bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Store‐Operated Ca2+ Entry in the Vascular Endothelium by the G Protein‐Coupled Estrogen Receptor
Authors: Terry Lara , TRAN KIM , VerMeer Mark , Giles Jennifer
Published: June 2021

The novel G protein‐coupled estrogen receptor (GPER/GPR30) has been found to participate in numerous cardiovascular functions. Store‐operated Ca2+ entry (SOCE) is an essential mechanism that is required for many endothelial cell functions. We found that activation of GPER using the GPER specific agonist G1 is associated with a dose‐dependent inhibition of SOCE in primary vascular endothelial cells. Interestingly, the GPER specific antagonist G15 increases SOCE in cells unstimulated by GPER intrinsic or exogenous ligand. Overexpression of GPER in HEK 293 cells is associated with a 40% decrease in the rate of SOCE, while knockdown of GPER in endothelial cells using shRNA or antisense oligomer directed against GPER increases SOCE by approximately 50%. In addition, coimmunoprecipitation revealed that GPER exists in endothelial cells as a glycosylated protein and forms a complex with the stromal interaction molecule 1 (Stim1). These data suggest that GPER may be an important regulatory input of store‐operated Ca2+ entry via its interaction with Stim1.
Knockdown of the D2 autoreceptor from dopaminergic terminals alters behaviors associated with impulsivity.
Authors: Bernosky‐Smith Kimberly A , Feeney Casey L , Bass Caroline E
Published: June 2021

High D2 receptor availability has been associated with high impulsivity. D2 receptors are found on both pre‐ and post‐synaptic sites in corticomesolimbic and nigrostriatal pathways. We have created a way to manipulate D2 receptor populations in different brain regions using a viral vector to deliver short interfering RNAs targeting the D2 receptor in rats. By injecting the virus in the midbrain, only the cell bodies will receive the shRNA, resulting in knockdown of the presynaptic D2 autoreceptor. When the D2 autoreceptor is knocked down only in the dorsal striatum (leaving post‐synaptic D2R intact) the rats become hyperactive and have altered responses to dopaminergic agents. We have also depleted the D2 autoreceptor from specific corticolimbic structures, including the prefrontal cortex. We hypothesized that enhanced dopaminergic tone will produce a similar hyperactive state and enhance impulsivity, specifically through alterations in the prefrontal cortex. We have employed the five‐choice serial reaction time task (5‐CSRTT), which has proven useful in elucidating one facet of impulsivity, the inability to wait. Our data suggest that D2 autoreceptor feedback inhibition mediates key aspects of impulsivity, including hyperactivity. In conclusion, we demonstrate a novel method to discriminate the role of the D2 autoreceptors in specific pathways and behavioral phenomena.
Induction of Autophagy with rapamycin overcomes Bcl‐2's deleterious effects on stroke outcome
Authors: Buckley Kathleen Mary , Hoda Nasrul , Herberg Samuel , Barrett John R , Periyasamy‐Thandavan Sudharsan , Kondrikova Galina , Hess Daniel L , Hess David C , Schoenlein Patricia V , Hill William D
Published: June 2021

Only one therapy is currently approved to treated ischemic strokes and the majority of patients do not have an opportunity to receive treatment, indicating a need for further treatment areas. One area that has been attractive is to target delayed cell death pathways in the penumbra. An emerging stroke literature indicates that in addition to apoptotic cell death, autophagy is seen after stroke. Autophagy is a process of regulated turnover of cellular constituents through an autophagosomal‐lysosomal pathway and plays a key role in cell homeostasis, development, normal growth control and during bioenergetic stress. At a crossroad between apoptosis and autophagy is the anti‐apoptotic protein Bcl‐2, which acts to block apoptosis by preventing mitochondrial leakage, but it is also anti‐autophagy as it binds the protein Beclin‐1 and prevents initiation of autophagy. The balance of Bcl‐2 activity after stroke and its effect on stroke outcome are not well understood. We show that normal basal autophagy is higher in brain than any other tissue and levels of Bcl‐2 are low in the brain. We demonstrate that autophagy is up‐regulated in the brain and that stroke injury increases expression of the anti‐apoptotic and anti‐autophagy protein, Bcl‐2. Treatment with rapamycin can help to overcome the Bcl‐2 initiated blockade of autophagy and improve stroke outcome.
Antihyperglycemic and antihyperlipidemic activity of MCy protein
Authors: Badri Kameswara Rao , Maddirela Dilip Rajasekhar , Marella Saritha , Kondeti Vinay Kumar , EGTV Kumar , Chippada Apparao
Published: June 2021

Previous studies reported the beneficial role of Momordica cymbalaria (MC) for diabetes mellitus (DM). The current study evaluates the efficacy of antihyperglycemic activity of MCy protein (MCP), which we recently reported from the fruits of MC. Here we studied the long‐term (6 weeks) treatment effects of MCP in streptozotocin induced diabetic rats on the levels of glucose, glycosylated hemoglobin, insulin, lipids and activities of carbohydrate metabolizing enzymes. MCP reduced the fasting blood glucose levels by increasing the plasma insulin accompanied by an improvement in the glycemic control. The results further showed that the MCP treatment has antihyperlipidemic activity. Administration of MCP to diabetic rats normalized the decreased activities of hepatic hexokinase and glucose‐6‐phosphate dehydrogenase. In addition MCP treatment normalized the altered activities of the gluconeogenic enzymes in diabetic treated rats. These findings conclude that MCP is therapeutic in experimental DM. However, further detailed toxicological studies need to be conducted to establish MCP as an alternate treatment for the management of DM.
Dynamic Model of Platelet Lifespan in Adult and Newborn Mice
Authors: Ait‐Oudhia Sihem , Mager Donald E. , Liu Zhi‐Jian , Veng‐Pedersen Peter , Sola‐Visner Martha
Published: June 2021

Objective

To develop a mathematical model of platelet survival (PLT‐S) in newborn and adult mice and identify factors controlling differences in PLT lifespan (PLT‐L) between these groups.

Methods

PLT‐S profiles were obtained from 23 mice (16 newborn and 7 adult) by measuring biotinylate labeled (LBL) and total PLT count (PLTtot=LBL+non‐LBL) using flow cytometry. Non‐LBL endogenous PLT were described with three transit‐compartments and a zero‐order production rate. In newborns, PLT‐L model parameters were time‐dependent and approached the constant values in adults. Biotinylation efficiency was assumed complete, and LBL and PLTtot were simultaneously analyzed using population‐based modeling. Between‐animal variability (BAV) was estimated.

Results

The final model well described LBL and PLTtot in all animals. Parameters were estimated with good precision. At time=0, PLT‐L was fitted at 4.92 and 3.76 days in newborn and adult mice. After 14 days, PLT‐L for all animals was constant at 3.76 days. PLT production was estimated to be similar between groups at 12.4 and 11.9 (103 cells/μL/h). BAV for PLT‐L parameters were below 30%.

Conclusion

Neonatal PLT‐L is greater than adults by 1 day, but PLT production rates are similar. Unlike traditional models assuming steady‐state, our dynamic PLT‐L model captured data well and allowed for accurate estimation of factors governing PLT counts. Grants HL046925 & GM 57980.
Histological studies on the hazardous effects of ethanol on liver and sple en in Swiss albino mice
Authors: ALDAHMASH BADR
Published: June 2021

Considering that ethanol caused number of health problems in the world, the present study was initiated to investigate the histological hazardous effects of ethanol on the liver and spleen. Animals were divided into 4 groups, the first group served as a control group, the second, third and fourth received 1,2 and 6 ml/kg/bw of 70% ethanol respectively. At the day 5 post treatment, the liver and spleen sections were prepared for the histological study. Ethanol intake induced marked histologyical alterations in the liver and spleen that correlated with the dose taken, low dose induced liver and spleen injury mainly as cytoplasmic degeneration in liver and abnormal structure of spleen, whereas, high doses of ethanol resulted in fibrosis in liver and splenomegaly.
Impact of concomitant use of opioids and benzodiazepines in opioid‐dependent pregnant women on the outcome of their newborns
Authors: Metz Verena , Comer Sandra D. , Fischer Gabriele
Published: June 2021

OBJECTIVE

The study objective was to compare neonatal outcome parameters of children born to opioid‐dependent women in opioid maintenance therapy with differing concomitant consumption patterns during the last trimester of pregnancy.

METHODS

An observational design and t‐tests were applied to analyze the outcome of 106 neonates born to opioid‐maintained, smoking mothers with (n=27) versus without (n=79) concomitant consumption of opiates and benzodiazepines.

RESULTS

Newborns of mothers with concomitant opiate and benzodiazepine consumption had significantly higher average withdrawal scores (8.27 vs. 6.15, p=0.007) and significantly longer postnatal hospital stays (26.5 vs. 18.6 days, p=0.011) compared to those of mothers without problematic concomitant consumption. A subgroup analysis confirmed the results for the 62 newborns whose mothers were maintained on methadone throughout pregnancy, but these differences could not be shown in the 44 children of buprenorphine‐maintained mothers.

CONCLUSIONS

These findings suggest that the opioid maintenance medication might have a stronger influence on neonatal outcome parameters than concomitant consumption of other substances during the last trimester.
Pregnancy outcomes following maternal exposure to second generation antipsychotics: considering polytherapy
Authors: Nulman Irena , Sadowski Alexander , Todorow Michelle , Brojeni Parvaneh Yazdani , Koren Gideon
Published: June 2021

Studies on the reproductive safety of second generation antipsychotics (SGAPs) are scarce, and none have compared monotherapy to polytherapy. A cohort study using a prospectively collected database was conducted. Pregnancy outcomes of 133 women exposed to SGAPs and other psychotropic drugs and 133 matched healthy controls were assessed. Psychotropic drug polytherapy was documented in 72% of exposed women. The exposed women had significantly higher pre‐pregnancy weight, smoked more cigarettes, experienced more associated comorbidities and instrumental deliveries and delivered more large for gestational age children. More of the exposed neonates were born premature, admitted to the intensive care unit and presented with poor neonatal adaptation signs and higher major malformations rates. In the monotherapy subgroup, no malformations were reported. In utero exposure to antipsychotic polytherapy is associated with adverse pregnancy outcomes for both mother and child. SGAP monotherapy appears to be associated with less risk to the fetus. Future studies should differentiate among the effects of psychotropic medication exposure and the underlying psychopathology and associated comorbidities.
Structure‐activity Relationships of Bicyclic Amine Heterocycles with α7 Nicotinic Acetylcholine Receptors (nAChR) and Related Ligand‐gated Ion Channels
Authors: Gomez Kimberly , Yamauchi John G. , Nemecz Akos , Ho Kwok‐Yiu , Talley Todd T. , Taylor Palmer
Published: June 2021

nAChRs are pentameric ligand‐gated ion channels (pLGIC) of the Cys‐loop receptor family located in the central and peripheral NS. The two most abundant subtypes in brain, α7 and α4β2, are targeted for development of selective ligands for managing Alzheimer's disease, schizophrenia, Parkinson's disease and tobacco cessation. Responses for human α7 and α4β2 nAChRs, as well as mouse 5‐HT3A pLGICs, were characterized by fluorescent functional assay using a genetically encoded indicator of Ca2+ flux through pLGICs. Pharmacological tests with α7 nAChRs in the presence of PNU‐120596 identified 19 of 21 compounds as agonists with sub‐μM IC50's. None were found to be antagonists. Two of 21 compounds were agonists for α4β2 nAChRs, whereas 6 were found to be antagonists. None of the compounds displayed agonist activity when tested with 5‐HT3A receptors, and 4 were found to be antagonists. The α7 nAChR agonists were characterized by concentration‐response curves with EC50 values ranging from 1nM to 30 nM. Competitive KA values for α4β2 nAChRs and 5‐HT3A receptors were calculated from dose‐response curves and Schild plots. Binding constants of several of these compounds were also determined with the acetylcholine binding protein, since structures of these complexes are amenable to crystallographic analysis and ascertaining binding poses and determinants of selectivity.
A new molecular sensor controlling cAMP activation of ERK
Authors: Emery Andrew Clayton , Eiden Maribeth V , Mustafa Tomris , Eiden Lee E
Published: June 2021

The signal transduction pathway linking cAMP elevation through GPCR‐Gs stimulation to ERK activation is incompletely understood. We recently reported that cAMP‐ and ERK‐dependent neuritogenesis in NS‐1 cells initiated by activation of the GPCR PAC1 occurs independently of both PKA and Epac activation, revealing the existence of an additional cAMP sensor (NCS, for neuritogenic cAMP sensor) (Emery and Eiden, FASEB J, 26: 3199, 2012). The adenylate cyclase (AC) inhibitor SQ22,536 blocks cAMP‐dependent ERK activation without affecting either CREB phosphoryation or Epac‐dependent Rap1 GTP loading, thus identifying NCS as a new target of SQ22,356 (see Emery et al., doi:10.1124/mol.112.081760). The pharmacological profile of the NCS has facilitated its molecular identification as a member of the Ras guanine nucleotide exchange factor superfamily. The characteristics of NCS cAMP binding studied in bovine chromaffin cells, and NCS‐dependent ERK signaling studied via gain‐of‐function in HEK293 cells, suggests a previously unremarked and neuroendocrine cell‐specific signaling pathway, GPCR→Gs→AC→cAMP→NCS→Rap1→Braf→MEK, connecting neuropeptide receptor occupancy to ERK activation.

Supported by NIMH‐IRP project ZO1‐MH002386.
Electrochemical investigation of a radical sadenosylmethionine enzyme: BtrN from Bacillus circulans
Authors: Maiocco Stephanie Jane , Grove Tyler , Goldman Peter , Booker Squire , Drennan Catherine , Elliott Sean
Published: June 2021

Radical s‐adenosylmethionine (SAM) enzymes catalyze a variety of reactions involved in biological pathways including the biosynthesis of antibiotics, cofactors, and biosynthesis and repair of DNA. Measurement of the electrochemical characteristics of radical SAM enzymes has been limited due to the typically buried location of the radical SAM cluster within the enzyme. The midpoint potential of a radical SAM cluster has only been determined for lysine aminomutase using spectroelectrochemical titrations with the use of mediators. This study presents the first direct electrochemical measurement of a radical SAM enzyme using the technique of protein film voltammetry (PFV). BtrN from Bacillus circulans is an emerging class of radical SAM dehydrogenases, which catalyze the third step in the biosynthetic pathway of the antibiotic butirosin. BtrN has been shown to contain a second [4Fe‐4S] cluster in addition to the canonical radical SAM [4Fe‐4S] cluster. Nonturnover PFV has characterized the electrochemical properties of these clusters and provided insight into the mechanism of electron transfer. Additionally, PFV has been used to characterize the effect of substrate binding on the clusters. These results provide insight into the catalytic mechanism of BtrN and the electrochemical characteristics of radical SAM enzymes in general. Research support was provided by NSF (MCB 1122977).
Inhibition of Topoisomerase IIα by Metal Complexes of Selected Thiosemicarbazones
Authors: Jiang Xiaohua , Lisic Edward
Published: June 2021

Uncontrolled cell proliferation is the main characteristic of all cancer types. To kill the over‐proliferated cells is one key to cure cancer. Before cell proliferation, the genetic material, DNA, needs to be replicated. Thus, DNA replication is the top target for many anti‐cancer drugs. Topoisomerase IIα (Topo IIα) is essential for DNA replication in order to release the supercoiled DNA daughter strands produced after replication (topology simplification) and reduce entanglements. Topo IIα breaks DNA strands, which make it one of the most important targets for anticancer drugs since the accumulation of DNA nicks from inhibition of Topo IIα induces apoptosis. We have synthesized and characterized several structurally different series of thiosemicarbazone ligands and their metal complexes, which show different inhibition activities towards Topo IIα. Our studies indicate that several of the new compounds exhibit high Topo IIα inhibition and are potential anticancer drugs.
Functional interactions of a monothiol glutaredoxin and an iron sulfur cluster carrier protein with the radical SAM enzyme MiaB
Authors: Butland Gareth , Boutigny Sylvain , Saini Avneesh , Baidoo Edward E.K. , Yeung Natasha , Keasling Jay D.
Published: June 2021

The biosynthesis of iron sulfur (FeS) clusters is a highly coordinated process enabled by multi‐protein systems such as Isc and Suf in E. coli. While these systems are believed to encode all factors required for initial cluster assembly and transfer to FeS carrier proteins, accessory factors of undefined function, including the monothiol glutaredoxin, GrxD and the FeS carrier protein NfuA, are located outside of these defined systems. These factors have been suggested to function both as shuttle proteins acting to transfer clusters between scaffold and carrier proteins, and in the final stages of FeS protein assembly by transferring clusters to client FeS apo‐proteins. Here we implicate both of these factors in client protein interactions. We demonstrate specific interactions between GrxD, NfuA and the methylthiolase MiaB, a radical SAM enzyme involved in tRNA maturation. We show that GrxD and NfuA physically interact with MiaB with affinities compatible with an in vivo function. We furthermore demonstrate that NfuA is able to transfer its cluster in vitro to MiaB, while GrxD is unable to do so. The relevance of these interactions was demonstrated by linking the activity of MiaB with GrxD and NfuA in vivo. We observe a severe defect in in vivo MiaB activity in cells lacking both GrxD and NfuA, suggesting that these proteins may play complementary roles in the MiaB catalytic cycle.

Supported by NIH award GM088196 to GB.
The effect of EMD 87580, an Na+/H+ isoform 1 inhibitor, on the rate of cell proliferation in mouse lung adenocarcinoma cells
Authors: Mraiche Fatima , Alasmar Aya Riyad , Al Marridi Wafa Ziad , Youssef Maiy
Published: June 2021

Background

Dysregulated pH is an emerging hallmark that maybe permissive for cancer progression. The Na+/H+ exchanger (NHE1) is one of the most notable pH regulating transporters and as such warrants further investigation for its role in non small cell lung cancer (NSCLC).

Methods

NHE1 protein expression and the rate of cell proliferation were examined in cultured mouse lung adenocarcinoma cells ±10μM NHE1 inhibitor (EMD 87580) or Etoposide for 24 or 48 hours using western blotting and the MTT assay, respectively.

Results

Western blotting demonstrated an increase in NHE1 protein expression in mouse lung adenocarcinoma cells. In the presence of EMD 87580 (N), NHE1 protein expression showed a trend towards reduction vs. control group (C) (100% C vs. 61.5% N). Treatment of mouse lung adenocarcinoma cells with EMD 87580 for 24 hours did not alter the rate of cell proliferation. Similarly, Etoposide, an anticancer topoisomerase inhibitor, had no effect on the rate of cell proliferation following 24 hours of treatment (92.8±6.3%). However, 48 hours of treatment with EMD 87580 revealed a trend towards reduction in the rate of cell proliferation in mouse lung adenocarcinoma cells (100±11.3% C vs. 77.4±2.6% N). The rate of cell proliferation was significantly less in mouse lung adenocarcinoma cells treated with Etoposide (34.9±7.3%).

Conclusion

Taken together, these data demonstrate that NHE1 protein was over expressed in the mouse lung adenocarcinoma cells. NHE1 inhibition regresses cell proliferation with prolonged treatment. It is evident the tumor progression and metastasis require NHE1.
Novel fluorescent reporters for the Stromal Interaction Molecule 1
Authors: VerMeer Mark , TRAN KIM
Published: June 2021

The stromal interaction molecule 1 (Stim1) is a Ca2+ sensor in the endoplasmic/sarcoplasmic reticulum whose Ca2+ sensing triggers its dimerization and subsequent activation of store‐operated Ca2+ entry via direct interactions with Ca2+ channels at the plasma membrane. We have generated a set of fluorescent reporters based on the intraluminal domain Stim1 where Ca2+ binding and dimerization take place. These fluorescent reporters consist of a FRET pair flanking the Ca2+‐binding loop of the canonical EF hand, the entire canonical EF hand, the hidden EF hand, and the sterile α motif (SAM), or a combination of these fragments. All reporters display enhanced FRET upon Ca2+ binding and FRET disruption upon Ca2+ chelation from a Ca2+‐saturated state. Reporters generated from the canonical EF domain and the Ca2+‐binding loop of this domain display the largest dynamic range in Ca2+‐dependent responses. Analyses of spectrofluorometric Ca2+ titrations from a nominally Ca2+‐free condition and Ca2+ chelator titrations from a Ca2+‐saturated condition yielded apparent dissociation constants in the millimolar range for these reporters. The highly quantitative nature of the responses allows studies of factors that affect the Ca2+ sensing capability of Stim1.
On The Mechanism Of The Cyclosporine‐Evoked Facilitation Of The Vasoconstrictor Activity Of Angiotensin II In The Rat Aorta
Authors: Nasser Suzanne A , Elmallah Ahmed I , Sabra Ramzi , Khedr Mohamed M , El‐Din Mahmoud M Mohy , El‐Mas Mahmoud M
Published: June 2021

Despite the established role of enhanced vasoconstrictor responsiveness to angiotensin II (Ang II) in the hypertensive action of the immunosuppressant drug cyclosporine (CSA), the precise mechanism remains unclear. In this contribution, pharmacological studies were undertaken to investigate the potential involvement of vasomotor pathways in the CSA‐Ang II vascular interaction. The chronic treatment of rats with CSA (25 mg/kg/day i.p., 7 days) increased the aortic contractile responses elicited by Ang II (1×10−10–3×10−7 M), with increases and decreases in Emax and EC50, respectively. The concurrent blockade of angiotensin AT1 receptors (losartan), or the inhibition of angiotensin converting enzyme (captopril), cyclooxygenase (indomethacin) or nitric oxide synthase (L‐NAME) abolished the CSA‐evoked augmentation in Ang II contractions. The combined, but not individual, treatment with endothelin ETA (atrasentan) and ETB (BQ788) receptors also abolished the CSA effect. The enhancement of Ang II contractions by CSA was preserved after selective blockade of thromboxane A2 receptors by terutroban. Together, these findings reveal a positive modulatory role for endothelin receptors and products of cyclooxygenase and nitric oxide synthase activity in the CSA‐Ang II vascular interaction.
TRPA1 channels play a critical role in cold‐induced vasodilatation
Authors: Graepel Rabea , Aubdool Aisah A , Kodji Xenia , Fernandes Elizabeth Soares , Bevan Stuart , Brain Susan D
Published: June 2021

The mechanisms involved in cold‐induced vasodilatation are unclear. Here, we have investigated an involvement of the cold‐sensitive channel TRPA1, known to be expressed in a subset of sensory neurons. Using laser Doppler flowmetry, peripheral blood flow was measured following cold exposure in the plantar skin in the anaesthetised mice before (5 min:baseline) and after local cold exposure of the hindpaw (30 min). This response consisted of a decrease in blood flow, followed by an increase in WT mice. This response was substantially reduced in TRPA1 KO as compared to WT mice (245.1 ± 39.6 vs 86.1 ± 11.0, 103 flux units, measured as area under the response curve, n=6, p<0.01) and antagonised by the TRPA1 antagonist HC030031 (100mg/kg, i.p., n=5). The cold‐induced response was also shown to be significantly reduced in WT mice pre‐treated with the calcitonin gene related peptide (CGRP) receptor antagonist, CGRP8–37 and the neurokinin‐1 receptor antagonist SR140333, suggesting a prominent role of neuropeptides in this response. These results suggest the major involvement of TRPA1 containing sensory nerves in local cold‐induced vascular responses.

Supported by a BBSRC‐led IMB capacity building award.
Nitric oxide‐dependent vasodilation is compromised in isolated pulmonary arteries from COX knockout mice
Authors: Wright Will , Mackenzie Louise , Kirkby Nicholas S , Mitchell Jane A
Published: June 2021

Cyclooxygenase (COX) has two isoforms and is essential for prostanoid synthesis. COX‐1 is constitutive whilst COX‐2 is induced in inflammation. Two COX products, prostacyclin (PGI2) and thromboxane (TxA2), regulate vessel tone; PGI2 mediates vasodilation and platelet inhibition, and TxA2 opposes this. PGI2 therapies are used in pulmonary arterial hypertension (PAH). Endogenous TxA2/PGI2 has been linked to PAH in animal models, but the mechanism and isoform involved is debated. We hypothesized that pulmonary artery (PA) from COX‐1−/− and COX‐2−/− mice would have altered vasodilatory function compared with wild‐type (WT; C57Bl6) mice. Vasomotor responses to contractile and relaxant agents were measured by myography. PA from all mice responded similarly to contraction by high potassium or the TxA2 mimetic, U46619. Relaxation to PGI2 receptor or PPARβ/δ agonists was also similar in all PAs. However, COX‐1−/− and, to a lesser extent, COX‐2−/− PA had impaired vasodilation to acetylcholine (ACh), which stimulates endothelial nitric oxide (NO) release, and COX‐1−/− PA also dilated less to sodium nitroprusside (SNP); an NO donor that works on smooth muscle (Fig 1). These data indicate an interaction between COX and NO sensing pathways in pulmonary vessels, and have implications for our understanding of PAH. Research funded by the Wellcome Trust. WRW receives a NHLI foundation studentship.
DETERMINATION OF BPA AND ITS METABOLITES BY HPLC‐UV‐Vis AND MALDI‐TOF
Authors: Naidu Naga , Munyu Sarah , Yakubu Momoh A.
Published: June 2021

Bisphenol A (BPA) is a weak estrogen and is of great environmental concern We have determined free BPA and its metabolites in urine, serum and feces samples using HPLC and MALDI‐TOF. Samples were collected from rats treated with 160 mg/kg BPA orally; BPA and its metabolites were extracted from urine, serum, and feces into acetonitrile and subjected to HPLC analysis. HPLC spectrum of standard BPA peak (RT 7.7) was determined and compared with the samples’ (R.T 7.5–7.7). HPLC spectra of samples confirmed the presence of BPA and its metabolites in different concentrations in urine (8.22 – 24.93ppm), serum (1225.30 – 1868.61ppm) and feces samples (3.48 – 29.95 ppm). No peaks corresponding to BPA was detected in controls. To confirm BPA and metabolites in the samples, eluent from R.T 7–9 was collected and analyzed on MALDI‐TOF. MALDI‐TOF analysis of standard BPA showed a peak at 223.8 m/z, urine samples showed three minor peaks at 223.8 m/z and 245 m/z and 415 m/z corresponding to free BPA, quinine and glucoronide conjugates; a major peak at 205.78 m/z representing BPF, a byproduct of BPA. Serum samples showed minor peaks at 223.68 m/z and 490.54 m/z corresponding to free BPA and BPA‐glu conjugate. Feces samples showed major peak at 215 m/z. Thus, HPLC‐UV‐Vis is a very sensitive tool for identifying BPA and its metabolites in urine and feces samples and confirmed by MALDI‐TOF.
Effects of alpha, beta‐unsaturated cinnamyl‐like aromatic compounds on the viability of a model human cancer (MCF‐7) cell line
Authors: Muhammad Yosra , Kerr Stephen G
Published: June 2021

Cinnamyl‐like aromatic compounds, like trans‐cinnamaldehyde (CA) and methyl cinnamyl ketone (MeCK), are α,β‐unsaturated carbonyl compounds that are believed to interact with cellular nucleophiles through a Michael addition. The Michael acceptor structure of CA and MeCK makes them potential therapeutic alkylating agents. This study investigated the effects of CA and MeCK on the viability of MCF‐7 cells, alone and in the presence of ethacrynic acid (EA), a diuretic and glutathione‐S‐transferase (GST) inhibitor, as well as glutathione (GSH). MCF‐7 cells were seeded in a 96‐well plate (104 cells/well/100μl), and treated with CA (10—250 uM) and MeCK (100—500 uM) alone, and in combination with EA (50 uM) or GSH (100 uM) for 24 h at 37 ºC in 5% CO2. MTS cell viability assays were performed to determine the percentage viability of the treated cells relative to that of the control cells. The results indicate that CA at low concentrations (10–50 uM) induced cell growth, while at concentrations of ≥ 100μM, it was cytotoxic to MCF‐7 cells in a dose‐dependent manner. In the co‐treatment studies, GSH provided protection to cells against MeCK activity, but not against CA. The presence of EA greatly enhanced the cytotoxicity of both CA and MeCK. These results suggest that the cinnamyl‐like aromatic compounds, CA and MeCK, have the potential to induce cell proliferation at low concentrations and to cause cytotoxicity at high concentrations. In addition, the cytotoxic effect was attenuated by co‐treatment of GSH with only MeCK, but enhanced in the presence of EA.
Amomum subulatum (Roxb.) seeds extract ameliorates paracetamol‐induced hepatoxocicity in rats
Authors: Parmar Mihir Y. , Shah Purvi A. , Al‐Rejaie Salim S , Al‐Hosaini Khaled A , Gandhi Tejal R
Published: June 2021

This study was designed to evaluate the Hepatoprotective activity of methanolic extract of Amomum subulatum seeds (MEAS) against paracetamol (3 g/kg, p.o. for 3 days) induced liver damage in rats using silymarin as standard. The degree of protection was estimated by measuring serum levels of alanine aminotransferas (ALT) and aspartate aminotransferas (AST), alkaline phosphatase (ALP), Moreover, biomarkers for oxidative stress such as malondialdehyde (MDA), reduced glutathione (GSH), super oxide dismutase (SOD) and catalase (CAT) were estimated in hepatic tissues. Significant higher levels of above serum biomarkers were observed in paracetamol control as compared to normal control. Treatments with MEAS and silymarin significantly inhibited paracetamol induced elevation in ALT, AST and ALP serum levels. Furthermore, there was a significant increase in MDA while decrease in GSH level, SOD and CAT activities in paracetamol group as compared to control animals. Again, treatment of the animals with MEAS and silymarin brought these stress parameters changes significantly towards normal levels as compared to paracetamol control group. These results suggest that MEAS may have potential therapeutic value to reduce the severity of paracetamol induced liver damage, probably by its antioxidative effect on liver by eliminating the deleterious effects of toxic metabolites of paracetamol.
Sildenafil restores endothelial function and reduces blood pressure in experimental hypertension
Authors: de Moura Balarini Camille , Gomes Isabele Beserra Santos , Dias Ananda Tissianel , Fahning Bernah Mathias , Cintra Amanda , Frossard Jessica , Gava Agata Lages , Meyrelles Silvana dos Santos , Vasquez Elisardo Corral
Published: June 2021

The aim of this study was to evaluate the effects of sildenafil on endothelial function of hypertensive mice. Male C57BL/6 mice (n=3–10) were subjected to renal artery clipping (2K1C). After 14 days, sildenafil (40 mg/kg/day) or vehicle was administrated for 14 days. At the end of the treatment, mice were anesthetized and catheterized to blood pressure recording and had the mesenteric arteriolar bed (MAB) and thoracic aorta (TA) removed to vascular function studies. Data are mean ± SEM. One‐way ANOVA was performed followed by Tukey's post hoc test. Sildenafil caused a significant reduction in the arterial blood pressure in treated mice (126±3 vs. 110±4mmHg). Endothelial dysfunction was observed in MAB of 2K1C mice, but not in the TA and it was restored by sildenafil (Rmax ACh: 44±2.6% vs. 59±4.5%). Ongoing studies are revealing an augmented participation of nitric oxide in this process in treated animals. Similarly, an increased maximal response to an alpha 1‐agonist was observed in MAB but not in TA in 2K1C mice, which was ameliorated by sildenafil (167±8 vs.117±9 mmHg). Both TA and MAB did not show impairment in smooth vascular relaxation to sodium nitroprusside. Thus, our data showed that sildenafil can restore endothelial function in hypertensive mice, which reflects in a significant reduction of arterial hypertension.

Financial Support: CNPq (Proc. 302582/2011–8) and FAPES/Universal 012/2011 (Proc. 54498465).
Enhanced coronary vasoconstriction in western diet‐induced obesity is associated with alterations in NHE1, SERCA2a and 3
Authors: Habibi Javad , Bender Shawn , Garro Mona , Mugerfeld Irina , Rehmer Nathan , Manrique Camila , Nistala Ravi , Whaley‐Connell Adam , Sowers James R
Published: June 2021

Cardiovascular disease (CVD) remains a major risk factor and cause of mortality in type 2 diabetes but, the effect of western diet (WD) on coronary artery vasoconstriction has not been well characterized. Therefore, the purpose of this study was to identify potential calcium (Ca2+) molecular mechanisms underlying alterations in coronary constriction in WD‐induced obesity. C57Bl/6J mice were fed a WD or control diet for 16wks. Coronary vascular function was assessed with wire myography and human coronary vascular smooth muscle cells (VSMC) were cultured. WD enhanced coronary constriction to the thromboxane analog U46619. This was associated with increased coronary VSMC expression of sodium‐hydrogen exchanger 1 (NHE1) and SERCA3 with decreased expression of SERCA2a. Oxidation of SERCA2a cysteine‐674, a signal for SERCA2a degradation, was increased in WD coronary VSMC. Exposure of cultured human coronary VSMC to hyperinsulinemic conditions elicited similar changes in SERCA2a, 3 and NHE1. This study suggests that enhanced thromboxane‐mediated coronary constriction in WD and insulin resistance/hyperinsulinemia may result from NHE1‐dependent changes in intracellular pH and/or impaired Ca2+ handling at the level of the SR. Supported by NIH HL107910, HL073101, and VA Merit Review 0018.
Circulating plasma factors contribute to age‐associated arterial stiffness
Authors: Johnson Lawrence Cody , Eng Jason S. , Fleenor Bradley S. , Seals Douglas R. , Sindler Amy L.
Published: June 2021

Previous findings from our laboratory showed that age‐related increases in large elastic artery stiffness in mice are associated with remodeling of the extracellular matrix (ECM), as indicated by increased collagen I expression, and oxidative stress, as indicated by increased nitrotyrosine staining. Here we tested the hypothesis that circulating factors in plasma contribute to arterial oxidative stress, ECM remodeling and stiffening. Nitrotyrosine, collagen I and elastic modulus, a measure of intrinsic mechanical stiffness, were determined in aortic rings from young (6 mo, n=4) male C57 mice cultured for 72 hours in media containing 10% plasma from either young (Yplasma) or old (Oplasma) mice. Compared with Yplasma, aortic rings incubated in Oplasma demonstrated increased NT abundance (3.11 ± 0.50 vs. 1.00 ± 0.30 AU, p<0.05), collagen I (1.66 ± 0.18 vs. 1.00 ± 0.12 AU, p<0.05) and elastic modulus (2780 ± 570 vs. 1768 ± 188 kPa, p=0.05). These preliminary results are consistent with the hypothesis that unknown factors in the circulating plasma induce “aging‐like” oxidative stress, ECM remodeling featuring increases in collagen I, and stiffening of the aorta. Identifying these circulating factors may lead to additional therapeutic targets for the prevention and treatment of ageassociated large elastic artery stiffening and clinical cardiovascular diseases.

Supported by NIH AG013038, AG000279, HL007822
Shortened life span in mice generated by in vitro fertilization
Authors: Rexhaj Emrush , Rimoldi Stefano , Pireva Agim , Robert Von Arx , Allemann Yves , Sartori Claudio , Scherrer Urs
Published: June 2021

In humans assisted reproductive technologies (ART) induce generalized vascular dysfunction in children. In line with this observation, ART induces vascular dysfunction and arterial hypertension in mice that is related to an epigenetic mechanism, but the long term consequences are unknown. To address this problem, we compared the long term survival of ART and control mice fed a normal chow diet (NC) or challenged with a high‐fat diet (HFD).

80 male FVB mice generated by ART and 80 control mice born and raised in our animal facility were observed in these studies. Three to four animals were placed in a cage in the clean conventional mouse facility. 6 ART and 7 control mice had to be sacrificed because of injuries related to fighting. 32 ART and 20 control mice were fed NC, 42 ART mice and 53 control mice were fed a HFD (49.5% fat, 31.5% protein and 0% carbohydrate). Kaplan‐Meier survival curves were constructed using known birth and death dates and the log‐rank test was used to evaluate statistical differences between groups.

We found that in mice fed with NC the life span was similar in the two groups. In contrast, HFD, while not having a detectable effect on life span in control mice, reduced the survival in ART‐mice by roughly 25%(P<0.0001).

ART is associated with a marked reduction of the life span in normal mice challenged with a Western style high‐fat diet.
In vitro structure/activity relationship studies of second generation derivatives of the translation initiation inhibitor desmethyl, desamino‐Pateamine A
Authors: KOMMARAJU SAI SHILPA , Li Jing , Zhu Mingzhao , Romo Daniel , Low Woon‐Kai
Published: June 2021

The natural product Pateamine A (PatA) from the marine sponge Mycale hentscheli has been found to be a highly potent antiproliferative agent with potential as a novel anti‐neoplastic agent. PatA binds to the eukaryotic translation initiation factor 4A (eIF4A) and prevents translation initiation. Previous structure/activity relationship (SAR) studies identified a rigid binding region on the molecule consisting of the “eastern” portion of the macrolide ring and a side chain terminating in a N,N‐dimethyl tertiary amine. The “western” portion of the macrolide serves as a flexible scaffold domain. Further SAR studies are presented here with a second generation of derivatives of the simplified Pateamine A analog desmethyl,desamino‐PatA (DMDA‐PatA). In vitro translation assays, and enzyme assays with purified recombinant eIF4AI were performed. The results presented here confirm the findings of the first SAR analysis and further clarify the structural requirements for activity. Rigidity of the binding domain was found to be critical for activity, and the tertiary amine found at the end of the side chain of the binding domain may serve as a potential site for future modifications to improve activity and specificity. Source of Funding, St. John's University.
Myosin II is important for endothelial barrier function but is not involved in sphingosine‐1‐phosphate‐induced endothelial barrier enhancement
Authors: Zhang Xun Elvis , Breslin Jerome W
Published: June 2021

Recently we discovered an association between local lamellipodia formation/turnover and endothelial barrier function. To determine cause‐effect, we tested the hypothesis that selective blockade of lamellipodia formation with the myosin II inhibitor (−)blebbistatin would disrupt endothelial barrier integrity. We also investigated whether myosin II inhibition affects sphingosine‐1‐phosphate (S1P)‐induced endothelial barrier enhancement. Transendothelial electrical resistance (TER) of confluent cultured human umbilical vein endothelial cells (HUVEC) served as an index of barrier function. HUVEC expressing GFP‐actin were used to study local lamellipodia dynamics. The results show that 100 μM (−)blebbistatin caused a significant decrease in TER, while (+)blebbistatin caused no change from baseline. In addition, (−)blebbistatin, caused local lamellipodia protrusion frequency to drop nearly 90%, while (+)blebbistatin caused no change. S1P (2 μM) increased TER and caused a brief, coordinated protrusion of the edges of individual endothelial cells. Pretreatment with (−)blebbistatin did not inhibit the S1P‐induced increase in TER. Our data indicate that MLC‐2‐driven local lamellipodia contribute to endothelial barrier function. However, an myosin II‐independent mechanism drives S1P‐induced edge protrusions and endothelial barrier enhancement. Supported by NIH R01HL098215.
Identification of a quinone compound inhibits human glutaminase activity and induces autophagy in carcinoma cells
Authors: Lee Shiow‐Ju , Lee Yue‐Zhi , Hsu Hsing‐Yu , Chang Hsin‐Yu , Chen Ih‐Sheng , Chao Yu‐Sheng
Published: June 2021

Natural product sources recently have been applied to discover drugs for being developed to treat cancer, immunosuppressive disorders, resistant bacteria and viruses. In many instances, the disease targeted natural products serve as tools to unravel the cellular molecular targets and pathways in the disease processes and thus facilitate further development. Here, we identified a natural quinone compound that inhibited human kidney glutaminase activity and the cell growth and proliferation of lung carcinoma A549 and hepatoma HepG2 as well as blocked the anchorage‐independent colony formation of HepG2. Autophagy but not apoptosis was found in A549 and HepG2 cells treated with the natural quinone compound. Further investigation demonstrated the natural quinone compound inhibiting glutaminase activity and conceivably depleting the cellular nutrient and energy to induce autophagy for anti‐cancer activity. (This work was funded by the National Health Research Institutes, Taiwan, R.O.C. and by the Ministry of Economic Affairs, R.O.C. “101‐EC‐17‐A‐02–04‐1099” and “102‐EC‐17‐A‐02–04‐1099”)
Calcium and its role in vascular smooth muscle cell cortical elasticity and adhesion
Authors: Zhu Yi , Li Min , Sun Zhe , Li Zhaohui , Hong Zhongkui , Meininger Gerald A.
Published: June 2021

Calcium (Ca2+) plays an important role in regulating vascular smooth muscle cell (VSMC) contractility and cell signaling. The goal of this study was to investigate the role of Ca2+ in the regulation of VSMC cortex stiffness or elasticity and adhesion in VSMC. In this report, we used 0.2 μM Ionomycin or 0.2 μM BAPTA‐AM to increase or decrease, respectively, the intracellular level of Ca2+ in non‐passaged, freshly isolated rat cremaster VSMCs. An atomic force microscopy (AFM) was used to measure VSMC cortical elasticity using a nano‐indentation protocol (300 nm indentation) and AFM probes were bio‐functionalized with fibronectin to measure cell adhesion. The results demonstrate that the stiffness of the cell cortical cytoskeletal components and adhesion of vascular smooth muscle cells increases after Ionomycin treatment to increase intracellular Ca2+, whereas it decreases after BAPTA treatment to lower Ca2+. Ionomycin treatment was also observed to enhance alpha 5 integrin expression on the VSMC membrane and the F‐actin content of the cells. In contrast, BAPTA reduced the expression and F‐actin content of the cells and reduced cell membrane alpha 5 integrin expression. Our study indicates that Ca2+ is one important signaling molecule that plays a role in regulation of cortical elasticity and adhesion of VSMC by influencing the actin‐integrin cytoskeletal axis. (NIH P01HL095486 to G.A.M).
Study of the roles of LPA3 on erythropoiesis and thrombocytopoiesis processes in zebrafish
Authors: LI MENG WEI , Huang Chang‐Jen , Lee Hsinyu
Published: June 2021

Study of the roles of LPA3 on erythropoiesis and thrombocytopoiesis processes in zebrafish

Hematopoiesis is a dynamic process regulated by complicated pathways. It is responsible for the production of hematopoietic stem cells, as well as their differentiation into more specific mature cell types. Lysophosphatidic acid (LPA), an extracellular lipid mediator, binds to its cognate G protein‐coupled receptors, through which can regulates differentiation and development of the circulation system. In previous study, we demonstrated that LPA regulates hematopoiesis determination in stem cells and erythropoiesis processes through activating LPA receptor 3 (LPA3). Since erythrocytes and thrombocytes were derived from common progenitors, we aim to investigate whether LPA3 also regulates thrombocyte development. Using zebrafish as a model, we found that morpholino (MO)‐mediated knockdown of LPA3 inhibited production of hemoglobin using o'dianisidine staining of zebrafish embryos. Furthermore, treatment with OMPT, a LPA3 agonist, and Ki16425, a LPA1 and LPA3 antagonists, were used to determine if the differentiation process of erythrocyte or thrombocyte is affected. Our results show that treatment with Ki16425 leads to erythropoiesis defects. In addition the expression levels of CD41 (αIIb, glycoprotein IIb [GPIIb]), a marker for thrombocyte, was increased by LPA3 MO and decreased by with OMPT treatments. In summary, our results demonstrated that LPA3 is an important regulator for erythropoiesis and thrombocytopoiesis process of hematopoietic stem cells.
The roles of LPA2 on erythrocyte and thrombocyte differentiation in zebrafish
Authors: Lin Shih hung , Lee Hsinyu
Published: June 2021

Lysophosphatidic acid (LPA) is a simple lipid growth factor regulates cell proliferation, migration, and survival. It binds to a family of trans‐membrane G‐protein‐coupled receptors. In our previous study, LPA receptors were expressed in hematopoietic stem cells (HSCs) and activation of LPA3 leads to erythropoiesis. However, the roles of LPA2 on erythrocyte differentiation remain unclear. During hematopoiesis processes, erythrocytes and thrombocytes have a common precursor. Therefore, we also attempted to determine whether LPA2 regulates thrombocyte differentiation in zebrafish. Anti‐sence morpholino oligonucleotide of LPA2 was injected into zebrafish embryos at different stages. We used o‐dianisidine staining to determine the levels of hemoglobin expression and Real‐Time PCR for mRNA expression of Gata1, an erythropoietic marker. The expression levels of CD41, a thrombocyte marker, were determined by Real‐Time PCR and in situ hybridization. Our results demonstrated that knocked down of LPA2 enhanced the expression of CD41 while no effect was observed for hemoglobin and Gata1 expression. Therefore, our results suggested that activation of LPA2 may decrease thrombocyte differentiation.
NMR Studies of the DNA Polymerase β L22P Variant
Authors: KIRBY THOMAS
Published: June 2021

The gastric cancer‐associated DNA Polymerase β (Polβ) variant with Lys22 replaced by Pro (L22P), has been shown to exhibit reduced DNA binding as well as reduced DNA lyase activity, and to be deficient in base excision repair [Dalal,S., Chikova,A., Jaeger,J., and Sweasy,J.B. (2008) Nucleic Acids Res., 36, 411–422]. The amino acid change is in the middle of helix1, the first of 4 helices that make up the Polβ lyase domain.

We have used NMR experiments with labeled L22P to probe the structure of the variant in solution. The 1H‐13C HSQC resonances of 13C methyl methionine‐labeled enzyme indicate that the L22P mutation appears to structurally disrupt at least part of the lyase domain, as judged by the chemical shift and intensity of the M18 resonance. The rest of the methionine methyl resonances, which arise from the polymerase domain, are unaffected by the mutation. The methionine methyl resonances of wild‐type Polβ exhibit characteristic perturbations upon formation of binary and ternary complexes with gapped DNA and a complementary dNTP. Under the same conditions, the methionine resonances of L22P show none of the changes associated with complex formation. Consistent with these observations, the 1H‐15N HSQC spectrum of the isolated 8K lyase domain of the L22P Polβ variant is characteristic of a disordered protein.