bluuebunny/crossref_metadata_2025 huggingface dataset:
COMPARATIVE EVALUATION OF PHAMACODYNAMICS AND NEURONAL OXIDATIVE STRESS ELICITED BY LARGE DOSES OF ARTESUNATE IN COMBINATION WITHMEFLOQUINE OR AMODIAQUINE IN Plasmodium berghei INFECTED MICE
Authors: Wilson Francis Daniel Temitope
Published: June 2021

Background: Artemisinins in combination with other conventional antimalarials have been found to elicit greater schizonticidal and gametocidal activity than other antimalarial drugs. However, their pharmacokinetic misfit has raised substantial concerns over their future pharmacodynamic potentials, which include restoration of neutrophil function during covalescence or at full recovery.

Method: A total of forty‐five mice (Mean weight = 22.1g) intraperitoneally infected with P. berghei at a dose of 5 × 107 parasitized erythrocytes, were randomized into 3 treatment groups of 15 mice per dosing regimen. The animals were scored for mortality, clearance of parasitaemia and pigmented neutrophils. .

Results: Administration of escalated doses of artesunate‐amodiaquine and artesunate – mefloquine resulted in dose‐dependent but non significant (P < 0.05) decreases in parasite clearance time (12 – 28.8 h for artesunate + amodiaquine; 16.5 – 27 h for artesunate + mefloquine) and parasite reduction rate (0.65 – 074 % parasite/h and 0.64 −0.78 % parasite/h). While parasitaemia correlated positively but non‐significantly (r = 0.065; P < 0.05), the neutropenia (4472.3 ± 240 vs. 1709.5 ± 17.8 cells/mm3; P < 0.05) and pigmented neutrophils (308.2±2.4 cells/mm3) elicited by P. berghei infection was also not corrected by the two artesunate – based regimens.

Conclusion: Based on the results of this study, it can be concluded that the artesunate based combination therapies lack neutrophil restoration ability and oxidative stress corrective function in spite of their antiparasitic efficacy.
pH Micro‐Environments Associated with Transport Activity of the Erythrocyte Membrane Cl−/HCO3− Exchanger, AE1
Authors: Johnson Danielle Elaine , Casey Joseph R.
Published: June 2021

Cl−/HCO3− exchange proteins (AEs) interact with carbonic anhydrases (CAs) to maximize HCO3− transport. CAs catalyse the hydration of CO2 to HCO3− and H+. We hypothesize that rapid AE1‐mediated HCO3− transport causes differences in pH between the region around AE1 and the rest of the cell (pH micro‐environments). To examine this possibility we have measured intracellular pH during HCO3− transport using pH‐sensitive fluorescent proteins (FPs) localized to unique cellular locations. We have identified a FP, deGFP4, that reports on pH without interference from other ions; deGFP4 had a Stern‐Volmer (Ksv) quenching constant of 5×107 M−1 for H+ and 0.5 M−1 for Cl−. We constructed deGFP4 fusion proteins to: AE1 N‐terminus (measures pH at the cytosolic surface of AE1), the nucleoside transporter hCNT3 (a spectator protein), calnexin C‐terminus (to probe pH at the cytosolic ER surface), a GPI‐linked construct (to probe pH at the extracellular surface), and deGFP4 alone (cytosolic pH). The AE1‐deGFP4 and cytosolic deGFP4 exhibit an average transport rate of 0.5 pH units/min with an overall change in pH of 0.8 pH units. A new pH‐sensitive red FP (RFP) was recently developed and has been used as a cytosolic pH sensor, and as a fusion to hCNT3. pH was measured simultaneously in distinct regions of the cell using both GFP and RFP fusion proteins. This research is supported by the Canadian Institutes of Health Research.
The divergent role of epoxyeicosatrienoic acids in the regulation of renal function in normotensive and two‐kidney one‐clip Goldblatt hypertensive rats
Authors: Kopkan Libor , Walkowska Agnieszka , Thumova Monika , Skaroupkova Petra , Imig John D. , Cervenka Ludek
Published: June 2021

To assess the renal function in anesthetized Hannover Sprague‐Dawley rats with induced renovascular hypertension for 27 days (2K1C) and normotensive sham operated control rats during acute inhibition of epoxyeicosatrienoic acids (EETs) generation in the kidney, epoxygenase inhibitor, methylsulfonyl‐6‐(2‐propargyloxyphenyl) hexanamide (MS‐PPOH, 12 μmol/L), was continuously administered directly into the nonclipped kidney at a rate 0.3 mL/hour for over 60 minutes. Glomerular filtration rate (GFR) and renal plasma flow (RPF) were determined by Inulin and PAH clearance, respectively.

Basal renal function of the nonclipped kidney was not different (GFR 0.79±0.07 vs. 0.74 ±0.03 mL/min/g; RPF 2.03±0.09 vs. 2.04 ±0.13 mL/min/g and sodium excretion 1.85±0.31 vs. 1.51 ±0.29 μmol/min/g) in hypertensive 2K1C rats (n=8) compare to sham operated control rats (n=8). Although administration of MS‐PPOH caused significant decreases in GFR (15±3%), RPF (18±4%) and sodium excretion (42±9%) in sham operated rats, MS‐PPOH did not significantly alter renal function in the nonclipped kidney of 2K1C rats.

These data further support the notion that EETs contribute in modulation of renal vasodilatation and natriuresis which might be impaired in 2K1C rats due to deficiency of intrarenal EETs in this hypertensive model.
Phospholipase C isoform expression in rat renal preglomerular microvascular smooth muscle cells
Authors: Wu Sheng , Langer William J. , Lane Pascale H. , Carmines Pamela K.
Published: June 2021

Expression of phospholipase C (PLC) isoforms varies in different tissues across species, and has not been thoroughly investigated in rat renal preglomerular microvascular smooth muscle cells (PVSMCs). The goals of this study were to compare mRNA expression of PLC isoforms in rat PVSMC and to confirm protein expression of these isoforms. PVSMCs (from interlobular artery and afferent arteriole) were cultured by the explant method. Total RNA and protein were extracted at passages 4–6. Quantitative real time RT‐PCR revealed PLC isoform mRNA expression according to the following rank order: δ1 = β4 = γ2 > β3 = β1 > γ1 = δ3 = δ4 ≫ β2. mRNA expression for the PLC isoforms ranged from 105 to 108 copies/ng total RNA (n=5–7 samples/isoform), indicating a 1000‐fold difference in expression of PLC β2 and the most highly expressed isoforms (δ1, β4 and γ2). Western blot analysis verified that PLC β3, β1 and γ1 existed in rat PVSMCs at the protein level. PLC β2 protein could not be detected. It was difficult to detect PLC β4 protein, although its mRNA level was high. We have not yet assessed protein expression of PLC δ1 and γ2. Preliminary experiments utilized siRNA to produce ≥80% decreases in PLC γ1 and PLC β3 protein levels in these cells. We conclude that at least the β3, β1 and γ1 isoforms of PLC exist in rat PVSMCs, and that siRNA should be a useful tool in investigating the roles of specific PLC isoforms in PVSMC function. (Supported by DK71152)
High Salt Diet Attenuates Juxtamedullary Afferent Arteriolar Autoregulatory Behavior and Responsiveness to P2X1 Receptor Stimulation
Authors: Inscho Edward W. , Cook Anthony K.
Published: June 2021

High salt diet reduces myogenic reactivity in resistance vessels. P2 receptors are important for intact afferent arteriolar (AA) autoregulation. We tested the hypothesis that AA autoregulation and responsiveness to P2 receptor activation is impaired in rats fed an 8% NaCl diet (HS) for 14 days. AA responses to β, γ‐methylene ATP, a P2X1 receptor agonist (0.01, 0.1, 1, 10 and 100 μM) were determined in kidneys perfused at 100 mmHg. Autoregulatory behavior was determined in 15 mmHg increments from 65 to 170 mmHg. Systolic blood pressure increased slightly from a baseline of 107±2 mmHg to 115 ± 2 mmHg after 14 days on a HS diet (P < 0.05; n = 34). In control kidneys from normal salt rats (NS), AA diameter decreased by 37 ± 2% (n=4) as perfusion pressure increased from 65 to 170 mmHg. In the HS group AA diameter decreased by 6 ± 4% (P < 0.05; n = 8) indicating marked reduction in pressure mediated vasoconstriction. In NS kidneys, the P2X1 agonist β, γ‐methylene ATP reduced AA diameter by 9 ± 2, 14 ± 8, 18 ± 7, 21 ± 9 and 28 ± 11% (n = 3). In HS kidneys, responses to β, γ‐methylene ATP were markedly impaired. AA diameter decreased by 0 ± 2, 3 ± 4, 4 ± 4, 7 ± 4 and 11 ± 4%, respectively (P < 0.05 vs. NS). These data demonstrate that a HS challenge impairs AA autoregulatory behavior. Loss of autoregulatory behavior is associated with impaired responsiveness to P2X1 receptor activation, consistent with P2X receptors mediating autoregulatory behavior. NIH DK44628
Direct demonstration of tubular fluid flow sensing by macula densa cells
Authors: Sipos Arnold , Toma Ildiko , Vargas Sarah , Peti‐Peterdi Janos
Published: June 2021

Macula densa (MD) cells in the cortical thick ascending limb (cTAL) detect variations in tubular fluid composition and transmit signals to the afferent arteriole (AA) that control glomerular filtration rate (tubuloglomerular feedback, TGF). Increases in tubular salt at the MD, which normally parallel elevations in tubular fluid flow rate, is a well accepted stimulus of TGF. The present study aimed to determine if MD cells can detect variations in tubular flow per se. Ratiometric calcium imaging of the in vitro microperfused isolated JGA‐glomerulus complex dissected from mice was performed using Fluo‐4/Fura‐Red and confocal microscopy. Increasing cTAL flow from 2 to 20 nl/min (constant 10 mM [NaCl]) rapidly produced a significant elevation in [Ca2+]i in AA smooth muscle cells (delta[Ca2+]i = 223.7 ± 21.5 nM) and AA vasoconstriction. Complete removal of the cTAL from around the MD plaque and application of laminar flow through a perfusion pipette directly to the MD apical surface essentially produced the same results even using low (10mM) salt solutions. Acetylated a‐tubulin immunohistochemistry identified single primary cilia in MD cells. Under no flow conditions, bending MD cilia directly with a micropipette rapidly caused significant elevations in AA smooth muscle [Ca2+]i and vasoconstriction. MD cells are equipped with a tubular flow sensing mechanism which may contribute to MD cell function and TGF.
PROXIMAL AND DISTAL LUNG MECHANICS IN ANTIGEN CHALLENGED ALLERGIC CYNOMOLGUS MONKEYS
Authors: Curran Aidan , Skeans Susan , Landers Devon , Chapman Richard
Published: June 2021

We used the forced oscillation technique (FOT) to measure impedance in the pulmonary system and the effects of antigen challenge on the proximal and distal lung. In addition, we treated animals with two oral doses of placebo or betamethasone (1 mg/kg) 18 and 2 hours prior to antigen challenge. FOT provides indices of proximal (Newtonian airway resistance RN) and distal airway status (tissue damping (G) and elastance (H)). Ascaris suum antigen challenge caused a significant increase in RN, G and H. Pretreatment with betamethasone significantly reduced the peak increase in RN and the area under the curve of the FOT response over 60 minutes. G and H responses were unaffected by steroid treatment. These data indicate that, using the FOT, we can dissociate the response of proximal and distal airways to an antigen challenge. Moreover, steroid pre‐treatment can reduce the bronchoconstrictor response to inhaled antigen but this effect is primarily via effects on proximal airways with little effect on the distal airways and parenchymal component of pulmonary impedance. These data may help to explain the apparent disconnect between the empirical and subjective measures of efficacy for some asthma treatments.
Hyperpolarized 3He magnetic resonance imaging of ventilation after bacterial lipopolysaccharide exposure in mice
Authors: Thomas Abraham , Voltz James , Fubara Boma , Driehuys Bastiaan , Zeldin Darryl
Published: June 2021

Introduction: Traditional lipopolysaccharide (LPS) inhalation studies focus on histology, lung function and inflammatory mediator release. This study utilizes hyperpolarized (HP) 3He MRI to elucidate regional differences in ventilation following LPS exposure in mice.

Methods: C57BL/6 mice were intratracheally dosed with high dose LPS (50 μg, N=2), low dose LPS (15–30 μg, N=7) or normal saline (NS) (N=2) and then imaged by HP 3He MRI.

Results: There is proximal (2nd or 3rd order) bronchial tree distension in 50 μg LPS dosed mice that is not evident at lower doses (Table 1). Preliminary results indicate subsegmental ventilation defects (VD) are distributed evenly throughout the lungs of LPS dosed mice. Further, VD number is dose dependent (Table 1).

Conclusions:

LPS induced VD and proximal bronchial tree distension are dose dependent. As proximal bronchi distend in LPS dosed mice, airway hyperresponsiveness likely results from more distal airway constriction.

Funding: NIEHS/NIH Division of Intramural Research and NCRR/NCI national resource funding.
Hyperoxia‐induced enhancement of lung parenchymal contraction is mediated via brain‐derived neurotrophic factor (BDNF)
Authors: Sopi Ramadan B , Martin Richard J , Dreshaj Ismail A , Yao Qin , Liu Shijian , Zaidi Syed I A
Published: June 2021

We have shown that hyperoxia (H) in rat pups upregulates expression of BDNF in airway smooth muscle. Therefore, we hypothesize that BDNF‐TrkB signaling plays a functional role in upregulation of cholinergic contractile mechanisms in hyperoxia exposed lungs. Five‐day‐old rat pups were exposed to room air (RA) or hyperoxia (>95% oxygen) for 7 days and administered daily K252a (50 μg/kg/day, i.p.) to block BDNF‐TrkB receptor signaling, or vehicle. After exposure acetylcholine (ACh) was measured in lungs and in vitro force measurements were performed in lung parenchymal strips. ACh content doubled in H‐lungs as compared to RA‐lungs. K252a treatment of H‐pups reversed ACh content to RA levels. H significantly increased electrical field stimulation‐induced contractile responses of strips as compared to RA‐pups (p<0.001). K252a treatment reversed the increased contractile responses in H animals to RA level. H also reduced IκBα expression as measured by Western blot when compared to RA exposure. IκBα is an inhibitory protein, degradation of which activates NF‐κB, a transcription factor of the bdnf gene. The data demonstrate that BDNF/TrkB receptor signaling appears to play a critical role in enhancing cholinergically mediated contractile responses of lung parenchyma. We speculate that H‐induced increase in BDNF expression and contraction of lung parenchyma is mediated via NF‐κB activation. Supported by HL‐56470.
Hormonal Influences on Airway Biology and Naphthalene‐ Mediated Clara Cell Injury
Authors: Mitchell Valerie Lee , Sutherland Katherine M , Edwards Patricia C. , DeAngelo Sharlee M , Van Winkle Laura S
Published: June 2021

Women have a greater chance of developing adult onset asthma, COPD, and some forms of lung cancer than men. Naphthalene (NA), a bioactivated cytotoxicant, is used to model distal Clara cell airway injury and repair. Our laboratory has shown that female mice are more sensitive than male mice to NA toxicity. The objective of the current study was to evaluate the role of hormones in normal airway biology and in NA mediated injury. This was achieved by 1) measuring estrogen receptors (ESR) gene expression by RT PCR in airways by estrus cycle stage, 2) measuring glutathione levels in airways by cycle stage, and 3) comparing airway damage in response to NA in intact vs. gonadectomized animals. Airway level specific responses were defined in microdissected airways. In comparison to non‐cycling mice, ESR expression was decreased in both diestrus and proestrus and increased in the estrus phase of the cycle. Glutathione levels were decreased in the airways of mice during diestrus and metestrus compared to male mice. Depletion of glutathione within the Clara cell plays a major role in NA cytotoxity. Confocal microscopy of microdissected airways as well as high resolution resin sections showed less extensive injury in ovariectomized mice compared to intact female mice. These findings support the hypothesis that sex differences in lung injury are influenced by hormones. Funded by NIH grants ES012720, HL007013‐29, and TRDRP 14RT‐0191.
Effectiveness of the Hybrid Model of Case Based Teaching in a Medical Pharmacology Class
Authors: Rajasekaran Senthil Kumar , Vaithilingam Andy , Armstrong Elizabeth
Published: June 2021

An alternative pedagogical strategy to the routine didactic format of lectures has long been sought after by instructors in medical schools. We designed this study to investigate the effectiveness of our hybrid model of case based teaching by comparing it with the traditional didactic form of delivery of lecture material. Students were divided into two groups and the study was conducted for two weeks. On week‐I group A students were taught a topic using the case based teaching methodology while group B students were taught the same topic using the didactic lecture format. On week‐II, the groups were crossed over in terms of teaching strategies. The learning outcome was measured by a MCQ test. As indicated in table‐1, the use of a hybrid model of case based teaching methodology showed a significantly enhanced student academic performance compared to the traditional didactic lecture format. As evidenced in table‐2 this performance appeared to be independent of subject matter, student group composition, order and type of topics presented. Students appreciated the significance of clinical application of basic science concepts in accordance with the Kolb's theory of experimental learning.

We conclude that such a hybrid format of delivering pharmacology content has greatly improved the learning outcome.
An investigation of learning styles in undergraduate students studying physiology.
Authors: Kay Ian Stuart , Kaur Raminder
Published: June 2021

It is well established that students learn in a variety of ways– visual (V), auditory (A), read/write (R) and kinaesthetic (K). Determining the preferred style of learning of an individual student can be assessed using a VARK questionnaire. VARK questionnaires were administered to 220 first year students reading for a degree in Biological or Biomedical Science (BS/BMS) and 60 students reading for a degree in Speech Therapy (ST) – completion rates were 44.5% (n=100) for the BS/BMS cohort and 60% (n=40) for the ST cohort.

Of the BS/BMS cohort, 64% were multimodal learners (67% were bimodal, 30% trimodal and 3% quadmodal) whilst 36% preferred a single learning style. For those who expressed a single preferred learning style, V learners accounted for 8%, A learners for 14%, R learners 25% whilst the remaining 53% were K learners.

Analysis of the ST cohort showed similar results to those of the BMS cohort – 65% were multimodal (54% were bimodal, 38% trimodal and 8% quadmodal) whilst 35% expressed the preference of a single mode. Of those who expressed preference for a single style, 31% were V learners, 23% A, 38% R whilst the remaining 8% were K. This data suggests differences in learning styles between these two cohorts of students, which requires consideration when they are taught physiology.
The muscular teritory of the posterior tibial artery angiozome at the level of the lower leg
Authors: MATUSZ Petru L. , PUSZTAI Agneta Maria , MASTACANEANU Mihaela , HORDOVAN Eniko‐Christine , DAESCU Ecaterina , HOGEA Bogdan
Published: June 2021

The necessity to perform complex surgical reconstructions at calf level represented a booster for surgical anatomy studies. The term of angiozome includes all the anatomical structures vascularized by a source artery. We studied the muscular teritory of the posterior tibial artery angiozome on 60 anatomical dissection casts and also on 10 lower legs amputated for surgical diseases. For the anatomical casts we used the macroscopic and the mezoscopic dissection. The amputated lower legs were first injected with China ink of different colours (specific for each source artery), followed by injection with Technovit 7143 to underline the arterial trunks and to facilitate their dissection. Correlations between dissection and injecting of the anatomical structures of the calf showed the muscular territory of the posterior tibial artery angiozome at calf level, represented by: the medial part of gastrocnemius, the medial part of soleus, the medial part of popliteal muscle, the medial part of flexor digitorum longus, the distal part of flexor halucis longus and distally the medial part of tibialis posterior muscle. Knowing these anatomical aspects regarding the extension of the muscular territory of the posterior tibial artery is the base for evaluation of the skin territory of this angiozome, constituting the theoretical base for a safe surgical approach of the posterior aspect of the calf.
Changes in zygapophysial (Z) joint synovial folds following induced segmental hypomobility in the rat
Authors: Cramer Gregory D. , Little Joshua W. , Henderson Charles N.R. , Daley Clover J.
Published: June 2021

Synovial folds (SFs) are part of the normal anatomy of the Z joints. Although degenerative changes and adhesions have been identified in hypomobile Z joints, putative changes in SFs have not been previously studied. Using an established, IACUC approved, rat model, 3 contiguous segments (L4, L5, L6) were fixed with a specially engineered, surgically implanted, vertebral fixation device. Spinal segments of control rats (n=24 animals, 88 joints) were compared with those of animals with 4, 8, 12, or 16 weeks of fixation (n=16 animals, 54 joints). At the end of the experimental period, deep anesthesia was induced, followed by transcardial perfusion with fixative. Tissue was embedded in paraffin, sectioned, stained, and examined and photomicrographed using bright field microscopy. The number of Z joint quadrants with SFs was determined for each joint (left and right L4/L5 and L5/L6). Average number of quadrants with SFs was calculated for each group. Results: SFs were found in all joints. Significant differences were found among and between groups (ANOVA, p<0.001) with joints of control animals of each time period having larger synovial folds than fixation animals of the same time period (Tukey post hoc, wks: 4, p<0.05; 8, p<0.001; 12, p<0.005; 16, p<0.001). We conclude that hypomobility of rat Z joints results in regression of synovial folds.

Support: NCMIC administered by FCER (Grant # 97‐10‐18).
Mechanisms of osteoblastic MG‐63 cell contraction and mRNA expression in stress‐relaxed collagen gels
Authors: Hart David A. , Parreno Justin , de‐Hemptinne Isabelle
Published: June 2021

Culture of osteoblast‐like MG‐63 cells within collagen gels results in the generation of intrinsic stress. Release of the gels from attachment results in contraction and significantly enhanced MMP‐1, MMP‐3, and α2 integrin mRNA levels. To understand the role of cytoskeletal elements and signaling pathways involved in contraction and gene expression, MG‐63 cells were cultured in collagen gels for 24 hours, released, and then immediately treated with cytoskeletal depolymerization agents or kinase inhibitors. Contraction was measured, RNA isolated, and real‐time PCR analysis was performed. The results showed that: (1) cytochalasin D treatment (microfilaments) inhibited contraction and depressed MMP‐1, MMP‐3, and α2 mRNA levels; (2) Nocodazole treatment (microtubules) enhanced early contraction and elevated mRNA levels for MMP‐3; (3) ROCK inhibitor treatment (Y27632) inhibited contraction and depressed MMP‐3 and α2 integrin mRNA levels; (4) the ERK1/2 inhibitor, U0126, did not affect contraction, but depressed MMP‐1, MMP‐3, and α2 mRNA mRNA levels; and (5) treatment with the p38 MAP kinase inhibitor SB203580 was ineffective. These results suggest that collagen gel contraction and gene expression is dependent on the tensegrity balance between microfilaments and microtubules, and the ROCK pathway, while gene expression but not contraction is also dependent on the ERK1/2 MAP kinase pathway.
In vitro sumoylation and ubiquitination of annexin I and helicase activity
Authors: Hirata Fusao , Hirata Aiko
Published: June 2021

Annexin I, a protein previously termed as lipomodulin or lipocortin I, is a member of the family that bind to phospholipids in a Ca2+ dependent manner. This protein is a major substrate of the oncogenic tyrosine kinases such as c‐met and c‐src, thus being proposed to be involved in cell proliferation and differentiation. While annexin I in the nuclei is thought to be involved in cell transformation and/or proliferation, the nuclear translocation of annexin I is facilitated by oxidative stress and possibly by phosphorylation (and Ca2+ signal). Since annexin I contains the consensus motif, ψKxE, for sumoylation, we tested if annexin I can be sumoylated by Ubc 9 in vitro. We found that purified bovine annexin I is indeed sumoylated by Ubc 9, and that E3 of the sumoylation system is not required. While nonmodified annexin I has no helicase activity, the sumoylation unmasked a helicase activity. On the other hand, the mono‐ubiquitination of annexin I was found to be catalyzed by Ubc H2a and H2b (hHR 6). This reaction required E3 of the ubiquitination system in the cytosolic fraction of HeLa cells. Difference in properties of their helicase activities will be discussed. These observations suggest that annexin I may play an important role in DNA damage bypass and mutagenesis mediated through modifications with ubiquitin and sumo. (Supported in parts by a grant from the Susan G. Komen Breast Cancer Foundation BCTR0600711)
Toxicogenomic analysis of human intestinal epithelial FHs 74 Int cell responsiveness to sulfide
Authors: Attene‐Ramos Matias Sebastian , Nava Gerardo Manuel , Muellner Mark G , Wagner Elizabeth , Plewa Michael , Gaskins H. Rex
Published: June 2021

Hydrogen sulfide (H2S), a metabolic endproduct of sulfate‐reducingbacteria in the large intestine, represents an environmentalinsult to the colonic epithelium linked with chronic disorders such as ulcerative colitis andcolorectal cancer. We demonstrated previously that sulfide, at concentrations similar to those found in the human colon, induced genomic oxidative DNA damage in mammalian cells. The present study sought to define the early (30 min) and late (4 h) response of non‐transformed intestinal epithelial cells (FHs 74 Int) to a genotoxic‐but not cytotoxic‐concentration of sulfide (500 μM Na2S) using pathway specific quantitative RT‐PCR arrays. Significant changes in gene expression were predominantly observed at 4 h, with the most notable being PTGS2 (Cox‐2; 7.9 fold upregulation), and IL‐1A (interleukin 1 alpha; 5.2 fold downregulation). Functional pathway analysis indicated that sulfide modulates cell cycle progression and triggers both an inflammatory and DNA repair response. The analysis also highlights p53, p21, c‐Fos, c‐Jun, NFκ‐B, and IL‐8 as potential key molecules in normal cellular responses to sulfide. Overall, the present study identifies functional pathways by which sulfide may perturb cellular homeostasis and contribute to the onset of chronic intestinal disorders.
Biochemical Characterization of SET and MYND Domain Containing Protein 3
Authors: Sirinupong Nualpun , Nico Lindsey , Yang Zhe
Published: June 2021

The goal of this study is to reveal the biochemical function of SET and MYND domain containing protein 3 (SmyD3), a histone methyltransferase. Histone methylation is a vital epigenetic mechanism that controls many fundamental processes such as transcription activation and repression. SmyD3 methylates histone H3 at lysine 4 and was found greatly upregulated in several cancers including colon, liver, and breast cancers. However, the biochemical function of SMYD3 remains poorly understood. To gain insight into the function of SmyD3, we overexpressed mouse SmyD3 in E.coli cells. We found that SmyD3 elutes from a gel filtration column as a monomeric protein. Circular Dichroism (CD) spectroscopy showed that SmyD3 has a large amount of alpha‐helical structures. Thermal denaturation experiments showed that SmyD3 undergoes an unfolding transition with a melting temperature of ~ 45 °C. Our future studies will focus on that: first, understanding of how DNA binding to N‐terminal MYND domain impacts the second and tertiary structures of SmyD3 using Isothermal Titration Calorimetry and fluorescence spectroscopy; second, examination of the effects of substrate histone peptide binding on the protein structure and stability; finally, investigation of the function of the conserved C‐terminal domain, especially focusing on its contribution to protein integrity.
Quantitation of transcription termination mediated by actin and hemoglobin polyadenylation sites using a novel tandem reporter
Authors: Sammarco Mimi C. , Ditch Scott , Banerjee Ayan , Wang Jeffrey , Grabczyk Ed
Published: June 2021

In the “torpedo” model of transcription termination by RNA polymerase II, transcript cleavage at the poly‐A addition site is thought to contribute to termination by creating a free 5′ end that permits the loading of a 5′ to 3′ exoribonuclease (Xrn2). Xrn2 degrades the nascent RNA, which guides it to the RNAP II complex. This model predicts that termination occurs at various distances downstream from the poly‐A site. To test aspects of this model we used a unique tandem reporter system designed for the study of transcription elongation and/or termination in live human cells. Self‐cleaving ribozymes produce two independent expression cassettes after successful transcription. Two luciferase reporters provide a ratiometric measure of termination that occurs within an insert cloned between the ribozymes. We compared the efficiency of termination by size‐matched fragments derived from two well characterized poly‐A regions (human beta actin, ACTB, and human beta hemoglobin, HBB) in our system. Constructs with only 200 bp upstream and 200 bp downstream of each poly‐A site showed a 16% (ACTB) and 40% (HBB) read through. In contrast, when an additional 1600 bp of downstream sequence is added both ACTB and HBB sequences allowed only about 1% of the transcribing polymerases to read through to the second reporter. Supported by NIH grant R01NS046567 and by a grant from the Friedreich's Ataxia Research Alliance (FARA).
The muscle‐specific transcription cofactor Vgll2 promotes myogenic differentiation through a casein kinase II dependent mechanism
Authors: Teng Allen CT , Belanger Melanie I , Stewart Alexandre FR
Published: June 2021

In D. melanogaster, the transcription factor of Scalloped (Sd) and its cofactor, Vestigial (Vg), control the development of indirect flight muscles and wings. Vestigial‐like 2 (Vgll2), a vertebrates homolog of Vg, is specifically expressed in skeletal muscle. Over‐expression of Vgll2 enhances myotube formation whereas Vgll2 knockdown blocks myogenic differentiation, demonstrating the important role of Vgll2 in skeletal muscle differentiation. However, the mechanisms that regulate Vgll2 interaction with TEA domain (TEAD) transcription factors (homologs of Sd) and MEF2 remain poorly characterized. An evolutionarily conserved casein kinase II (CKII) phosphorylation site (Serine 96) in all vestigial‐like (Vgll) proteins. Bacterially‐expressed purified GST‐Vgll2 protein was selectively phosphorylated by CKII in vitro. Two mutant constructs generated by site‐directed mutagenesis, S96A and S96E, were tested in mammalian‐two hybrid assays. The alanine mutant completely abolished the Vgll2‐TEAD1 interaction whereas the glutamate mutant enhanced this interaction. Immunocytochemistry showed that the alanine mutant was inappropriately localized in the periphery of the nucleus while the glutamate mutant was associated with euchromatin. These results imply that phosphorylation of the serine 96 residue is necessary for the Vgll2‐based myogenic differentiation.
TATA box binding protein (TBP) recruitment to the U1 gene promoter is disrupted by substituting a U6 proximal sequence element (PSEA) for the U1 PSEA
Authors: Barakat Nermeen , Stumph William E.
Published: June 2021

Most of the spliceosomal small nuclear RNAs (U1, U2, U4, and U5) are synthesized by RNA polymerase II, but U6 snRNA is synthesized by RNA polymerase III. In Drosophila melanogaster. transcription of all snRNA genes requires a unique proximal promoter element, the PSEA. Surprisingly, the RNA polymerase specificity of the snRNA genes is determined by a few nucleotide differences between the PSEAs in the two classes of genes. In both classes, the PSEA is recognized by the small nuclear RNA activating protein complex (DmSNAPc). Previous transfection assays demonstrated that Pol II was unable to initiate transcription from a U1 promoter that contained the U6 PSEA. We have now used chromatin immunoprecipitation assays to determine at which step Pol II pre‐initiation complex assembly is disrupted when the U1 promoter contains a U6 PSEA. Interestingly, we found that DmSNAPc stably assembled on a U1 promoter that contained either the U1 or the U6 PSEA. However, although TBP was recruited to the wild type U1 promoter, TBP failed to assemble in vivo on the U1 promoter that contained the U6 PSEA. These results indicate that changing the U1 PSEA to a U6 PSEA had no effect on the binding of DmSNAPc, but it instead suggests that DmSNAPc is in the wrong conformation when bound to a U6 PSEA to recruit TBP to the U1 promoter.

N.B. is a recipient of the Arne N. Wick Predoctoral Research Fellowship from the California Metabolic Research Foundation.
Thrombin activates transcriptional factors NF‐kappaB and NFAT to induce optimal ICAM‐1 expression in endothelial cells.
Authors: Xue Jiaping , Bair Angela , Christman John W , Malik Asrar B , Tiruppathi Chinnaswamy
Published: June 2021

Thrombin‐induced increase in intracellular Ca2+ activates transcription factors NF‐?B and NFAT in endothelial cells (ECs). We investigated the possible role of NF‐?B and NFAT in the signaling mechanism of thrombin‐induced ICAM‐1 expression in ECs. We identified the presence of two NF‐?B binding sites in the intron‐1 (+ 70, NF‐?B site 1; +611, NF‐?B site 2) of human ICAM‐1 gene. We observed the binding of both RelA and NFATc1 to the intronic NF‐?B site 1 of ICAM‐1 gene in thrombin‐stimulated ECs by chromatin immunoprecipitation. To delineate the role of intronic NF‐?B site 1 in the ICAM‐1 gene, we transiently transfected ECs with ICAM‐1‐promoter‐intronic NF‐?B site 1 reporter construct. Thrombin increased the reporter expression several‐fold. Exposure of transfected cells to calcineurin inhibitor FK506 markedly reduced thrombin‐induced reporter expression. Introduction of mutation in the intronic NF‐?B site 1 significantly reduced thrombin‐induced reporter expression. Knockdown of either RelA or NFATc1 with siRNA inhibited ~80% of thrombin‐induced ICAM‐1 expression; however, NFATc1 knockdown with siRNA had no effect on TNF‐a‐induced ICAM‐1 expression in ECs. These findings suggest that both RelA and NFATc1 bind to the intronic enhancer elements in the ICAM‐1 gene and signal the transcription of ICAM‐1 gene in response to thrombin in endothelial cells.
C‐terminal binding protein 1 (CTBP1) and the redox control the PEPCK‐C gene transcription
Authors: Yang Jianqi , Kong Xiaoying , Hanson Richard W
Published: June 2021

Transcription of the hepatic gene for the cytosolic isoform of phosphoenolpyruvate carboxykinase (GTP) (PEPCK‐C) (EC 4.1.1.32) is acutely regulated by a number of transcription factors and co‐regulators in response to stimuli such as hormones and nutrients. Since the cellular redox status controls the function of various transcription factors, we investigated the possibility of transcription factor‐mediated redox control of the PEPCK‐C gene transcription. When the cellular NAD+/NADH ratio was decreased after treating cells with ethanol, the expression of PEPCK‐C was attenuated, while cells treated with pyruvate, which increases the cellular NAD+/NADH ratio, showed an elevated expression of the gene for PEPCK‐C. Cobalt chloride, which induces hypoxia in cells and decreases the cellular NAD+/NADH ratio, also inhibited transcription from the PEPCK‐C gene promoter in HepG2 cells. The C‐terminal binding protein 1 (CTBP1), which is activated by NADH, has been implicated as a redox sensor in cells. We investigated its effect on transcription from the PEPCK‐C gene promoter. CTBP1 markedly repressed transcription of PEPCK‐C gene promoter in the presence of the catalytic subunit of protein kinase A. We conclude that a decrease in NAD+/NADH ratio activates CTBP1, which in turn represses PEPCK‐C gene transcription. Supported by grants DK058620 and DK025541 from the NIH.
Small interfering RNA‐mediated silencing of mitochondrial NADP+‐dependent isocitrate dehydrogenase enhances the sensitivity of HeLa cells toward tumor necrosis factor‐a and anticancer drugs
Authors: Park Jeen‐Woo , Kil In Sup , Kim Sung Youl
Published: June 2021

Tumor necrosis factor‐α (TNF‐α and several anticancer drugs induce the production of reactive oxygen species, which play an important causative role in apoptotic cell death. Recently, we demonstrated that the control of mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of mitochondrial NADP+‐dependent isocitrate dehydrogenase (IDPm) by supplying NADPH for antioxidant systems. In the present report, we show that silencing of IDPm expression in HeLa cells greatly enhances apoptosis induced by TNF‐α and anticancer drugs. Transfection of HeLa cells with an IDPm small interfering RNA (siRNA) markedly decreased activity of IDPm, enhancing the susceptibility of anticancer agent‐induced apoptosis reflected by morphological evidence of apoptosis, DNA fragmentation, cellular redox status, mitochondria redox status and function, and the modulation of apoptotic marker proteins. These results indicate that IDPm may play an important role in regulating the apoptosis induced by TNF‐α and anticancer drugs and the sensitizing effect of IDPm siRNA on the apoptotic cell death of HeLa cells offers the possibility of developing a modifier of cancer chemotherapy.
A simple assay for detection of small‐molecule redox activity
Authors: Lor Leng Anne , Schneck Jessica , McNulty Dean E , Diaz Elsie , Brandt Martin , Thrall Sara H. , Schwartz Benjamin
Published: June 2021

In our research, we found that high throughput screens against certain targets, such as cysteine proteases and metal containing enzymes, often yield undevelopable hits because of their lack of specificity and irreversible nature. Weeding through these undesirable hits to get to those of interest can often be difficult, negatively impacting the chance of success in drug discovery. In order to better understand the mechanism of this inhibition, LC/MS/MS analysis was performed with cathepsin L (cat L), a cysteine protease. Incubation of these undesirable compounds with cat L resulted in specific oxidation of the active site cysteine. Based on this mechanism of inhibition, a surrogate high throughput fluorescence assay was developed to measure the tendency of compounds to oxidize enzyme targets. In this assay, reactive compounds catalyze two 1 electron transfers from DTT to resazurin, resulting in the formation of the fluorescent resorufin. Using this assay, we were able to show that a fluorescence increase was observed only with suspected redox inhibitors, and not with known tool compounds. Finally, we tested 400 compounds from a recent drug discovery project, and compared their reactivity in this assay against their potency with the target of interest. Compounds that clearly inhibit via this mechanism could easily be picked out from those that specifically inhibit the target. Hence this detection assay serves as a valuable tool for quickly triaging redox active compounds from HTS campaigns.

GlaxoSmithKline Pharmaceuticals, Collegeville PA
A novel mutation (G389E) in p53 “extreme C‐terminus”
Authors: Giardina Bruno , De Rosa Maria Cristina , Alinovi Cristiana Carelli , Concolino Paola , Vendittelli Francesca , Capoluongo Ettore
Published: June 2021

The human tumor suppressor protein p53 consists of several functional domains, including an N‐terminal activation domain, a DNA‐binding core domain and C‐terminal tetramerization and basic regions. The basic region (residues 367–393), termed the “extreme C‐terminus”, is a negative regulatory domain whose post‐translational phosphorylation and acetylation occurs in vivo and activates the tumor suppressor. A novel mutation (G389E) in the p53 “extreme C‐terminus” was identified in a patient with congenital adrenal hyperplasia (CAH). This study describes experimental and computational chemistry studies of wild type and mutant p53 “extreme C‐terminus” to gain insights into the structural changes which may occur upon G389E mutation and to investigate the molecular basis of the desease. Site‐directed mutagenesis experiments followed by in vitro protein expression, supported by molecular dynamics trajectories, outline the effect of the mutation on the phosphorylation sites which are clustered within the p53 “extreme C‐terminus” and the importance of intermolecular interactions in biological systems.
Evaluation of Short Peptide Inhibitors to Counteract Botulinum Neurotoxin A (BoNT/A) Poisoning In Vitro.
Authors: Andres Devon K , Keyser Brian , Zhang Peng , Benton Betty , Hauck Stephanie , Ray Prabhati , Ray Radharaman
Published: June 2021

Botulinum neurotoxins (BoNTs) inhibit stimulated neuroexocytosis, resulting in flaccid paralysis. Among the seven serotypes (A–G), serotype A (BoNT/A) is the most toxic. BoNT/A consists of a 100 kD heavy chain and a 50 kD light chain (LC). The LC, a zinc endopeptidase, hydrolyzes the vesicle fusion protein SNAP‐25 and thus blocking neuroexocytosis. A prospective countermeasure against BoNT/A poisoning is to inhibit the LC‐induced hydrolysis of SNAP‐25 using a short peptide inhibitor. A high affinity synthetic peptide inhibitor N‐acetyl‐CRATKML‐amide has been reported. We compared this peptide inhibitor and two other derivatives, which include an aldehyde or a fluoromethyl ketone (an irreversible inhibitor) side group, with respect to their inhibitory potencies against SNAP‐25 hydrolysis and blockade of neuroexocytosis induced by BoNT/A in a human neuronal model, M‐17. Using SNAP‐25 hydrolysis and high K+ (80 mM) stimulated (3H)‐glycine release assays, we observed that all three inhibitors significantly inhibited BoNT/A effects. We also observed using an Alexa555 fluorescent tag that the peptide inhibitors were permeable through neuronal cell membranes. These peptide inhibitors show promise as therapeutic intervention(s) against BoNT/A poisoning. Research supported by JSTO‐DTRA Award # T.T.0009_06_RC_B to R. Ray. The views represented are those of the authors and are not necessarily endorsed by the US Army.
Synthesis and Structural Analysis of a Novel Series of Non‐beta‐lactam Inhibitors of AmpC Beta‐lactamase
Authors: Tomlinson Jenna Marie , Mishra Uma J , Kubiak Rachel L , Schroeder William , Davis Chris J , Smart Robert P , Powers Rachel A
Published: June 2021

(‐lactams are the most widely prescribed class of antibiotics. However, their continued utility is threatened by the expression of (‐lactamase enzymes, which hydrolyze the defining lactam ring of these antibiotics, rendering them useless. Current clinical inhibitors for these enzymes also contain a lactam ring, allowing resistance to develop rapidly. Inhibitors that do not resemble (‐lactams would require bacteria to develop novel resistance mechanisms. Previous research identified a novel, non‐(‐lactam inhibitor for the class C (‐lactamase AmpC (3‐[(4‐chloroanilino)sulfonyl]thiophene‐2‐carboxylic acid; Ki 26 (M). In an effort to improve the binding affinity of this inhibitor, a series of sulfonylthiophene carboxylic acid derivatives were synthesized and tested for inhibition of AmpC. Several of these inhibitors were co‐crystallized with AmpC, and the structures of the complexes were determined using X‐ray crystallography.
Comparison of Protein‐Protein Interactions between BLIP and Class A Beta‐Lactamases
Authors: Nguyen Toan D , Wang Jihong , Mai Tu T , Pham Ngoc , Zhang Zhen , Palzkill Timothy , Chow Dar‐Chone
Published: June 2021

The goal of our project is to determine and compare the binding thermodynamics of the interaction between β‐lactamase Inhibitor Protein (BLIP) mutants and Class A β‐Lactamases using ITC. We have previously determined the thermodynamics of binding interactions between TEM‐1, class A β‐lactamase family, and a set of alanine substituted contact residue mutants of BLIP (Wang J., et al. JBC. 2007; 282:17676–84). We have extended our approach to other members of class A β‐lactamases. Here, we report the binding thermodynamics between the inactive TEM‐1 mutant (S70A) and BLIP. We find that the alanine substitution of the active site Ser70 of TEM‐1 results in the lowering of the iso‐enthalpic temperature, which is more enthalpically favorable. The binding between BLIP and Bla1, another class A β‐lactamase, is dominated by an entropy driven force with an extrapolated iso‐enthalpic temperature of 42oC. Its binding heat capacity change is ∼ −450 cal/mol.K (∼ 20% less negative than that of BLIP/TEM‐1). However, the BLIP mutant W150A interaction with Bla1 shows a non‐linear relationship between the binding enthalpy and temperature with a positive binding heat capacity change at low temperature and a negative binding heat capacity change at high temperature, which is contrary to the general observation that the heat capacity change is negative for protein‐protein interactions.

Support from AHA & UH to D.C. and NIH AI32956 to T.P.
Arsenic (+3 oxidation state) methyltransferase and the methylation of arsenicals in the invertebrate chordate Ciona intestinalis
Authors: Nava Gerardo Manuel , Lee David Y. , Cai Shi‐Ying , Boyer James L. , Hernández‐Zavala Araceli , Thomas David J. , Gaskins H. Rex
Published: June 2021

The biotransformation of inorganic arsenic (iAs) involves methylation by an arsenic (+3 oxidation state) methyltransferase (AS3MT), yielding methyl arsenic (MA), dimethyl arsenic (DMA), and trimethylarsenic (TMA). To identify molecular mechanisms that coordinate arsenic biotransformation, we are using a comparative genomic approach focused on the invertebrate chordate, Ciona intestinalis. Bioinformatic analyses were used to annotate an AS3MT gene in Ciona. RT‐PCR analysis from replicate animals demonstrated constitutive expression of AS3MT in branchial sac (BS), gastrointestinal tract (GIT), and heart. Also, replicate animals were exposed to zero (control) or 1 ppm of iAs (as arsenite) in sea water during 24 h or 5 d. Hydride generation‐atomic absorption spectrometry was used to measure iAs, MA, DMA, and TMA concentrations in BS, GIT, and heart. Methylated metabolites were detected in each of the tissues at both 24 h and 5 d in Ciona specimens exposed to iAs compared with controls (P < 0.01). Average total speciated As concentrations were highest in the BS (3705 ng/g) followed by heart (1019 ng/g) and GIT (835 ng/g) after 5 d. Thus Ciona metabolizes iAs to its methylated metabolites, consistent with the presence of an AS3MT ortholog in its genome, making this basal chordate a useful model to examine the evolution of As detoxification. (This abstract does not reflect U.S. EPA policy.)