bluuebunny/crossref_metadata_2025 huggingface dataset:
MicroRNA‐21 increases the resistance to CDDP in Hela cervical carcinoma cells by targeting PDCD4 and MSH2
Authors: Cheng JiaoYing , Nishi Hirotaka , Yonekawa Chinatsu , Takara Kohji , Isaka Keiichi
Published: June 2021

Objectives

MicroRNA‐21 (miR‐21) has been found to be over‐expressed in many human cancers, including cervical cancer. Our aim is to investigate the association of miR‐21 with the chemosensitivity of HeLa cervical carcinoma cells to cis‐Diaminedichloroplatinum (CDDP).

Methods

With cervical carcinoma cell lines, HeLa and CDDP‐resistant HeLa (HeLa/CDDP), the cell growth was examined by MTT assays with miR‐21 or miR‐21 inhibitor transfection. TaqMan RT‐PCR was performed to detect the miR‐21 and mRNA expression of Programmed Cell Death 4 (PDCD4) and mutS homolog 2, colon cancer, nonpolyposis type 1 (MSH2). The protein expression of PDCD4 and MSH2 was detected with western blot assay. Luciferase assay was performed to analyze the potential targets of miR‐21.

Results

Cell growth results suggested miR‐21 inhibitor inhibit CDDP‐resistant HeLa growth significantly. Compared with HeLa, MiR‐21 and mRNA of PDCD4 and MSH2 expression in HeLa/CDDP were higher, but the protein expression of PDCD4 and MSH2 in HeLa/CDDP were downregulated in western blot results. Transfection of miR‐21 inhibitor for HeLa/CDDP with PDCD4 3′ untranslated region (3′ UTR) or MSH2 3′UTR vector showed significant increase in luciferase activity compared with control.

Conclusions

Over‐expression of miR‐21 may play critical roles for the CDDP‐resistance in cervical cancer, at least in part via targeting PDCD4 and MSH2.
Antibacterial and anti‐proliferative activity of Isolated Fractions of Aqueous extract from the rhubarb stem
Authors: Bommareddy Praveen K , Mrinal Bhattacharjee K , Depass Anthony L
Published: June 2021

Medicinal uses of rhubarb roots have often been reported. The aim of this current research is to evaluate the anti‐bacterial and anti‐proliferative effects of the rhubarb stem. Aqueous extract from rhubarb stem was fractionated using DEAE Sephadex (anion exchange) followed by Sephadex G25 (gel filtration). Fractions were monitored by assaying for their antibacterial activity on the periodontal pathogen Aggregatibacter actinomycetemcomitans. The fraction from gel filtration column having antibacterial activity was used for further studies. Purity of the active compound was confirmed by the presence of a single major peak in HPLC. The purified active compound was found to have broad spectrum antibacterial activity. It inhibited growth of the Gram negative bacteria, E. coli and A. actinomycetemcomitans as well as the Gram positive bacteria, B subtilis. Its anti‐proliferative activity was tested on the breast cancer cell line, MCF‐7. It was observed that proliferation of the tumor cells was inhibited by 60 % while the cell viability was not affected. The purified compound was partially characterized based on its 1H and 13C NMR spectra. Preliminary analysis indicated that this compound is carbohydrate but the exact identity of the sugar has not yet been determined. This research helped to discover a compound in rhubarb stem that has antibacterial and anti‐tumor activity but very little cytotoxic effect. This research was supported by the Long Island University Intramural Research Support Program.
Photoprotective Effects of Cycloheterophyllin Isolated from Artocarpus heterophyllus against UVA‐Induced Damage and Oxidative Stress in Human Dermal Fibroblasts
Authors: Hung Chi‐Feng , Li Hsin‐Ju , Lin Chun‐Nan
Published: June 2021

Ultraviolet (UV) radiation is considered a cause of skin aging and may result in pathological changes in the skin. Particularly, ultraviolet A (UVA), a long known aging ray and a significant source of oxidative stress, plays a major role in the photoaging of human skin. In the past, many studies have documented that skin cells produce reactive oxygen species after UV exposure. The mitogen‐activated protein kinase (MAPK) pathway would be activated, resulting in phosphorylation of JNK, ERK, and p38 kinase in human skin cells. Previous studies also demonstrated that cycloheterophyllin, a prenylflavone isolated from the root bark of Artocarpus Heterophyllus, has an antioxidant effect and can effectively scavenge free radicals. Although cycloheterophyllin has been studied, the molecular mechanisms involved in the protection of human skin cells from photoaging have not been investigated. Therefore, we used human dermal fibroblasts to investigate protective effect of cycloheterophyllin on UV‐induced damage. We found that cycloheterophyllin significantly increased cell viability and attenuated the phosphorylation of MAPK after UVA exposure. Furthermore, cycloheterophyllin could reduce H2O2 generation. In the in vivo studies, we found that the topical application of cycloheterophyllin before UVA irradiation significantly decreased the value of trans‐epidermal water loss (TEWL), erythema, corneometer and the speed of blood flow. These results indicate that photoprotective effects of cycloheterophyllin inhibited UVA‐induced oxidative stress and damage, and may be beneficial in the prevention of skin photoaging.
Discovery, synthesis and biological evaluation of substituted urea: new activators of heme regulated inhibitor
Authors: Chen Ting , Takruri Khuloud , Hee‐Hwang Sung , Rana Sandeep , Chorev Michael , Natarajan Amarnath , Morisseau Christophe , Hammock Bruce , Aktas Bertal H
Published: June 2021

HRI plays critical roles in proliferation and maturation of red blood‐cell precursors, responding to cytoplasmic stress and adaptation to hemoglobin disorders. N,N′‐diarylureas are potent activators of the heme‐regulated inhibitor kinase (HRI), an eIF2‐alpha kinase. We hypothesized that other substituted ureas with potentially more favorable activity and physic‐chemical properties would also activate HRI. Initially, we screened a ~2000 member library containing a diverse array of substituted ureas in the surrogate assay for eIF2‐alpha phosphorylation. We identified di‐substituted aryl‐cyclohexylarylurea as an active scaffold. We explored contributions of substituted aryl and cyclohexylaryl groups to the activity of this scaffold by assembly of a focused library and follow‐up synthesis. We tested N‐aryl, N′‐cyclohexylarylureas in the surrogate assay for eIF2‐alpha phosphorylation and cell proliferation assays, demonstrating significantly improved bioactivities. A random group of these compounds was further characterized in secondary assays for activity and specificity. These agents may become leads for the development of potent, non‐toxic, and target specific anti‐cancer agents.
Endothelin ETA Receptor‐Mediated Nitric Oxide Synthase Inhibition Underlies Cyclosporine Impairment Of Cholinergic Vasorelaxations In Rats
Authors: Nasser Suzanne A , Elmallah Ahmed I , Sabra Ramzi , Khedr Mohamed M , El‐Din Mahmoud M Mohy , El‐Mas Mahmoud M
Published: June 2021

Impairment of endothelium‐dependent relaxations contributes to vascular dysfunction caused by cyclosporine (CSA). We tested whether vasoconstrictor pathways of angiotensin II, endothelin‐1 (ET‐1), and/or thromboxane A2 (TXA2) modulate the attenuating effect of cyclosporine (CSA) on acetylcholine (ACh) vasorelaxations in the rat aorta. Cumulative relaxations caused by ACh (1×10−9–1×10−5 M), but not sodium nitroprusside (1×10−10–3×10−6 M), were blunted in phenylephrine‐precontracted aortas obtained from CSA (25 mg/kg/day i.p., 7 days)‐treated rats. This detrimental effect of CSA was preserved in rats treated simultaneously with terutroban (TXA2 receptor blocker), losartan (AT1 receptor blocker), captopril (angiotensin converting enzyme inhibitor) or losartan plus captopril. In contrast, selective blockade of ETA receptors by atrasentan significantly increased ACh relaxations and virtually abolished the reductions in ACh responses caused by CSA. The atrasentan effect disappeared in rats treated with L‐NAME (NOS inhibitor) but not BQ788 (ETB receptor blocker) or indomethacin (cycloxygenase inhibitor). It is concluded that NOS downregulation by ETA receptors contributes to the depressant effect of CSA on cholinergic vasorelaxations. Neither ETB receptors nor cycloxygenase‐derived prostanoids are involved in the favorable effect of atrasentan on the aortic CSA‐ACh interaction.
Electrostatic potential surface in the aldehyde binding site of aldehyde dehydrogenases (ALDHs) is a determinant for substrate specificity
Authors: Julián‐Sánchez Adriana , González‐Segura Lilian , Riveros‐Rosas Héctor , Díaz‐Sánchez Ángel G. , Muñoz‐Clares Rosario A.
Published: June 2021

Within the ALDH superfamily, proteins belonging to the ALDH9, ALDH10, ALDH25, ALDH26 and ALDH27 families display activity as ω‐aminoaldehyde dehydrogenases (AMADHs). Active‐site acidic and aromatic residues are involved in binding the ω‐aminoaldehydes in plant ALDH10 enzymes. In order to evaluate their possible relevance in determining the aminoaldehyde specificity, the degree of conservation of these residues among every ALDH family that have at least one known crystal structure, including the AMADHs. We also compared the electrostatic potential surface of the aldehyde binding sites of these structures. Results showed that 3 or 4 aromatic residues form a similar “aromatic box” in the active site of the AMADHs, being the equivalents to Phe170 and Trp177 (human ALDH2 numbering) strictly conserved in all of them, which supports their relevance in binding the aminoaldehyde by cation‐π interactions. In addition, all AMADHs exhibit a negative electrostatic potential surface in the aldehyde‐entrance tunnel, due to side‐chain carboxyl and hydroxyl groups or main‐chain carbonyl groups, which steer the positivelycharged substrates toward the active site. In contrast, ALDHs that have non‐polar or negatively charged substrates exhibit neutral or positive electrostatic potential surfaces, respectively, in this region. Supported by PAPIIT‐UNAM (IN208510 & IN216513) and CONACYT (167122 & 101986).
Endogenous acetylcholine contributes to endothelium‐dependent relaxations induced by mild hypothermia in the SHR aorta.
Authors: Zou Qian , Leung Susan W.S. , Vanhoutte Paul M.
Published: June 2021

The present study was designed to investigate whether or not endothelial endogenous acetylcholine contributes to endothelium‐dependent relaxations induced by mild hypothermia. Aortic rings of spontaneously hypertensive (SHR) and Wistar‐Kyoto normotensive (WKY) rats were incubated with antagonists of m‐ and n‐acetylcholine receptors (atropine and tubocurarine, respectively) or enzyme and inhibitors that can interfere with the synthesis, degradation and transport of acetylcholine (acetylcholinesterase, bromoacetylcholine, hemicholinium‐3, and vesamicol) and exposed to progressive mild hypothermia (from 37°C to 31°C) to trigger relaxations. Mild hypothermia induced endothelium‐dependent relaxations which were inhibited significantly by all the inhibitors used in SHR but not in WKY aortae. The endothelium of both SHR and WKY aortae were able to take up extracellular choline and synthesize endogenous acetylcholine. Thus, the present study demonstrates that endothelium of both normotensive and hypertensive rats can produce acetylcholine. However, mild hypothermia causes endothelium‐dependent relaxations which can be reduced by inhibiting acetylcholine receptors or interfering with the metabolism of acetylcholine only in SHR rat aortae. Thus, in the hypertensive rat, endothelial endogenous acetylcholine can in an autocrine manner activate acetylcholine receptors and elicit endothelium‐dependent relaxations.
Development of Capabilities for Simultaneous Measurement of Pulmonary and Systemic Pressure to Support Evaluation of Pulmonary Hypertension Therapies
Authors: Zhao Huawei , Sun Shu‐Yu , Shen Xiaolan , Hong Xueninh , Yang Liming , Ping Xiaoli , Wang Bo , Davis Christopher A , Metzger Joseph M , Small Kersten M , Madwed Jeffrey B
Published: June 2021

Pulmonary arterial hypertension (PAH) is a life threatening disease with few proven treatment options. It is important that new therapies directed at decreasing pulmonary pressure demonstrate vascular selectivity on the pulmonary bed over the systemic bed. Therefore, we establised methods to directly measure pulmonary arterial pressure (PAP) and systemic blood pressure (systemic BP) simultaneously in conscious freely moving rats using novel HD‐S21 dual pressure transmitters from DSI. Transmitters were implanted in normotensive male Sprague Dawley rats, with one catheter of transmitter placed in the pulmonary artery and the other in abdominal aorta. To create a more clinically relevant PAH model and to increase the assay window, rats were exposed to hypoxic conditions (10% O2) over two weeks which increased baseline sPAP from ~25 mmHg to ~80 mmHg, with minimal effects on systemic BP. Validation studies were performed using standard of care PAH therapies: Tadalafil and Sildenafil (PDE5 inhibitors). Both compounds had similar maximal effect on systolic PAP (sPAP) reduction (~18–22 mmHg) and modest effects on the systemic BP (10–15 mmHg) on hypoxia‐induced PAH. In conclusion, innovative capabilities to directly measure PAP and systemic BP simultaneously have been established in conscious freely moving rat, and these methods provide a powerful approach to directly compare pulmonary vs. systemic hemodynamic activities minimizing cross‐study variability with different cohort of animals and to identify PAH therapies that demonstrate pulmonary selectivity.
2K‐1C Hypertensive Rats Treated With Tempol Show Decreased MMP‐2 Activity Possibly Due To Oxidative Stress Blockage
Authors: Antonio Raquel Carros , Ceron Carla Speroni , Rizzi Elen , Santos José Eduardo Tanus , Gerlach Raquel Fernanda
Published: June 2021

Hypertensive structural and functional modifications are associated with increased MMP‐2 activity and reactive oxygen species (ROS). ROS can induce post‐translational modifications in proteins, such as nitrosylation, that are known to affect anzyme activity. ROS can also directly activate MMPs. This study examined whether the treatment with Tempol prevents raised systolic blood pressure (SBP) and the following biochemical findings of hypertensive heart tissue: ROS activity, total gelatinolytic activity, MMP‐2 activity, and presence of nitrotyrosine residues.

Study protocol:

2kidney‐1clip (2K‐1C) rats and Sham‐operated rats (Sham) were treated with vehicle or Tempol (T) (18 mg/kg/day). Tested used were in situ oxidative stress; in situ zymography; immunofluorescence for MMP‐2 and nitrotyrosine. Tempol attenuated hypertension (2K‐1C: 193± 3mmHg; 2K‐1C+T; 159± 2mmHg; p<0,05), and led to decreases in ROS, total gelatinase activity, MMP‐2 activity and amount of stained nitrotyrosine in cardiac tissue (p<0,05 for all comparisons between the 2K‐1C group versus 2K‐1C+T). Thus, nitrosylation of MMP‐2 or of its substrates may change MMP‐2 activity in hypertension. Results show one important benefit of the treatment with the antioxidant Tempol that needs better understanding.

This study was supported by Conselho Nacional de Pesquisa (CNPq) Brasil.
MicroRNA modulation of cardioprotective pathways
Authors: Luther Kristin , Mallick Faryal , Jones W. K.
Published: June 2021

Every year, tens of thousands of Americans suffer a myocardial infarction. Cardioprotection (CP), the reduction of myocardial injury, involves the induction of pro‐survival processes and genes. Preconditioning of the myocardium can bring about CP through activation of an NF‐kB dependent gene program, in which research shows a key regulatory role for miRNA. Our data indicate the existence of an interconnected regulatory network of miRNAs and protective genes from multiple pathways, which are crucial to the adaptive stress responses of CP. The hypothesis of this study is that preconditioning causes a decrease in several regulatory miRNAs, facilitating an increase in protective protein expression. We preconditioned HL‐1 cardiomyocytes through oxygen and glucose deprivation, which significantly decreased cell death upon subsequent ischemia‐reperfusion injury. Levels of miRNAs and their predicted target genes were assayed by Realtime PCR and western blot. The results confirmed that the miRNAs decreased, while expression of the proteins increased. The data support that through their ability to target multiple genes, miRNAs act to coordinate multiple CP pathways. Insight into the role of miRNAs in CP will allow therapeutic modulation of miRNA levels using antagomirs, which represents an attractive clinical approach to reduce infarct size. This research was supported by NIH R01HL091478.
Identification of a novel, in vitro O‐glucuronide metabolite of a MEK inhibitor, AR00340509, linked by way of a carbonyl oxygen
Authors: Franklin Ronald Brian , Allen Andrew , Kraser Christopher F , Dinkel Victoria L.
Published: June 2021

While studying the metabolism of unique MEK inhibitors, including AR00340509 (4‐((4‐bromo‐2‐fluorophenyl)amino)‐N‐(2‐hydroxyethoxy)‐1,5‐dimethyl‐6‐oxo‐1,6‐dihydropyridine‐3‐carboxamide), we identified a novel O‐glucuronide linked through the carbonyl oxygen of the hydroxamate moiety. This glucuronide metabolite was observed in UDPGA‐supplemented hepatic microsomes and hepatocytes from a number of animal species including humans and was formed by rUGT1A1 and rUGT1A3. The terminal O‐linked glucuronide on the hydroxamate terminal OH side chain and N‐glucuronide of the amide metabolite were observed. The identity of the O‐linked carbonyl glucuronide metabolite was supported by the synthesis of the peracetate derivative of the glucuronide. Within this structural class of MEK inhibitors, some but not all, of the O‐linked carbonyl glucuronides were unstable and identification of the pathway was carried out via the more stable peracetate. Accurate mass determination of the peracetylated glucuronide of AR00340509 was carried with LC‐QTOF instrumentation, which yielded an M+H ion of 730.1258 and a structural formula of C29 H34 Br F N3 O13. The structural class of MEK inhibitors studied lend themselves to enhanced tautomer enolization, thereby facilitating the formation of these unique O‐glucuronides. We are investigating factors affecting the formation of carbonyl glucuronides, including substitutions with both electron withdrawing and electron donating entities.
Activators of CAR and PXR Rapidly Alter Chromatin Accessibility in Mouse Liver
Authors: Lodato Nicholas J , Rampersaud Andy , Waxman David J
Published: June 2021

CAR and PXR are important xenobiotic sensors that rapidly induce and repress genes regulating diverse liver metabolic processes, including drug, steroid and lipid metabolism. The effects of specific activators of CAR and PXR on mouse liver chromatin accessibility were determined using DNase‐Seq to identify global changes in DNase hypersensitive sites (DHS) 3 hr after treatment of mice with TCPOBOP (CAR activation) or PCN (PXR activation). Differential DHS analysis (ΔDHS) identified 928 TCPOBOP‐induced DHS and 161 TCPOBOP‐repressed DHS, as well as 1,062 PCN‐induced DHS and 390 PCN‐repressed DHS, with some overlap between the two sets of ΔDHS. Thus, CAR and PXR rapidly induce large numbers of localized changes in chromatin accessibility. Several ΔDHS were confirmed by qPCR, including ΔDHS upstream of the CAR target gene Cyp2b10. The ΔDHS were not randomly distributed: mapping ΔDHS to their presumed targets (i.e., the nearest gene within 100 kb) revealed strong (11.4‐fold) enrichment (p<E‐19) of the TCPOBOP‐induced DHS in the set of TCPOBOP‐induced genes, identified in the same livers. The gene targets of TCPOBOP‐induced DHS also showed strong enrichment (5.9‐fold, p<0.005) in the set of TCPOBOP‐repressed genes. Thus, chromatin opening is associated with rapid activation as well as rapid repression of CAR target genes. We also found strong enrichment (11.7‐fold, p<E‐29) of target genes of PCN‐induced DHS in the PCN‐inducible gene set, whereas genes repressed by PCN were significantly enriched (6.4‐fold, p<E‐04) in PCN‐repressible DHS gene targets. Thus, CAR and PXR may repress genes by fundamentally different mechanisms: DHS opening for CAR vs. DHS closing for PXR.
Analysis and Impact of Mainstream Tobacco Smoke from Regular and Reduced Exposure Products on Blood Brain Barrier: Are we clearing this smoke cloud yet?
Authors: Naik Pooja , Cucullo Luca
Published: June 2021

Active and passive tobacco smoking (TS) are associated with dysfunction of vascular endothelial physiology (including the blood‐brain barrier) in a causative and dose dependent way largely related to the relative content of reactive oxygen and nitrogen species (ROS/RNS). ROS and RNS can mediate progressive endothelial damage causing alteration of extracellular matrix and apoptosis of endothelial cells. Accordingly, the tobacco industry is marketing several lines of “reduced‐exposure” (RE) tobacco products claimed to be less harmful. For this purpose we measured the ROS and nitrogen content of mainstream TS from different brands (including tobacco free and ultralight cigarettes) and assessed their effect on BBB endothelial cells. Our results showed that RE products have a higher content of hydrogen peroxide and RNS which correlated with higher release of MMP‐2 and IL‐6 from the BBB endothelium. These results were further supported by gene array analysis which also showed alterations in the gene expression of CYP enzymes, vascular adhesion molecules and signal transduction proteins (SHC4). Alteration in gene expression of hydroxysteroid (11‐beta) dehydrogenase 1 and 2, insulin induced gene 1, also suggests that TS could play a role in the pathogenesis of type 2 diabetes. Our results suggest that ‘light’ and ‘tobacco free’ cigarettes are equally or potentially more toxic than the regular products.

This work was supported by NIH/NIDA R01‐DA029121–01A1 and A.R.D.F. to Dr. Luca Cucullo.
Characterization of the coronary vascular transcriptome in a rat model of metabolic syndrome
Authors: Bender Shawn B , Davis J. Wade , Rector R. Scott , Laughlin M. Harold , Sowers James R
Published: June 2021

Obesity, insulin resistance and the metabolic syndrome (MetS) cause coronary microvascular dysfunction and defects of coronary flow control. Although myriad mechanisms contribute to this dysfunction no study has comprehensively determined MetS‐associated differential coronary vascular gene expression (the coronary transcriptome). Coronary arteriolar function and gene expression was determined in vessels from obese Otsuka Long‐Evans Tokushima Fatty (OLETF) and lean Long‐Evans Tokushima Otsuka (LETO) rats. Coronary endothelial dysfunction was present in OLETF rats by 5–8wk of age. At 32wk of age, OLETF exhibited obesity, hyperglycemia, and hypertriglyceridemia but normal blood pressure compared to LETO. At this age, coronary arteriolar dysfunction was characterized by vasoconstriction to insulin and impaired dilation to acetylcholine. Vasoconstriction to endothelin‐1 and the thromboxane analog U46619 was increased while constriction to 80mM KCl or the L‐type calcium channel opener BayK 8644 was unchanged. The coronary transcriptome was determined by RNA deep sequencing in OLETF and LETO septal artery samples and revealed dramatic differences in gene expression between groups. This is the first study to comprehensively characterize the coronary transcriptome in an established model of MetS. Supported by NIH HL107910 and HL36088, VHA‐CDA2 IK2BX001299, MU Internal Medicine Research Council.
Voluntary Aerobic Exercise Destiffens Arteries of Old Mice Via Inhibition of NADPH Oxidase‐ and Superoxide‐Dependent Oxidative Stress
Authors: Eng Jason Samuel , Pham Bryant Thanh , Johnson Lawrence Cody , Giosca‐Ryan Rachel A , Seals Douglas R , Fleenor Bradley S
Published: June 2021

We tested the hypothesis that voluntary aerobic exercise (wheel running) reverses age‐related large elastic artery stiffening by inhibiting NADPH oxidase‐ and superoxide‐dependent oxidative stress. Intrinsic mechanical properties were examined in thoracic (TA) and abdominal (AA) aortic segments from young (Y, 8 mo, n=8) and old (O, 26 mo, n=7) cage‐control male C57BL/6 mice, and old animals with access to voluntary running wheels (OVR, 26 mo, n=6) for 10 weeks. Compared with Y, O had greater elastic modulus (EM), an indicator of stiffness, in the TA (5899 ± 731 vs. 3307 ± 324 kPa, p<0.05) and AA (7796 ± 794 vs. 5268 ± 371 kPa, p<0.01), and increased aortic nitrotyrosine abundance (NT, 2.78 ± 0.61 vs. 1.00 ± 0.23 AU, p<0.05), a marker of oxidative stress. Voluntary running decreased EM in the TA (to 3712 ± 878 kPa, p<0.05) and AA (to 5988 ± 507 kPa, p<0.05), and reduced NT abundance (to 0.91 ± 0.25 AU, p<0.05). Additional TA segments were cultured for 72 hr with TEMPOL (100μm), a superoxide scavenger; apocynin (Apo, 1μmol), a NADPH oxidase inhibitor; or vehicle control (C). Treatment with either TEMPOL or Apo normalized EM in O mice to that observed in Y and OVR animals, without affecting EM in the latter groups. Our results demonstrate that voluntary aerobic exercise normalizes age‐associated stiffening of the TA and AA in mice via reversal of NADPH oxidase‐ and superoxide‐dependent oxidative stress.

NIH AG013038, AG000279, HL007822
The role of differential oxygen distribution between the brain and peripheral tissues on tolerance to induced central hypovolemia
Authors: Lee Kay Victoria , George Mebin , Soller Babs R. , Ryan Kathy L. , Hinojosa‐Laborde Carmen , Convertino Victor A. , Rickards Caroline A.
Published: June 2021

Background

Maintenance of cerebral perfusion and oxygenation is essential for survival from hemorrhage. As individuals vary in tolerance to reduced blood volume (e.g., central hypovolemia), we hypothesized that subjects with high tolerance (HT) will maintain cerebral oxygenation compared to low tolerant (LT) subjects, which will be associated with greater peripheral vasoconstriction.

Methods

13 human subjects were instrumented for cerebral (ScO2) and muscle (SmO2) oxygenation (near‐infrared spectroscopy, NIRS), then completed a step‐wise presyncopal‐limited lower body negative pressure (LBNP) protocol. Total hemoglobin (HbT) was calculated as an index of local peripheral resistance (inverse relationship) at the muscle.

Results

Progressive decreases in ScO2 were observed in LT subjects (n=5) from −45 mmHg LBNP (P≤0.01 compared to baseline), while ScO2 did not change throughout LBNP for the HT subjects (n=8; P≥0.44). SmO2 decreased at −30 and −60 mmHg LBNP (P≤0.01) in the HT group with concomitant increases in resistance (decreases in HbT; P≤0.03). In contrast, both SmO2 (P=0.01) and HbT (P=0.06) only decreased at −60 mmHg LBNP in the LT group.

Conclusions

In support of our hypothesis, increased tolerance to progressive central hypovolemia was associated with maintained cerebral oxygenation, due, in part, to earlier onset of peripheral vasoconstriction.

Source of Funding: US Army MRMC
Mice Produced by the Use of Assisted Reproductive Technologies from Dams Provided a High‐Fat and ‐Fructose Diet Have Reduced Arterial Vasodilation Responses to Acetylcholine
Authors: Ramirez‐Perez Francisco I. , Schenewerk Angela L. , Foote Christopher , Zhao Guiling , Rivera Rocio M. , Martinez‐Lemus Luis A.
Published: June 2021

Maternal obesity or the use of assisted reproductive technologies (ART) such as superovulation, and embryo culture and transfer are associated with unfavorable cardiovascular outcomes in offspring. We hypothesize that maternal obesity in combination with ART causes impaired vascular responses in offspring early in life. We tested this hypothesis in isolated mesenteric arteries from eight week‐old mice offspring of natural birth (NoART) or ART dams fed normal chow or a high fat and fructose diet (HF). Offspring were fed the same diet as their dam. Experiments were performed in pressure myographs with arteries kept at 37C and pressurized to 70 mmHg. Arteries from offspring of ART‐HF dams had significantly reduced vasodilation responses to acetylcholine (10–5 M) vs. NoART‐HF (19.7 ± 3.2 vs. 38.2 ± 7.1%, respectively). These arteries also had greater passive wall to lumen ratios indicative of inward remodeling (0.16 ± 0.01 vs. 0.13 ± 0.01, respectively). Responses to phenylephrine or sodium nitroprusside between ART‐HF and NoART‐HF were not different. ART had no effect in mice fed normal chow. These results suggest that ART in conjunction with maternal obesity is detrimental to vascular function and structure in young offspring. (Supported by NIH HL‐088105 to LAM and F21C to RMR)
Disruption of catenin‐cadherin interaction decreases tail regeneration in Lumbriculus variegatus.
Authors: Henry Charmaine , Reid Megan
Published: June 2021

Lumbriculus variegatus is an aquatic oligochaete that can regenerate new tail segments within two to three weeks of tail removal. While many studies have investigated this ability to regenerate, not much is known about the molecular pathways that are activated during the regeneration process in Lumbriculus. Regeneration of animal tissue must also involve the formation of adherens junctions between new cells in the proliferating tissue. Cadherins are calcium‐dependent adherens molecules that bind adjacent cells together. Cadherins in turn bind to catenins. The β‐catenin has been shown to be important for cell adhesion as well as for the Wnt signaling pathway which influences the polarity of developing organisms. As a first step in determining if β‐catenin is involved in regeneration of new tissue in Lumbriculus, we tested the effect of the β‐catenin inhibitor quercetin, on tail regeneration rate. Low Dose groups were exposed to 25 mM of quercetin and High Dose groups were exposed to 50 mM of quercetin for two weeks. Controls were not exposed to this inhibitor. Results showed that, while all groups experienced tail segment regeneration during the first week of treatment, the rate of regeneration was significantly slower in groups exposed to both low and high doses of quercetin from the second to the fifth day. We suggest that the β‐catenin‐cadherin signaling pathway plays a role in Lumbriculus tissue regeneration.
Effect of Cardiotonic Steroids on Monolayer Permeability and Junction Proteins in Lymphatic Endothelial Cells
Authors: Horvat Darijana , Cromer Walter , Kuehl Thomas , Zawieja David , Uddin Mohammad Nasir
Published: June 2021

Objective

Preeclampsia (preE) is a hypertensive disorder unique to pregnancy. Cardiotonic steroids (CS) such as marinobufagenin (MBG), cinobufotalin (CINO), and ouabain (OUB) are Na+/K+ ATPase inhibitors. MBG is elevated in a rat model and patients with preE. MBG causes a vascular leak syndrome in vivo and increases endothelial cell monolayer permeability. We evaluated the effect of these CS on monolayer permeability of lymphatic endothelial cells (LECs).

Methods

LECs were isolated from a rat mesenteric collecting lymphatic vessel. The cells were treated with DMSO (basal), MBG, CINO, or OUB. Monolayer permeability of CS‐induced LECs was measured. The expression of β‐catenin and VE‐cadherin was measured by immunofluorescence. Western blot was used to measure β‐catenin and VE‐cadherin protein levels.

Results

MBG (≥ 1 nM, p<0.05) and CINO (≥ 10 nM, p<0.05) significantly increased the monolayer permeability of LECs compared to basal (DMSO) while OUB had no effect. MBG (p<0.05) and CINO (p<0.05) treatment significantly downregulated β‐catenin protein expression. However, CS did not cause any disruption of the LEC tight junctions. CINO downregulated the VE‐cadherin protein expression, but MBG did not.

Conclusions

We have demonstrated that bufadienolides MBG and CINO caused an increase in the monolayer permeability and downregulated the β‐catenin protein expression with no effect on tight junctions.
Hsp70 distribution patterns in rat skeletal muscle and vasculature post high and low intensity exercise
Authors: Kowalchuk Hana , Melling C.W. James , Noble Earl
Published: June 2021

Contracting skeletal muscle works as an integrated unit where increased muscle blood flow, myofiber (MF) activation and elevated temperature represent stresses which result in induction of the cytoprotective heat shock protein 70 (Hsp70) in blood vessels (BV) and MF. The effect of exercise intensity on Hsp70 expression within, and relationships between, these structures has yet to be examined. Tested hypotheses were 1) with increasing exercise intensity, more BVs, progressing from large to small would express Hsp70 2) MFs expressing Hsp70 would be located adjacent to Hsp70 expressing BVs. Male rats served as controls or were run for 1h on a treadmill (2% incline) at 15m/min (LoEx) or 30m/min (HiEx). 24 hr post‐exercise the white portion of the vastus muscle was harvested for localization of Hsp70 (n=10/group). The ratio of BVs expressing Hsp70/total vessels was greater in HiEx vs LoEx as a consequence of increased expression in small vessels (P<0.05). Hsp70 integrated density was also greater in large vs small BVs (P<0.05). Finally, the ratio of Hsp70 expressing MFs/total MFs was higher around smaller vs larger BVs (P<0.05). These observations demonstrate that HiEx produces a greater and more extensive Hsp70 response than LoEx in the vasculature, possibly as a result of vessel recruitment. The greater Hsp70 response in MF surrounding small BVs suggests an intensity‐dependent linkage of MF recruitment with blood flow.
Transcriptome and microRNA Signatures of Staphylococcal enterotoxin B Exposure under the Background of Battlefieldlike Stress
Authors: Muhie Seid , Hammamieh Rasha , Jett Marti
Published: June 2021

Leucocytes isolated from pre‐ and post‐training whole blood samples of US Army Ranger Cadets were exposed in vitro to Staphylococcal enterotoxin B (SEB) to analyze SEB‐induced regulation of gene expressions both in the presence and absence of severe‐stress. Gene profiling data showed suppression of immune response genes that are important in pattern recognition receptors, phagocytosis, antigen presentation, chemotaxis and activation of lymphocytes. Immune response genes that were suppressed by the training stressors remained suppressed after SEB exposure. Post‐training leucocytes seemed to be anergic towards SEB. Stress‐regulated microRNAs were identified as potential up‐stream regulators of many transcripts important in chemotaxis, antigen presentation, inflammation, lymphocyte activations and transcription factors important in immune response. Despite the complications due to suppressed immune responses seen at the end of the intensive Army Ranger Training, we have also identified other groups of genes that showed consistent differential expression towards SEB both in pre‐ and post‐training leukocytes. Some of these genes were SEB‐specific and have the potential to serve as diagnostic markers of SEB exposure irrespective of other pathophysiologic conditions.
E. coli a‐haemolysin leads to ATP release and P2‐dependent calcium influx in human erythrocytes
Authors: Skals Marianne Gerberg , Leipziger Jens , Praetorius Helle
Published: June 2021

The E. coli toxin á‐haemolysin (HlyA) lyses erythrocytes by inserting as pores in the membrane. Recently, we demonstrated that HlyA‐induced haemolysis is a sequential process with an initial volume reduction that precedes swelling and lysis. The initial volume reduction is triggered by an abrupt increase in [Ca2+]i.

Moreover, we have shown that HlyA‐induced haemolysis requires extracellular ATP and P2 receptor activation. The present study investigates the mechanism for ATP release and [Ca2+]i increase that precede the volume changes in erythrocytes. By the luciferin/luciferase assay, we show that HlyA leads to ATP release within 5 minutes after toxin is added. ATP release was independent of pannexin‐ and volume‐dependent anion channels; two suggested ATP exit pathways. Thus, ATP may simply be released via the toxin pore. Through a combination of flow cytometry and live cell imaging, we show that the [Ca2+]i response consists of an abrupt increase followed by at slower, sustained increment. P2 antagonists do not interfere with the initial acute [Ca2+]i increase, but significantly reduces the slow phase of the HlyA‐induced influx of [Ca2+]i and cause a marked delay in the swelling of the cells. We suggest that ATP may be released via the toxin pore, which activates P2 receptors in the erythrocyte membrane. The influx of cations through P2 receptors contributes to net ion influx and thereby cell swelling and lysis.
CORONARY VASOCONSTRICTION INDUCED BY POLYMERS OF ANG II IS PROPORTIONAL TO LUMINAL ENDOTHELIAL AT1 RECEPTORS DENSITY
Authors: Torres‐Tirado David , Rubio Rafael
Published: June 2021

We have shown that intravascular AngII does not diffuse across the coronary vessel wall and acts solely in coronary endothelial luminal membrane AngII receptors (CELM‐AT1R) and its sustained intracoronary infusion causes transient vasoconstriction effects (+V). This contrasted with the sustained +V by a large size, non‐diffusible polymer of AngII (15,000 kDa, AngIIPol) that unquestionably activate solely CELM‐AT1R. We also show that AngII transient +V paralleled a decrease of CELM‐AT1R density (AT1RD), but, for AngIIPol the AT1RD remained constant. HYPOTHESIS; transiently of +V effects depends directly on AT1RD both are inversely determined by the molecular weight (MW) of AT1R agonist. To test this hypothesis we synthesized AngIIPol's (included AngII) of various MW (kDa): 1.3, 2.7, 11, 47, 527, 3270 and 15000. In isolated constant flow (8ml/min) perfused Langendorff guinea pig hearts AII‐Pol's were separately intracoronary given for 16 min and coronary perfusion pressure measure as an index V. All AngIIPol's caused the same maximal +V (+Vmax) that decayed with time at a rate and to a stable level of +V, both inversely proportional to MW. During AngIIpol's infusion AT1RD decayed parallel to +V; the relationship +V‐‐AT1RD was linear. Further dependence of +V on AT1RD was demonstrated by two successive administrations of AngIIPol's, the primary dose caused a +Vmax but the +Vmax to the secondary dose was depressed; a tachyphylaxis which was inversely proportional to the logarithm of MW. These results support our hypothesis and indicate that AT1R internalization is dependent on MW.
Functional characterization of Atg24 domain in autophagy regulation
Authors: Yu Li‐En D , Huang Wei‐Pang
Published: June 2021

Autophagy, a degradation pathway, is highly conserved in eukaryotes. It is associated with many physiological processes and regulated by various Atg proteins. In autophagy, a double‐membrane autophagosome engulfs cytoplasmic cargoes and fuses with lysosome/vacuole, which results in the breakdown process through the actions of hydrolases. Generally, autophagy is considered a non‐specific process, but there are selective autophagy functioning in degradation of excess organelles. In yeast, nonselective autophagy is highly induced under starvation environment, but a particular type of selective autophagy, the cytoplasm‐to‐vacuole targeting (Cvt) pathway, is constitutively active and operates in nutrient‐rich condition. Although these two pathways share the same core machinery, specific regulators for each pathway are also required. Atg24, specifically regulates the Cvt pathway, but is dispensable for the bulk autophagy. Except the PI3P‐binding domain, domain structure analysis of Atg24 identifies two coiled‐coil domains and a BAR domain associated with an amphipathic helix. The BAR domain is considered a membrane‐curvature sensor. Therefore, BAR‐domain proteins regulate membrane trafficking through facilitating lipid transfer and tethering of vesicles. In this study, we functionally characterized Atg24 in autophagy regulation. First, we found that Atg24 interacts with Atg20 through a region between the two coiled‐coil domains. This interaction is critical to the Cvt pathway, because Atg24 and Atg20 promote Cvt vesicle formation in a mutually dependent manner. Next, we confirmed that the coiled‐coil domains and the BAR domain of Atg24 are required for endosome trafficking, vacuolar protein sorting, and the Cvt pathway. Taken together, we propose that Atg24 functions after PI3P signaling and regulates the Cvt pathway through its BAR domain.
Lumbar denervation attenuates the sympathetic‐cardiovascular response to acute increases in cerebrospinal fluid sodium concentration
Authors: Simmonds Sarah Shayne , Stocker Sean D.
Published: June 2021

Increased concentration of sodium in cerebral spinal fluid elevates sympathetic nerve activity (SNA), heart rate (BPM), and mean arterial pressure (MAP). The present study aims to identify the contribution of the sympathetic nervous system to this response. In anaesthetized male Sprague Dawley rats, infusion of 1M NaCl (5μL/10min) into the lateral ventricle increased MAP (Δ 7±1 mmHg), heart rate (Δ 14±4 BPM), lumbar SNA (Δ 115±3%), splanchnic SNA (Δ117±4%) and adrenal SNA(Δ111±3%) but decreased renal SNA (Δ 89±3%). Pretreatment with the ganglionic blocker chlorisondamine (5mg/kg, IV) prevented the rise in MAP(Δ1±1mmHg) and heart rate (Δ4±3BPM). In a final set of experiments, the increase in MAP was significantly attenuated by lumbar denervation (area under the curve: control 5.3±0.8; lumbar 3.1±1.0) but unaffected by renal denervation (area under the curve: 5.6±1.5) or celiac ganglionectomy (area under the curve: 7.1±2.0). These findings indicate that an increase in sodium concentration in cerebral spinal fluid activates the lumbar nerve, at least partly, to increase MAP and heart rate.
Oxygen consumption during gravidity compared to digestion and exhaustive activity in the viviparous Checkered Garter Snake
Authors: Jackson Alexander G.S , Hicks James W
Published: June 2021

The goal of this research was to measure oxygen consumption (i.e. VO2) associated with exhaustive activity, digestion, and gravidity in the viviparous Checkered Garter Snake. To determine gravidity, female snakes were imaged with a portable ultrasound. Gas analysis was performed using flow through respirometry with carbon dioxide and oxygen sensors. Current results indicate that gravid female Checkered Garter Snakes increase resting VO2 up to 1.6 fold the values the non‐gravid state. Furthermore, gravid females demonstrate a higher VO2 during digestion (scope = 1.5), but demonstrate a lower peak VO2 during fasting exhaustive activity (scope= .83). Both gravid and non‐gravid females exhibit a higher VO2 during post‐prandial exhaustive activity compared peak VO2 during either exhaustive activity or digestion. Therefore, this species demonstrates an additional capacity for oxygen consumption that is revealed only when simultaneously digesting a large meal and maximally active. Funding was provided by NSF grant IOS 0922756 to JWH.
Differential impacts of environmental salinity on gastrointestinal sodium transport of the invasive crayfish Procambarus clarkii and Orconectes rusticus
Authors: Rhoads Megan Kathleen , Morrisey Caitlin , Sunnenberg Lexi , Osborn Jeffrey L
Published: June 2021

Procambarus clarkii regulates internal hemolymph osmolality in response to changes in environmental salinity enhancing its ecological plasticity. Orconectes rusticus is an invasive species beyond the Ohio River Basin but its osmoregulatory processes are unstudied. Animals were acclimated to freshwater or brackish water medium (0.5% and 1.5%NaCl, respectively). Brackish water acclimation increased the hemolymph osmolality of P. clarkii by 92±10.6 mOsm/l and O. rusticus by 91±8.4 mOsm/l. After acclimation, gastrointestinal (GI) tracts were perfused with control (0.5% NaCl), 2.0% NaCl, and recovery (0.5% NaCl) solutions. Following brackish water acclimation, GI Na+ excretion of P. clarkii increased in response to high NaCl by 35.9±3.7μmol/min while that of O. rusticus increased by only 28.4±2.73 μmol/min (p<0.05). After fresh water acclimation, changes in Na+ absorption after 2.0% NaCl perfusion were not different. The improved osmoregulation of P. clarkii after brackish water exposure may enhance the ability of the species to survive environmental osmotic challenges while O. rusticus has yet to adapt to altered environmental salinity. Supported by UK Biology Gertrude & Flora Ribble Foundation.
Tylophorine induces c‐Jun protein accumulation through two discrete prolonged pathways NF‐kappaB/PKCdelta/JNK and PI3K/PDK1/PP2A/eEF2 to promote G1 arrest in carcinoma cells
Authors: Yang Cheng‐Wei , Lee Yue‐Zhi , Hsu Hsing‐Yu , Wu Chia‐Mao , Chang Hwan‐You , Chao Yu‐Sheng , Lee* Shiow‐Ju
Published: June 2021

Herein, we report that c‐Jun protein, a component of AP‐1, was elevated in the carcinoma cells by tylophorine treatment and the c‐Jun phosphorylation was mainly through activated JNK. Moreover, ectopically overexpressed c‐Jun significantly facilitated G1 arrest in the presence of tylophorine analyzed by flow cytometry analysis. Ultimately, the participation of c‐Jun in downregulated cyclin A2 by tylophorine in carcinoma cells was dissected by ChIP and element reporter assays. Upon increase in binding of c‐Jun to the deregulation AP‐1 site and decrease in binding to up‐regulation ATF site in c‐Jun promoter, the tylophorine elevated c‐Jun downregulated cyclin A2 promoter activity and thus its expression thereby leading to G1 arrest. Further biochemical studies using pharmacological inhibitors and RNA silencing approaches demonstrated that tylophorine elevated the c‐Jun protein level is mainly through two discrete prolonged signaling pathways: 1) a prolonged activation of NF‐kB_PKC?_(MKK4)_JNK cascade to phosphorylate c‐Jun and increase its stability; 2) a prolonged activated PI3K_PDK1_PP2A_eEF2 cascade to sustain eEF2 activity and thus the c‐Jun protein translation. (This work is founded by National Health Research Institutes, Taiwan, R.O.C. and National Science Council of Taiwan, R.O.C. [99–2320‐B‐400–009‐MY3])
Hepatic proteome analysis in mice following 90 days of spaceflight
Authors: Fuller Charles A. , Fuller Patrick M. , Strollo Felice , Masini Maria Angela
Published: June 2021

Long‐term exposure to altered gravitational environments leads to altered intermediary metabolism and concomitant changes in body adiposity. It is unclear to what extent changes in hepatic function might underlie these metabolic shifts. Using proteomic technology, we examined liver protein profiles in mice exposed to long‐term microgravity via spaceflight. The analysis was performed on livers obtained from male mice (n=3) that were exposed to 90 days of microgravity on the International Space Station (ISS). Control livers were obtained from mice (also n=3/group) housed in either simulated ISS flight cages or standard vivarium cages on Earth. Liver proteins were extracted, separated on a 1‐D gel, trypsin digested and analyzed by LC MS/MS. Proteins were searched and validated with X! tandem and Scaffold Software. ANOVA was used to determine significant differences in expression (spectral counts) between groups. Roughly 4,000 proteins with a 7.7% protein and 0.1% peptide false discovery rate were identified, of which approximately 500 demonstrated significant changes (p<0.05). Of these, approximately 250 proteins were uniquely different between flight and ground groups, including a number of proteins involved in fatty acid metabolism. This work was supported in part by NASA Grant NNX08AR88G (CAF).