as therapeutics but also produces well‐documented deleterious effects on cognitive endpoints. In the present study, CB1 agonists—THC (0.03–1.8 mg/kg), WIN 55,212 (0.3–1.8 mg/kg), AM4054 (0.003–0.1 mg/kg), anandamide (3.0–32 mg/kg alone and after the FAAH inhibitor URB 597)—were further evaluated by comparing their effects on cognition in nonhuman primates. Drugs were studied using touchscreen procedures to assay learning (repeated acquisition, reversal), motivation (progressive ratio), and, with novel methodology, attention (titrating vigilance). THC, WIN, and AM4054 produced dose‐related impairment of each type of complex behavior. However, drug potency varied across tasks, i.e., particular cognitive endpoints appeared more vulnerable to drug action (e.g., learning<reversal). Anandamide had negligible effects and, even after URB597, produced only moderate dose‐related impairments. These data suggest that anandamide may offer a therapeutically advantageous alternative to other CB1 agonists for further development. (supported by DA031020, DA007252)
Discriminative stimulus effects of direct‐ and indirect‐ acting dopamine receptor agonists in free‐feeding and food‐restricted mice.
Authors: Collins Gregory T , Jackson Jonathan A , Henson Cedric , Koek Wouter , France Charles P
Published: June 2021

In rats, the discriminative stimulus effects of direct‐ (e.g., quinpirole) and indirect‐ (e.g., cocaine) acting dopamine receptor agonists are mediated by multiple dopamine receptor subtypes and the relative contribution of D2 and D3 receptors to these effects can be altered by manipulating feeding conditions (e.g., food restriction). In these studies two‐lever discriminations were trained with either 10.0 mg/kg cocaine or 0.032 mg/kg quinpirole in free‐fed and food‐restricted mice using a fixed ratio 10 schedule of reinforcement for 50% sweetened condensed milk. Although free‐fed mice responded at lower rates under training conditions and were more sensitive to the rate suppressant effects of their respective training drugs, feeding condition did not affect the acquisition of either discrimination and did not alter the potencies of cocaine or quinpirole to increase cocaine‐ and quinpirole‐appropriate responding, respectively. These novel discriminations will allow for the identification of the specific receptors that mediate the interoceptive effects of cocaine and quinpirole and will serve as the basis for future studies of the influence of diet on drug effects in mice. CPF is supported by a NIDA Senior Scientist Award (K05 DA017918).
Zinc Oxide Nanparticles Induce Apoptosis and Necrosis in Hepatocellular Carcinoma HepG2 Cells
Authors: Tadinada Satya Murthy , Lai Maria B , Idikuda Vinaykumar , Mukka Krishnaveni , Singh Ratan Mani , Pfau Jean , Bhushan Alok , Leung Solomon , Lai James C.K.
Published: June 2021

Targeted drug delivery systems with minimal potential adverse/side effects are gaining utility in diverse applications. Uses of nanomaterials to design such targeted drug delivery are escalating exponentially. Nevertheless, the biocompatibility of nanomaterials is inadequately addressed. Consequently, among our systematic investigation of putative cytotoxic effects of nanomaterials in mammalian cell types, this study aims to elucidate mechanisms underlying the putative cytotoxic effects of ZnO nanoparticles on hepatocellular carcinoma HepG2 cells. Our results demonstrate that these nanoparticles exerted dose‐related decreases in survival of HepG2 cells. Findings from flow cytometry analysis and lactate dehydrogenase release assays reveal that the mode of cell death underlying the effects of the nanoparticle was a combination of apoptotic and necrotic cell death, while the percentage of necrotic cells predominated at higher treatment concentrations. Thus, our findings may have pathophysiological implications in biocompatibility of ZnO nanoparticles.
Effects of dabigatran on bleomycin‐induced pulmonary toxicities and its safety profile in mice
Authors: Shrestha Ninu , Sookvanichsilp Nongluck
Published: June 2021

Chronic inflammation causes pulmonary fibrosis. Bleomycin‐induced murine pulmonary toxicity is generally used as a model of interstitial lung disease (ILD). Thrombin is involved in the initial phase of ILD. This study investigated the benefits of dabigatran, a direct thrombin inhibitor on bleomycin‐induced pulmonary changes and its safety profile on hemostasis. Male ICR mice were divided into 3 groups, i.e. saline, bleomycin (50 mg/kg, i.v. in two divided doses on day 1) and dabigatran (bleomycin‐pretreated mice, received 10 mg/kg/day dabigatran etexilate, orally on days 2–9). Tail vein bleeding time was measured on day 10 just before euthanatization. The drug effects on the lung were assessed by the lung coefficient for edema and histological score for inflammation and fibrosis. The results indicated that bleomycin caused lung injuries with significant increases (p<0.05) in all the parameters tested. Dabigatran treatment caused a significant decrease in edema but not in inflammation and fibrosis, while tail vein bleeding time was significantly increased at the dose. Dabigatran at the dosage mentioned had a limited benefit on bleomycin‐induced pulmonary toxicities in mice with bleeding risk. Therefore, for an effective management, dose of dabigatran cannot be increased and may require drug combinations. The authors wish to thank the Thailand International Development Co‐operation Agency for the financial support.
Genetic ablation of aquaporin‐2 in the mouse connecting tubules results in defective renal water handling
Authors: Kortenoeven Marleen L.A. , Pedersen Nis B. , Miller R. Lance , Rojek Aleksandra , Fenton Robert A.
Published: June 2021

Body water balance is regulated via the water channel aquaporin‐2 (AQP2), expressed in the renal connecting tubule (CNT) and collecting duct (CD). To study the role of AQP2 in the CNT we generated a mouse model with CNT‐specific AQP2 deletion (AQP2‐CNT‐KO). Confocal laser scanning and immunogold electron microscopy demonstrated an absence of AQP2 in the CNT of AQP2‐CNT‐KO mice. 24 hour urine output was significantly higher and osmolality lower in AQP2‐CNT‐KO mice compared to controls. After 24 hour water restriction, urine osmolality was still significantly lower. A significant difference in urine osmolality between groups before dDAVP was not apparent 2 hours after injection, with urine osmolality increased significantly in both groups. AQP2‐CNT‐KO mice had a significantly lower cortical AQP2 abundance, with no changes in NKCC2, AQP3, AQP4 or medullary AQP2. Lithium chloride, known to cause nephrogenic diabetes insipidus, increased urine volume and decreased osmolality in both groups. After 8 days, the AQP2‐CNT‐KO mice still had a significantly higher urine volume, lower osmolality and less cortical AQP2 compared to controls. Our studies show that the CNT plays a role in regulating body water balance under basal conditions, but not for maximal concentration of the urine during antidiuresis. Besides, our studies suggest that unlike CD principal cells, CNT principal cells are not severely affected by lithium.
Indoleamine‐2,3‐Dioxygenase Restrains Hypertension Induced by Angiotensin II in Rats Fed a High Salt Diet
Authors: Yiew Kan Hui (Nicole) , Yoon Sook Kyung , Huang Lei , Jin Chunhua , Mellor Andrew L. , Pollock David M.
Published: June 2021

Inflammation and T‐cell activation has been shown to contribute to the pathogenesis of angiotensin II (AngII) dependent hypertension. Indoleamine‐2,3‐dioxygenase (IDO), the rate‐limiting enzyme involved in the degradation of tryptophan (TRP) to kynurenine (KYN), has a central role in increasing T regulatory cells and decreasing T effector cells. In this study, we examined the role of the IDO pathway in the hypertensive response to chronic AngII infusion in rats fed a high salt diet (HS; 4%NaCl). We hypothesized that inhibiting IDO with 1‐Methyl‐D‐Tryptophan (1‐MT) will exacerbate hypertension in this model due to loss of IDO‐mediated suppression of inflammation and T effector cells. To test our hypothesis, we administered 150 ng/kg/min Ang‐II (osmotic minipump) to male Sprague‐Dawley rats fed a 4% HS chow for two weeks. After two weeks of treatment, 1‐MT had no effect on MAP in control groups on either a normal salt (110 ± 3 mmHg) or HS (112 ± 2 mmHg) diet. Interestingly, 1‐MT significantly exacerbated MAP in Ang II/HS hypertensive rats (146 ± 4 mmHg vs. 161 ± 5 mmHg; p<0.05). There were no differences in food and water intake or in urinary protein and creatinine excretion among groups. These findings support the hypothesis that IDO activity contributes to blood pressure regulation by limiting Ang‐II dependent hypertension.
A Case for a Standardized Method for the Normalization of Muscle Sympathetic Nerve Activity (MSNA) Amplitude
Authors: White Daniel Wade , Kay Victoria L , Moralez Gilbert , Eubank Wendy L , Raven Peter B
Published: June 2021

MSNA has no agreed upon standard for analysis. For example, mean burst strength (MBS), total activity (TA) and total MSNA (TMSNA) rely on amplitude measurements assigned arbitrary units which are highly dependent on the method of normalization. We investigated the hypothesis that computer algorithms used for automatic normalization of MSNA amplitudes differ from those obtained with manual normalization leading to erroneous interpretations. MSNA from healthy human subjects (n=8, 2 female) was obtained by microneurography of the peroneal nerve while resting quietly in a 30o semi‐recumbent position. Data was analyzed by 3 different normalization methods (Min‐Max Auto (MM), Mean Auto (MA), and Rectified Manual (RM)). MSNA is quantified as burst frequency (BF), burst incidence (BI), MBS, TA, and TMSNA. There were no differences in BF or BI between the three analysis methods. However, significant differences were detected between MM and RM in MBS, TA and TMSNA (p=0.002, p=0.009, and p=0.004). Rankings according to activity were not different between MM and RM but were different in MA compared to either MM or RM. There is an overwhelming need to standardize the analysis of MSNA. The current study points out that the variables BF and BI can be compared across studies. However, our data identify that the RM normalization is the only method to quantify MBS, TA and TMSNA for cross‐study comparisons.
Evaluation of muscle sympathetic nerve activity in patients with chronic severe aortic insufficiency
Authors: SOUZA SILVIA B P C , Accorsi Tarso A D , Katz Marcelo , Bezerra Otavio C , Neves Gizele a , Grinberg Max B P , Consolim‐Colombo Fernanda M , Tarasoutchi Flavio B P
Published: June 2021

The aim of this study is to evaluate the activity of sympathetic nerve sistem by microneurography in asymptomatic patients with aortic insufficiency (AoI) chronic important.

Were included 10 asymptomatic patients with anatomically important AoI of rheumatic etiology with normal left ventricular function (ejection fraction>; 55%) by echocardiogram.

Exclusion criteria

diabetes mellitus, renal failure, neuropathy, or beta‐blockers. A control group of normotensive volunteers (control group, n=11) with age, sex and body mass index‐ matched was included. MNSA by microneuragraphy, and blood pressure (BP) by FINOMETER® were evaluated in supine rest position. Continuous variables are described as mean ± SD and categorical variables as relative frequencies. Student t test and a multivariate analysis model were used, p <0.05 was considered statistically significant.

AoI group and control group showed no statistically significant regarding age, sex and BMI. Systolic BP in AoI group was 148 +16 mmHg vs. 120±4 mmHg in control group (p = 0.0001), Diastolic BP was 60±6 mmHg in AoI and 73±3 mmHg in control group (p = 0.0001). The MSNA was in the AoI group 25±3 vs. 15±2 in the control group (p <0.001). Aortic insufficiency was associated independently with a higher MSNA (p = 0.02).

AoI important chronic and asymptomatic is associated with increased MSNA.
Elevated brain‐derived neurotrophic factor (BDNF) levels in the paraventricular nucleus (PVN) increase blood pressure, heart rate and indices of sympathetic activity
Authors: Erdos Benedek , Backes Iara , McCowen Michael L , Scheuer Deborah A
Published: June 2021

BDNF expression increases in the PVN during stress, but its role in mediating stress‐induced rises in blood pressure (BP) and heart rate (HR) is unclear. We tested the hypothesis that overexpression of BDNF within the PVN would elevate baseline BP and HR and would augment BP and HR responses to stress. BP was recorded in male Sprague‐Dawley rats using telemetry. At 11wks of age, adeno‐associated viral vectors [AAV2‐GFP (n=8) or AAV2‐BDNF (n=9)] were injected bilaterally into the PVN. BP recording continued for 4 wks, and responses to a 15 min water stress (WS, 1cm deep, 25 °C) and to a 60 min restraint stress (RS) were evaluated during wks 3 and 4. BDNF overexpression significantly increased BP and HR. At the end of week 4, daytime BP was 99±2 (GFP) and 122±3 mmHg (BDNF, p<0.01), while daytime HR was 314±4 (GFP) and 394±9 bpm (BDNF, p<0.01). BDNF treatment also significantly (p<0.05) increased sympathetic tone as indicated by HR variability (LF/HF ratio was 0.32±0.05 in GFP, 0.86±0.20 in BDNF rats) and BP variability (LF power was 1.6±0.1 mmHg2 in GFP, 5.0±1.2 mmHg2 in BDNF rats). However, BP and HR increases to WS and RS were significantly (p<0.05) reduced in BDNF (e.g. 25±1 and 28±2 mmHg) compared to GFP rats (33±3 and 36±2 mmHg). We conclude that BDNF is an important regulator of BP, HR and sympathetic tone in the PVN, but may not augment stress‐induced BP and HR responses. Supported by AHA 11SDG7560022, an AFAR research grant, and by NIH #HL76807, #HL93186.
Sympathoinhibition in men during paced breathing at 0.25 Hz
Authors: Coverdale Nicole , Usselman Charlotte W. , Gimon Tamara I. , Nielsen Chantelle A. , Luchyshyn Torri A. , Shoemaker J. Kevin
Published: June 2021

This study tested the hypothesis that paced breathing (PB) would inhibit muscle sympathetic nerve activity (MSNA). Integrated MSNA (peroneal nerve) and hemodynamics were assessed in healthy young men (26 ± 2 years; n=8) while supine during spontaneous breathing over 2 to 5 minutes and then at 0.25 Hz for 4 minutes with a recovery period. Breathing rate was not different between spontaneous and PB (15.6 ± 2.0 vs 15.0 ± 0.5 breaths/minute). Compared to baseline, PB reduced (~25%) burst frequency (19 ± 7 vs 14 ± 8 bursts/minute, p<0.01) and incidence (35 ± 12 vs 27 ± 13 bursts/100 heart beats, p<0.01). These returned to baseline levels during recovery. Total MSNA, (mean burst amplitude x burst frequency) also was reduced with PB (2.5 ± 1.3 a.u.) compared with spontaneous breathing (3.3 ± 1.1 a.u.)(p<0.05). No differences were observed in heart rate, mean arterial pressure, burst amplitude, or stroke volume between conditions but cardiac output was higher (~10%) during PB (3.6 ± 0.9 L/min) versus baseline (4.0 ± 0.9 L/min)(p<0.05). Therefore, PB inhibited MSNA in a manner that was not accounted for by concurrent changes in blood pressure or cardiac output. Supported by CIHR and NSERC.
Neurocardiovascular Effects of V1b Receptor Blockade within Paraventricular Nucleus in Conscious Sprague Dawley Rats
Authors: El Werfali Wafa M. , Maliszewska‐Scislo Maria , Chen Haiping , Rossi Noreen F.
Published: June 2021

The paraventricular nucleus (PVN) plays a critical role in both renal sympathetic nerve activity (RSNA) and vasopressin (AVP) secretion by the neurohypophysis. AVP is also released from dendrites of magnocellular neurons within PVN. In addition to V1a receptors, data indicate V1b receptors are present on PVN neurons. We tested the hypothesis that inhibition of V1b receptors within the PVN inhibits increases in mean arterial pressure (MAP) and RSNA induced by AVP injection into PVN. Male Sprague Dawley rats were instrumented. Studies were performed in awake unrestrained rats at least 24 hr after recovery from anesthesia. Baseline MAP, heart rate or RSNA did not differ among the groups. Microinjection of 100 ng AVP into the PVN increased MAP from 138.2 ± 3.1 (baseline) to 151.0 ± 3.2 mmHg (10 min, P<0.01). RSNA increased 47 ± 6.6%baseline (P<0.01). Pre‐injection with 100 ng of the V1b blocker nelivaptan significantly inhibited the pressor, tachycardic and RSNA responses (P<0.05 vs control). Experiments performed after ganglionic blockade with chlorisodamine indicate that the pressor response to central AVP is also in part due to an increase in plasma AVP. Vehicle did not block any of the responses. These findings support the concept that V1b receptors within PVN may be activated by dendritic release of AVP that stimulates both RSNA and peripheral release of AVP.
Continuous and intermittent electrical stimulation of the aortic depressor nerve in conscious rats: time course of the hemodynamic responses
Authors: Salgado Helio , Brognara Fernanda , Dias Daniel Penteado Martins , Castania Jaci Airton , Fazan Rubens
Published: June 2021

We investigated the time course of the hemodynamic responses to continuous (CONT) or intermittent (ON/OFF) electrical stimulation of the aortic depressor nerve (ADN) in conscious rats. Rats were implanted with electrodes around the ADN and a catheter into the femoral artery. On the next day the baseline systolic (SAP), diastolic (DAP), mean arterial pressure (MAP) and heart rate (HR) were recorded during 30 minutes and the rats were randomly assigned to CONT (0.5 mA; 0.25 ms; 30 Hz) or ON/OFF (5s ON/3s OFF; 0.5 mA; 0.25 ms; 30 Hz;) stimulation during 60 min. Changes were greater under CONT as compared to ON/OFF stimulation (SAP: −21 ± 4 vs −10 ± 2 %; DAP: −23 ± 4 vs −14 ± 3 %; MAP: −22 ± 4 vs −12 ± 3 %). In addition, under CONT stimulation the HR was reduced within the first 5 min. The hypotensive and bradycardic responses were maintained over the entire period of stimulation in the ON/OFF protocol. Nevertheless, as compared to the first 5 min, CONT stimulation showed a rapid transitory increase in DAP (72 ± 3 vs 64 ± 3 mmHg) and MAP (87 ± 3 vs 78 ± 4 mmHg). In conclusion, CONT stimulation of the ADN elicits greater hemodynamic responses as compared to the ON/OFF protocol. This finding indicates that the CONT stimulation is more effective to reduce the arterial pressure during long period of electric baroreflex activation.

Financial support: FAPESP, CNPq and CAPES.
Technique‐dependent considerations when assessing racial differences in arterial baroreflex function
Authors: Keller David M , Holwerda Seth W , Credeur Daniel P , Fadel Paul J
Published: June 2021

Examination of the arterial baroreflex (ABR) may provide insight into underlying racial differences in the development of hypertension. We compared the ability of a commonly used open loop method to assess ABR gain (i.e., modified Oxford) with a standard closed loop “spontaneous” method (sequence technique) in detecting potential racial differences. Fifteen non‐hypertensive men (7 black and 8 white, ages 18–27 yr) participated. Mod‐Ox trials were performed using 100μg iv bolus of sodium nitroprusside (NTP) followed 1min by 150μg bolus of phenylephrine (PE) during supine rest (3X per subject). Sequence technique was performed during 10min of supine rest. Overall ABR gain, while not different using modified Oxford analysis (p=0.64), was greater in blacks with sequence technique (p<0.05). In addition, ‘hypotensive’ responses were not different using the mod‐Ox, yet enhanced in blacks using sequence technique (p<0.05). For hypertensive responses, a greater gain was also found with the sequence technique (black: 28±11 vs white: 18±5 ms/mm Hg, p<0.05); however, the ABR gain for the mod‐Ox was in complete opposition with a significantly lower gain in blacks (10±4) compared to whites (16±5 ms/mm Hg, p<0.05). These findings demonstrate technique‐dependent outcomes that warrant consideration when assessing group differences in ABR function.

Supported by NIH R15HL096103 (DMK) and RO1 HL093167 (PJF)