bluuebunny/crossref_metadata_2025 huggingface dataset:
Using the NCBI web site to understand GPCR structure
Authors: Scofield Margaret A. , Huggett Kathryn D.
Published: June 2021

Pharmacology educators have discussed the need for future PhD scientists to have diverse training, to think critically and question assumptions, to acquire communication skills, to critically evaluate articles, and demonstrate independence. Many students enter pharmacology doctoral programs with diverse backgrounds and high GRE scores but lack these skills. The required doctoral courses provide a vast array of scientific knowledge and students must apply this information to their field of specialty. All of our graduate students take an entry level course titled “Molecular Biology in Pharmacology” where the above skill sets are integrated into the curriculum. The course objective is to provide students with knowledge of basic concepts of molecular biology and more importantly, to use this information to understand pharmacological principles. One course component introduces students to the tools and information available on the National Center for Biotechnology Information (NCBI) web site. Using bioinformatics, students must complete an assignment that integrates principles of molecular biology and biochemistry into an investigation of a G‐protein coupled receptor gene, its disease states, and protein structure. Upon completing this exercise students demonstrate the ability to use data base resources and apply that information to understand the structure and function of receptors in pharmacology.
Ionic mechanisms for bumetanide enhancement on aminoglycoside ototoxicity: a study with MDCK cells
Authors: Wang Tian , Wang Qi , Yang Yu‐Qin , Ma Ke‐Tao , Steyger Peter , Jiang Zhi‐Gen
Published: June 2021

It is well known that loop diuretics such as bumetanide dramatically enhances ototoxicity of aminoglycosides (AG) when they are administered together to mammalians including humans. The mechanism(s) to cause such enhancement remain poorly understood. MDCK cells share many membrane properties with cochlear marginal cells and have been used to investigate the AG‐permissive channel mechanisms. Using gramicidin‐perforated patch‐clamp techniques and fluorescent gentamicin (GTTR) imaging analysis, we found: 1) MDCK uptake of GTTR was concentration‐dependently enhanced by bumetanide and furosemide; the enhancement was suppressed by La3+, the Cl−‐channel blocker N‐phenylanthranilic acid (PAA) or extracellular low Cl− (60 mM) but not by TEA, 4‐AP or glipizide. 2) La3+ (5 mM) reduced input conductance in all cells tested with a net current reversal potential of ~−10 mV. 3) Bumetanide hyperpolarized MDCK cells for 10.5 mV from a mean of −32.1 (p<0.01). 4) Bumetanide caused an increase in slope conductance of the whole‐cell I/V curve (from ~0.2 to ~1.5 nS at −60 mV). Bumetanide‐induced net current I/V curve reversed its polarity at ~ −45 mV. 5) The membrane action of bumetanide was suppressed by PAA and low Cl but not by FAA, NFA, 4AP, TEA, glipizide, La3+ or Gd3. We conclude: (1) Bumetanide enhances the La3+‐sensitive AG‐uptake by MDCK cells; (2) The enhancement is likely due to bumetanide‐induced hyperpolarization thus an increased driving force for cationic AG influx; (3) Bumetanide causes the hyperpolarization via its activation of a PAA‐sensitive Cl−‐channel; (4) The Cl− equilibrium potential is a main source of membrane potential of the MDCK cells we used. Funded by NIDCD R01 04716 (ZGJ) R01 04555 (PSS) and P30 05983.
Incompletely activated CD4+ T cells induce granzyme B+ regulatory B cells in an interleukin 21‐dependent manner
Authors: Dahlke Karen , Hagn Magdalena , Sontheimer Kai , Beyer Thamara , Lunov Oleg , Fabricius Dorit , Tron Kyrylo , Nienhaus G. Ulrich , Simmet Thomas , Jahrsdörfer Bernd
Published: June 2021

Immune regulation is central for the development of an efficient cellular immune response. Both Treg cells and plasmacytoid DC can suppress T cell proliferation in a granzyme B (GzmB)‐dependent manner. In the present study we found that, depending on stimulation with interleukin (IL−) 21, B cells (BC) can also express GzmB and suppress T cell proliferation. GzmB expression in BC is enhanced by BC receptor engagement, and is suppressed by CD40 ligation. Since CD4+ T cells are a main source of IL‐21, we tested whether they can induce GzmB in BC. We found that incompletely activated CD4+ T cells, but not fully activated T cells can induce GzmB in co‐cultured BC. Using confocal microscopy, we showed that BC‐derived GzmB is enzymatically active and that GzmB+ BC transfer GzmB to CD4+ T cells. Furthermore, GzmB+ BC decreased CD4+ T cell expression of the TCR‐zeta chain, a GzmB target, which is required for T cell proliferation. Our results suggest BC may regulate cellular adaptive immune responses by Treg cell‐like mechanisms. Inhibition of BC‐derived GzmB may represent a novel strategy to induce more effective and comprehensive cellular immune responses.
Novel Allosteric Properties Exhibited by SgrAI, a Restriction Endonuclease Native to Streptomyces griseus
Authors: Ghare Imran , Agrawal Alka , Bitinaite Jurate , Park Chad K. , Horton Nancy C.
Published: June 2021

An 8‐bp recognition sequence, secondary activity, and allosteric activity for the primary sequence make SgrAI a rather peculiar endonuclease. Recent structural evidence suggests that primary site recognition is dependent upon dimerization of SgrAI. However, secondary site and allosteric activity have remained elusive until now. Through a series of kinetic and binding assays we have reason to believe that dimeric kinetic activity is significantly lower than the activity of the HMWS (high molecular weight species). Native gel electrophoresis suggests that increasing concentrations of PCP (precut primary sequence, 40‐mer) increases formation of the HMWS. Furthermore, our kinetic assays are also accelerated by the addition of PCP. Kinetics were measured via endpoint quench methods, substrates and products were separated by denaturing gel electrophoresis and quantized by computer integration. It appears that the HMWS is an activated form of the enzyme. In order to reaffirm our findings we tested kinetics and binding of two mutants, P27G/W. Both mutants were designed to disrupt tetramer formation. Crystal structures show that the tetramer has an N‐terminal region which is domain swapped with another dimer to form the tetramer. Native gel electrophoresis suggests that P27W/G do not form a HMWS to the same extent as wtSgrAI. Additionally, P27W/G does not exhibit the same acceleration as wtSgrAI, which leads us to believe that formation of the tetramer is necessary for formation of the HMWS. It also further supports the notion that the active conformation of the enzyme can be found in the HMWS. The Howard Hughes Medical Institute and the NIH, General Medical Sciences, support this work.
gp130 cytokines stimulate proteasomal degradation of tyrosine hydroxylase in sympathetic neurons by ERK‐dependent pathway
Authors: Shi Xiao , Habecker Beth A.
Published: June 2021

Heterogeneity of sympathetic transmission in the heart after myocardial infarction contributes to ventricular arrhythmias and sudden cardiac death. Tyrosine hydroxylase (TH), the rate‐limiting enzyme in norepinephrine synthesis, is depleted in the left ventricle after myocardial infarction, but elevated in other parts of heart. Inflammatory cytokines acting through gp130 cause local depletion of TH in cardiac sympathetic nerves post infarction. However, the mechanism of cytokine‐induced TH loss remains unknown. We used cultured sympathetic neurons to investigate the mechanism of gp130 cytokine suppression of TH protein. Cytokines stimulate TH ubiquitination, and cytokine‐induced TH loss was prevented by MG‐132, suggesting that the proteasomal degradation pathway was involved. Cytokine activation of gp130 stimulates Janus kinase (JAK) which leads to activation of STAT3, ERK1/2, and PI3K/AKT. Blocking ERK activation by U0126 prevented cytokine‐induced TH ubiquitination, while inhibiting STAT3 phosphorylation with STAT3 inhibitor V enhanced cytokine‐induced TH ubiquitination. Inhibiting STAT3 enhanced ERK1/2 phosphorylation, suggesting that ERK was a major regulator of TH ubiquitination. Blocking JAK activity inhibited all downstream signaling and prevented cytokine‐induced ubiquitination of TH. Preliminary data implicate SOCS3 as a mediator of ERK‐STAT3 crosstalk.
Mechanism of Peripheral NMDA Receptor‐Mediated Pressor Response in Conscious Rats
Authors: McGee Marie A , Abdel‐Rahman Abdel
Published: June 2021

Despite its expression in vascular and cardiac tissues, the role of peripheral NMDARs in blood pressure control in conscious animals has not been examined. This study is the first to report a dose (125, 250, 500 and 1000 μg/kg)‐related pressor response elicited by systemic NMDA in male Sprague‐Dawley rats. Femoral vessels were catheterized 2 days prior to NMDA injection and NMDA was administered i.v. at 15 min intervals (n=6). In a subsequent experiment, we tested the hypothesis that an NMDA‐evoked generation of vascular reactive oxygen species (ROS) underlies the pressor response. A submaximal dose (250 μg/kg) of NMDA or its vehicle (saline) was administered i.v. in 2 groups of conscious rats and vascular (aorta) and cardiac tissues were collected at the conclusion of the experiment. The NMDA‐treated group displayed a significant increase in MAP when compared to the control group (n=8 each, p<0.05). Tissue ROS and NO levels were analyzed using dihydroethidium (DHE) and 4‐amino‐5‐methylamino‐2′7′‐dichlorofluoroscein diacetate (DAF‐FM) fluorometry. The pressor response elicited by peripheral NMDAR activation was associated with elevated vascular ROS. The increase in vascular ROS, which approached statistical significance when compared with control values, seems to support the tested hypothesis. Interestingly, however, vascular NO was increased significantly by peripheral NMDAR activation. These findings are the first to demonstrate a peripheral NMDAR‐evoked pressor response and yield insight into possible molecular mechanisms that contribute to this response.

This research was supported by East Carolina University and NIH grant R01 AA07839.
MODULATION OF SYMPATHETIC TRANSMISSION BY NITRIC OXIDE IS MAINTAINED IN RATS WITH PULMONARY HYPERTENSION
Authors: ORIOWO MABAYOJE , EL‐HAJJ HANADI
Published: June 2021

Pulmonary hypertension (PH) is a chronic disabling disease that largely affects the pulmonary vasculature. In this study, we have examined the effect of blocking NOS on sympathetic neurotransmission in the pulmonary artery and how this is influenced by PH. Rats treated with MCT developed pulmonary hypertension. Electrical field stimulation (EFS) of the pulmonary artery produced frequency‐dependent contractions. The frequency‐response curve was shifted to the left in artery segments from pulmonary hypertensive rats. The non‐selective NOS inhibitor, L‐NAME (0.1 mM), potentiated EFS‐induced contractions at all frequencies of stimulation in both groups with no difference between the groups. 7‐NI, an inhibitor of neuronal NOS had no effect. Noradrenaline produced concentration‐dependent contractions of pulmonary artery segments from control and MCT‐treated rats. L‐NAME potentiated the contractions in control but not MCT‐treated rats. It was concluded that endothelial NO modulated adrenergic neurotransmission in the rat pulmonary artery and this was not affected by pulmonary hypertension.
Mechanism of target specialization of prokaryotic transcription factors GreA, GreB and DksA
Authors: Parshin Andrey V. , Lee Jookyung , Borukhov: Sergei
Published: June 2021

Bacterial transcription factors GreA, GreB and DksA regulate RNA polymerase (RNAP) function by acting through its secondary channel. GreA and GreB stimulate RNAP intrinsic nucleolytic activity, thereby suppressing transcriptional pause and arrest, enhancing transcription fidelity and helping promoter escape. DksA activates and/or inhibits transcription both in vivo and in vitro by destabilizing RNAP open promoter complexes. We characterized the molecular interfaces of GreA‐RNAP and DksA‐RNAP complexes using site‐specific protein‐protein crosslinking and site‐directed mutagenesis. We also determined the binding affinities of Gre/DksA towards different transcription complexes and analyzed their functional activities by in vitro transcription assays. We identified the key residues in Gre, DksA and RNAP secondary channel required for specific Gre/DksA‐RNAP interactions. Based on our data, we created plausible structural models of Gre‐ and DksA‐RNAP complexes. In the models, Gre and DksA bind to RNAP β’ N‐terminal coiled‐coil element. However, the contact residues for Gre and DksA on β’ are distinct. DksA coiled‐coil interacts with the β’ conserved G‐loop element. The conformational changes in the G‐loop define the binding of GreA to RNAP. Unlike Gre factors, DksA does not bind to elongation complexes and does not compete with GreA during transcription. These results indicate that Gre and DksA target different classes of transcription complexes in vivo, most likely, depending on the conformation of the flexible G‐loop element of RNAP.

This work was supported by National Institute of Health.
A high‐resolution crystal structure of ligand‐free cytochrome P450 2B4 (H226Y) in a closed conformation
Authors: Shah Manish B , Zhang Qinghai , Stout C. David , Halpert James
Published: June 2021

Recent structural studies from our laboratories on the xenobiotic metabolizing cytochrome P450 2B subfamily have provided a wealth of information on the basis for the broad substrate specificity of these enzymes. We have recently determined the structure of ligand‐free cytochrome P450 2B4 (H226Y) to 1.9 Å resolution. Use of facial amphiphiles during crystallization helped to achieve diffraction quality crystals and higher resolution. In the absence of the facial amphiphiles, the crystals were observed in only very limited conditions and yielded poor diffraction. The closed conformation observed was in contrast to the open conformation of 2B4 (H226) reported previously by our group. The new structure is very similar to other closed 2B4 structures with small imidazoles bound. It positions many of the key active site residues in close proximity to the heme and may enable a direct contact with the substrate once it is sequestered in the active site. The results suggest that cytochrome P450 2B4 may exist in heterogeneous and energetically accessible conformations in solution between an open and a closed state. Recent evidence from hydrogen‐deuterium exchange mass spectrometry suggests that the open conformation predominates in solution. (This research was supported by NIH grant ES003619).
Effects of Diet‐induced Obesity on Protein Expressions of Insulin Signalling Pathways in the Skeletal Muscle of Male Wistar Rats
Authors: Fatani Sameer , Itua Imose , Naderali Ebrahim
Published: June 2021

Effects of Diet‐induced Obesity on Protein Expressions of Insulin Signalling Pathways in the Skeletal Muscle of male Wistar Rats

The exact mechanism involved in insulin transduction in skeletal muscle is not well understood. Protein expressions of PI 3‐kinase and MAP‐kinase insulin signalling pathways in skeletal muscle of diet induced obese rats were investigated.

Male Wistar rats (190 ± 3g) were randomly allocated to control, vehicle (diet induced obese animals), diet‐to‐chow and fenofibrate treated group. Expression of proteins involved in insulin signalling was determined by western blotting. Triglyceride (TG) content of skeletal muscle was determined by homogenising 160–200mg gastrocnemius muscle in 500μl distilled water and assaying the supernatants. Results were normalised and compared to control using student t test or repeated measures one‐way ANOVA or Mann‐Whitney test. Data are expressed as mean ± SEM and considered significant at p<0.05.

Vehicle animals were significantly heavier than control, diet‐to‐chow and fenofibrate animals. TG levels in vehicle were significantly increased compared to other groups. IR‐β and IRS‐1 were unaffected. PKC‐β, regulatory subunit p85 of PI 3‐kinase and eNOS respectively were significantly increased in vehicle and fenofibrate groups. Akt in diet‐to‐chow were significantly lower than vehicle group. Shc and ERK1/2 were significantly increased in vehicle.

Thus in diet induced obesity, insulin resistance in skeletal muscle is achieved by increasing expression of p85 of PI 3‐kinase.
Enhanced nitration of several mitochondrial proteins during cardiac ischemia and reperfusion injury
Authors: Yang Meiying , Camara Amadou K.S. , Gadicherla Ashish K. , Wakim Bassam , Aldakkak Mohammed , Boelens Age D. , Heisner James S. , Stowe David F
Published: June 2021

Superoxide (O2•−) produced during cardiac ischemia‐reperfusion (IR) injury can react with nitric oxide (NO•) to form peroxynitrite (ONOO−) and induce protein tyrosine nitration to alter protein structure and function. Our objective was to investigate if nitration of selective mitochondrial proteins by ONOO− occurs during IR and to identify the proteins. Isolated Guinea pig hearts were perfused with KR buffer without (40 min of time control) or with 30 min of ischemia and 10 min reperfusion (IR). Mitochondria were isolated immediately after each treatment. Tyrosine nitration of mitochondrial proteins was measured by Western blot with anti‐nitrotyrosine antibody (anti‐NT). We found that compared to time control, IR treatment displayed an anti‐NT immunoreactive band at about 30 kDa with a 50% increase in band intensity. To identify proteins in the anti‐NT immunoreactive band, mitochondrial lysate from IR treated mitochondria was subjected to immunoprecipitation (IP) with anti‐NT, and precipitated proteins were separated by SDS‐PAGE followed by silver staining. Silver stained anti‐NT immunoreactive bands were analyzed by LC/MS/MS. Three tyrosine nitrated mitochondrial proteins: 3‐oxoacid CoA‐transferase, E2K and ADP/ATP translocase 1 were identified. Knowledge of tyrosine nitration in specific proteins during IR could provide an opportunity to target these proteins for therapeutic intervention.
Expression of nAChRs subunits within rostral nucleus ambigus (rNA) of ferrets
Authors: Neziri Burim , Krasniqi Shaip , Devaja Shqipe , Sopi Ramadan , Jakupaj Muharrem
Published: June 2021

The role of nicotine acetylcholine receptors (nAChRs) in regulating cholinergic outflow to the airways is not fully elucidated. Furthermore, the origin of acetylcholine (ACh) or choline, as endogenous and selective agonists for nAChRs is not clear. The neurotransmitter ACh exerts its effects on the central nervous system (CNS) and peripheral nervous system (PNS) through two distinct types of receptors: the muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively). The nAChRs are involved in a wide range of physiological and pathophysiological processes. In the CNS, the nAChR system participates in multiple regulatory brain functions, including neuronal signaling, synaptic transmission and plasticity, transmitter release, development and survival of neurons, including spinal cord motor cells. In the central nervous system (CNS), there are at least 9 α (α 2–α 10) and three β (β 2– β 4) subunits. The homomeric assembly of subunits such α 7, α 8, and α 9 has been shown to be functional in mammalian cells, whereas α 2– α 6 and α 10 become operational only when co‐expressed with – β subunits. Our studies have been focused to determine the precise subunit composition and physiological roles of α 7, α 4 and β2 nAChRs in regulating upper airway dilator muscle tone. The expression of α 7, α 4, or β 2 was analyzed at the protein level, in the tissues of the rNA region, where AVPNs are located. The findings of the present study done on ferrets rNA brain region, show that AVPNs express α 7, α 4 and β 2 nAChR subunits that could assemble into functional receptors with homologous or heterologous pentameric channels.
Upregulation in the Expression of Tryptophan Hydroxylase 2 (TPH2) in the Lower Brainstem in Depression
Authors: Kerman Ilan A , Amilineni Nina , Jones Edward G. , Bunney William E. , Huda Akil , Watson Stanley J.
Published: June 2021

In addition to affective disturbances depression is characterized by physical symptoms, which suggest dysfunction of motor, autonomic, and pain regulatory systems. These functions are modulated by serotonin (5‐HT), and extensive evidence implicates dysfunction in its neurotransmission in the pathophysiology of depression. In the current study we examined gene expression of TPH2, a key enzyme in 5‐HT synthesis, within brainstem regions that contain spinally‐projecting 5‐HTergic neurons. We collected post‐mortem human brainstem samples from depressed and control subjects, and used a combined neurochemical and histological staining approach to identify 5‐HT cell groups in the lower brainstem that are homologous with those in other species. Using radioactive in situ hybridization we detected an upregulation in TPH2 expression within: raphe obscurus, gigantocellular nucleus pars α, and raphe magnus. These regions are well‐known to regulate somatomotor, sympathetic, and nociceptive functions, and a similar increase in TPH2 expression has been reported within ascending 5‐HTergic cell groups. Taken together, these findings suggest shared molecular alterations within distinct 5‐HT circuits that mediate affective and physical manifestations of depression.

Support: Pritzker Neuropsychiatric Disorders Research Consortium, 5K99MH081927‐02 (to IAK), and NARSAD Young Investigator Award (to IAK).
Blockade of the Renin‐Angiotensin System Improves Glucose Intolerance in Pre‐diabetic Rats on a High Glucose Diet
Authors: Rodriguez Ruben , Viscarra Jose , Nakano Daisuke , Nishiyama Akira , Ortiz Rudy M.
Published: June 2021

The benefits of an angiotensin receptor blocker (ARB) on glucose tolerance in pre‐diabetic, obese (OLETF) rats were examined. After 5 weeks of treatment, an oral glucose tolerance test (oGTT) was used to evaluate the impairment of glucose tolerance in the following groups of rats: 1) untreated LETO (lean control strain; LETO), 2) untreated OLETF (OLETF), 3) OLETF + 5% glucose water (OLETF HG), and 4) OLETF + HG + ARB (olmesartan; 10 mg/kg/d; OLETF HG/ARB). Additionally, oGTT was measured in a separate group of OLETF (OLETF bl) prior to initiating treatments to serve as a baseline control. Plasma [glucose] was measured prior to oral infusion and at 15, 30, 60 and 120 min post‐infusion. Area under the curve was used to evaluate the response to oGTT. The response to oGTT increased 63% in OLETF compared to LETO, suggesting that glucose tolerance was compromised, establishing the model. There was no difference in response between LETO and OLETF bl, and a 36% increase in OLEFT compared to OLETF bl, demonstrating the manifestation of glucose intolerance in this model. The oGTT response increased 17% in OLETF HG compared to OLETF was reconciled with ARB treatment, suggesting that blockade of RAS improves glucose tolerance in a pre‐diabetic, high glucose intake condition. These data suggest that activated RAS may impair glucose tolerance, which may contribute to the manifestation of diabetes in a model of the metabolic syndrome. (UCSC‐MHIRT)
Tetrahydrobiopterin does not affect end‐organ responsiveness to norepinephrine‐mediated vasoconstriction in aged skin
Authors: Lang James A , Bruning Rebecca S , Holowatz Lacy A , Kenney W Larry
Published: June 2021

We have recently found that tetrahydrobiopterin (BH4), a cofactor in norepinephrine (NE) biosynthesis, offsets the attenuated reflex VC in aged skin. We hypothesize that localized BH4 supplementation would not affect end‐organ VC responsiveness to exogenous NE after localized sympathetic nerve blockade. Two microdialysis fibers were placed in bretylium tosylate pretreated (presynaptically blocks neurotransmitters release from sympathetic adrenergic nerve terminals; iontophoresis, 200 μA for 20 min) forearm skin of 10 young (Y) and 10 older (O) subjects for perfusion of 1) Ringer's (control) and 2) 5 mM BH4. While local skin temperature was clamped at 34oC, 6 randomized concentrations of NE (10−12, 10−10, 10−8, 10−6, 10−4, 10−2 M) were infused at each drug‐treated site. Cutaneous vascular conductance was calculated (CVC = laser Doppler flux / MAP) and normalized to baseline (%ΔCVCbase). Despite prejunctional adrenergic blockade, NE‐mediated VC was blunted in aged skin at each NE dose (10−12: −12 ± 2 vs. −21 ± 2, 10−10: −15 ± 2 vs. −27 ± 1, 10−8: −22 ± 2 vs. −32 ± 2, 10−6: −27 ± 2 vs. −38 ± 1, 10−4: −52 ± 3 vs. −66 ± 5, 10−2: −62 ± 3 vs. −75 ± 4 %ΔCVCbase; P<0.01), and this response was not affected by pretreatment with BH4 (P>0.05). Localized BH4 did not affect end‐organ responsiveness to exogenous NE suggesting that BH4 augments VC primarily through the NE biosynthetic pathway in adrenergic nerve terminals.
A Helix‐Loop‐Helix Motif within a Genomic DNaseI Footprint of the Runx2 Gene Promoter is Essential for Basal Bone Tissue‐Specific Transcription
Authors: Zhang Ying , Hovhannisyan Hayk , Hassan Mohammad Q. , Glackin Carlotta , Lian Jane B. , Stein Janet L. , Montecino Martin , Stein Gary S. , van Wijnen Andre J.
Published: June 2021

Transcriptional mechanisms controlling Runx2 levels are critical for its osteogenic function during osteoblast differentiation. Here we examine the chromatin organization of the genomic Runx2 locus in intact cells. Chromatin immuno‐precipitations were performed with antibodies of modified histones and PCR probes spanning 120 kbp surrounding its bone‐specific (P1) promoter. The results show that a ~10 kbp genomic segment around the P1 promoter interacts with highly acetylated histones H3 and H4 reflecting an open chromatin conformation. Within this acetylated domain, we find a single major DNaseI hypersensitive (DHS) region that resolves into a cluster of smaller sites (−0.4 ~0.0 kbp) and coincides with the promoter segment that controls basal Runx2 transcription in osteoblasts. Genomic DNase I footprinting indicates that the DHS region contains several protein/DNA interaction sites (e.g., at AP1, HLH/E‐box and Runx motifs). HLH/E‐box occupancy of the DHS region persists in all mesenchymal cell types examined. Occupancy of the AP1 binding site occurs only during S phase when Runx2 expression is minimal. Point‐mutation of the HLH/E box causes a dramatic reduction of the P1 promoter activity. We propose that protein/DNA interactions at the AP1 and HLH/E box motifs may control Runx2 transcription when osteoblasts transit into a quiescent and differentiated state. Supported by NIH grant AR039588.
Mitochondrial‐targeted antioxidants attenuate immobilization‐induced skeletal muscle atrophy
Authors: Min Kisuk , Smuder Ashley J , Kwon Oh‐sung , Kavazis Andreas N , Szeto Hazel H , Powers Scott K
Published: June 2021

Prolonged periods of muscular inactivity (e.g., limb immobilization) result in skeletal muscle atrophy. Although is established that reactive oxygen species (ROS) play a role in inactivity‐induced skeletal muscle atrophy, the pathway(s) responsible for inactivity‐induced ROS production remain unclear. To investigate this important issue we tested the hypothesis that elevated mitochondrial ROS emission is essential for immobilization‐induced increases in oxidative stress and proteolysis in skeletal muscle. Cause and effect was determined by administering a novel mitochondrial‐targeted antioxidant (SS‐31) to prevent immobilization‐induced mitochondrial ROS emission in skeletal muscle fibers. Compared to ambulatory controls, 14‐days of immobilization resulted in decreased skeletal muscle mass along with increased mitochondrial ROS production, muscle oxidative damage, and protease activation. Importantly, treatment with SS‐31 attenuated the inactivity‐induced increase in mitochondrial ROS emission and prevented oxidative stress, protease activation, and fiber atrophy. These results support the hypothesis that redox disturbances are required for immobilization‐induced skeletal muscle atrophy and that mitochondria are a major source of ROS production in muscle fibers during prolonged periods of inactivity. Supported by NIH R01 HL087839 awarded to SKP
The ATM activator chloroquine stimulates phosphorylation of AMP activated protein kinase (AMPK) and acetyl CoA‐carboxylase (ACC) independent of ATM
Authors: Spears Larry D. , Qin Charles , Zhang Zhihong , Ralston Lyle , Fisher Jonathan S.
Published: June 2021

Ataxia Telangiectasia mutated (ATM) is a kinase that has been shown to be necessary in the phosphorylation of AMPK in fibroblasts stimulated by IGF‐1, and we have previously found that the ATM activator chloroquine (CQ) causes an increase in the phosphorylation of AMPK. Based on these findings, we hypothesized that CQ would cause an increase in the phosphorylation of AMPK in C2C12 myotubes in an ATM dependent manner. Myotubes were incubated with 0‐750 μM CQ. 750 μM CQ increased phosphorylation of ATM at S1981, a marker for ATM activation (P<0.05). To test whether ATM was necessary for CQ's effect of increasing phosphorylation of AMPK, C2C12 cells were lentivirally transduced with short hairpin RNA (shRNA) against ATM. Cells transduced with shRNA against ATM had an 80% decrease of ATM expression compared to wildtype cells and a cell line transduced with green fluorescent protein (GFP). 500 μM CQ increased phosphorylation of AMPK in cell lines expressing either the shRNA against ATM or GFP (P<0.01, main effect of CQ). A specific ATM inhibitor, KU55933 (KU), was used to discover whether ATM was involved in increasing phosphorylation of a downstream target of AMPK, ACC. Even in the presence of KU, 500 μM CQ caused phosphorylation of ACC to increase (P<0.01, main effect of CQ). Our results suggest that ATM is not required for CQ‐related stimulation of AMPK phosphorylation and ACC phosphorylation in C2C12 myotubes.
The Effects of Hindlimb Suspension on Proteins Essential to Cholesterol Metabolism in Rat Skeletal Muscle
Authors: Lee Teak Veng , Swift Joshua M , Chen Vincent CW , Lee Chang Woock , Bui Steve , Riechman Steven E
Published: June 2021

Introduction

Hindlimb suspension (HS) results in significant deleterious effects on numerous physiological systems, including atrophy of weight‐bearing muscles. However, HS and its effects on proteins essential for cholesterol metabolism have not been investigated in unloaded skeletal muscle. Cholesterol serves as a critical constituent of all cell membranes, including skeletal muscle. The purpose of this study was to examine the effects of 28 days of HS on sterol regulatory element‐binding protein 2 (SREBP‐2), which is a transcriptional factor necessary for endogenous cholesterol synthesis. We hypothesized that HS causes a downregulation of this protein in rat skeletal muscle.

Methods

Twelve male Sprague‐Dawley rats, 6 months of age, were hindlimb suspended for 28 days (HS; n=5) or served as cage controls (CC; n=7). After 4 weeks, plantarflexor muscles were harvested, weighed and analyzed by Western Blotting for SREBP‐2.

Results

Soleus muscle mass and soleus muscle mass to body mass were significantly different between groups (HS: 97.0 ± 14.2 mg and 0. 22 ± 0. 03 mg/g; CC: 244.3 ± 38.1 mg and 0.49 ± 0. 06 mg/g, P < 0.05). However, mature SREBP‐2 was not significantly different between groups (HS: 0.66 ± 0.31 arbitrary units (AU) and CC: 1.06 ± 0.39 AU, P=0.09).

Conclusion

After 28 days, HS resulted in significant decreases in soleus muscle mass, but not mature SREBP‐2, as compared to cage controls.
Decreased klotho expression in early aldosterone‐induced hypertension
Authors: Takenaka Tsuneo , Inoue Tsutomu , Okada Hirokazu , Miyazaki Takashi , Suzuki Hiromichi
Published: June 2021

Aldosterone activates mineralocorticoid receptor in distal nephron. Klotho is expressed in distal nephron, and suppresses oxidative stress. However, the effects of aldosterone on klotho expression have not adequately examined. In the present study, the expression of klotho was assessed in 4 groups of rats; Wistar rats as a control (C), aldosterone‐infused (0.75 μg/hr SQ) rats (A), rats fed high (6%) salt diet (S), and rats loaded with both aldosterone and salt (A+S). Initially, left nephrectomy was performed in all rats. After a week of recovery, aldosterone and/or salt load was started. A week later, rats were killed with over‐anesthesia, and harvested right kidney for analysis. Mean blood pressure in A+S group (111±6 mmHg, n=5, p<0.05) was higher than C group (97±3 mmHg, n=5). Significant changes in blood pressure were seen in neither A nor S group. In addition, creatinine clearance and albumin excretion were similar among 4 groups. However, 8‐epi‐prostaglandin F2α excretion was markedly increased in A+S group (13±3 ng/24hr, p<0.01), compared to C group (5±1 ng/24hr). RT‐PCR and immunoblot using monoclonal antibody against klotho (KM2076) revealed that compared to C group, renal klotho expression was impaired in A+S group, assessed by mRNA (45±8 %, p<0.05) and protein level (50±11 %, p<0.05). Group A and S did not show significant changes in klotho expression. Our data indicated that blood pressure was mildly elevated in early aldosterone salt‐induced hypertension. The present findings provided the evidence that klotho expression was reduced before developing overt nephropathy. Our results suggest that a reduced klotho expression may accelerate oxidative stress in aldosterone salt‐induced hypertension.
Connecting tubule glomerular feedback (CTGF) antagonizes tubuloglomerular feedback (TGF) in vivo
Authors: Wang Hong , Garvin Jeffrey L. , Carretero Oscar A.
Published: June 2021

We recently described that there is crosstalk between the connecting tubule (CNT) and the Af‐Art that causes dilation. CTGF is mediated by CNT Na transport via epithelial Na channels (ENaC). We hypothesized that CTGF opposes TGF. We performed renal micropuncture studies in male rats. We induced TGF by increasing late proximal perfusion rate (0, 10, 20, 40, 80 nL/min). Stop‐flow pressure (PSF) was used as an index of TGF response. First, we showed that TGF was reversible and reproducible. Next, we tested whether inhibition of CTGF with benzamil (ENaC inhibitor; 1 μM) potentiated TGF. In control, high flow (80 nL/min) increased PSF indicating that CTGF increased Af‐Art dilatation. CTGF inhibition with benzamil potentiated the TGF response, especially at high flow rates. When NaCl transport in the distal tubule was inhibited with hydrochlorothiazide (HCTZ, 1 mM) to increase delivery of Na to the CNT, benzamil potentiated the TGF response at lower (20 nL/min) flow rates (Table). We conclude that CTGF antagonizes the vasoconstrictor effect of TGF.

Perfusion rate (nL/min)
0
10
20
40
80

Control PSF (n=5)
39.6 ± 0.8
39.2 ± 0.6
36.4 ± 0.8
32.4 ± 0.9
34.2 ± 0.8†

Benzamil PSF
39.1 ± 0.8
38.7 ± 0.7
35.7 ± 0.9
30.1 ± 0.8**
30.1 ± 0.7***

HCTZ PSF (n=4)
40.0 ± 0.6
40.3 ± 0.6
37.4 ± 0.5
33.5 ± 0.8
35.5 ± 0.8†

HCTZ+Benzamil PSF
39.8 ± 0.4
39.9 ± 0.5
35.8 ± 0.4#
31.0 ± 0.6#
30.9 ± 0.6#

p < 0.01 and

p < 0.001 vs. control;

p < 0.05 vs. HCTZ;

p < 0.001 40 vs. 80 nL/min

Source of Funding: This study was supported by NIH Grants HL028982 and HL088036.
Synchrotron radiation exposure of mice head leads to reduction of spleen weight, but not testicular weight, nor spleen cytokine levels
Authors: Shao Jiaxiang , Liu Yewei , Liu Na , Xia Weiliang , Ying Weihai
Published: June 2021

Synchrotron radiation has wide biomedical applications yet one key issue is its safety concerns for use in medical procedures. In this study we carried out pilot experiments to explore the safety dosage by assessing cellular/organ damages in mice that underwent exposure of synchrotron radiation for varying durations (15, 10, 5, 3, 1 and 0 min, n=6 for each time point). ICR male mice (age: 6 wk) were anaesthetized with sodium pentobarbital before their head being exposed to radiation under the same parameters (32 keV, 1.5*E6~5*E7 mA chamber currency which is most widely used for Barium sulfate imaging in medical applications) in Shanghai Synchrotron Radiation Facility. The animals were euthanized 1 or 3 days post irradiation; spleen and testicles were removed, weighed and frozen in liquid nitrogen. It was found that paired testes weight did not change significantly 1 or 3 days afterwards with increasing duration of irradiation; however, 3 days afterwards spleen weight reduced significantly with 1 min exposure, and ~55% reduction was observed for 3 min and 5 min exposure group. Additional analysis of spleen cytokine levels (TNFα and IL6) by real‐time PCR found that no significant changes occurred either 1 or 3 days after irradiation. Further study is warranted to the understanding of synchrotron irradiation damage to other organs when the brain is the target irradiation site.
Hypocitraturia and alkalinuria: Risk factors for nephrolithiasis in topiramate‐treated subjects.
Authors: Sours‐Brothers Sherry B. , Maalouf Naim , Sakhaee Khashayar , Moe Orson
Published: June 2021

Topiramate (TPM) is widely prescribed for seizures and migraine prophylaxis. TPM has multiple and yet uncharacterized mechanisms of actions, including inhibition of carbonic anhydrase (CA), and its use is associated with increased urinary pH (UpH), hypocitraturia and mild hypercalciuria, contributing to increased risk of calcium phosphate urolithiasis. Our data show that stone prevelance is higher than initially reported. In 75 TPM‐treated patients, 9.3% had symptomatic stones. An additional 3 of 15 had asymptomatic stones when examined by CT, for a combined prevalance of 13%. In 52 TPM‐treated patients, 81% had hypocitraturia and 63% had UpH >6.5. The relative saturation ratio for brushite was >2 in 69%. The mechanism by which TPM induces these changes is undefined. We generated a rat model of TPM‐mediated hypocitraturia that will be crucial to understanding this pathophysiology. Male sprague‐dawley rats were given 100 mg/kg body weight TPM or vehicle (Veh). 24‐hr urines were collected at baseline and 1, 2, 7, and 14 days of treatment. 24‐hr citrate excretion was significantly lower with TPM after the day 1 (1.521 ± 0.231 mg citrate/mg Cr vs 2.850 ± 0.125 Veh) and continued throughout treatment. UpH was significantly higher with TPM, as seen in humans. These data establish TPM treatment in rats as a model to study the mechanism leading to increased stone risk in TPM‐treated patients.
REGULATION OF MIRNAS BY BISPHENOL A IN CARDIOMYOCYTES
Authors: Castaneda Julie Theresa , Waikel Rebekah
Published: June 2021

Bisphenol A (BPA) is an organic compound commonly used in the production of plastics and plastic additives. BPA is an endocrine disruptor that can mimic hormones, including estrogen, and can cause severe health problems. Studies have consistently shown an association with BPA urine concentrations and heart disease. It has been well documented that estrogen has cardioprotective effects. Therefore, it is seemingly contradictory that BPA, which activates the estrogen receptor, would be detrimental to the heart. In order to gain greater insight into the biochemical response to BPA in comparison to estrogen, we characterized miRNA signatures in cardiomyocytes of miRNAs previously documented to influence cardiovascular disease. Following treatment of primary neonatal rat cardiomyocyte cultures with either estradiol or BPA, miRNAs levels were analyzed by Taqman Real‐Time PCR. Exposure to BPA did produce a unique miRNA signature as compared to exposure to estradiol.
Helix 8 for ER Export of Leukotriene B4 type‐2 Receptor (BLT2)
Authors: Yasuda Daisuke , Hirota Nobuaki , Hashidate Tomomi , Shimizu Takao , Nakamura Motonao
Published: June 2021

Many G protein‐coupled receptors (GPCRs) possess a putative cytoplasmic helical domain, termed helix 8 (H8), at the proximal region of the C‐terminal tail. However, the significance of this domain is not fully understood. Here, we demonstrate the requirement of H8 for the proper folding of GPCRs for passage through the quality control mechanism in the endoplasmic reticulum (ER). In human leukotriene B4 type‐2 receptor (hBLT2), lack of H8 led to an accumulation of the receptor (hBLT2/ΔH8) in the ER. Similar results were obtained in two representative human GPCRs, dopamine type‐1 and lysophosphatidic acid type‐2 receptors, which were engineered to lack H8. Treatment with the several ligands, which act as pharmacological chaperones, facilitated the surface expression of hBLT2/ΔH8. The surface‐trafficked hBLT2/ΔH8 exhibited an agonist‐evoked increase in Ca2+, demonstrating that H8 is not critical for ligand binding and activation of coupled G proteins. Thus, these results suggest that the H8 region of hBLT2 plays an important role in transport‐competent receptor folding. Supported in part by a grant‐in‐aid from Ministry of Education, Culture, Sports, Science, and Technology of Japan
De novo disulfide bridges cause pleiotropic effects on phytase characteristics other than stability
Authors: Weaver Jeremy D. , Ripoll Daniel , Lei Xin Gel
Published: June 2021

De novo disulfide bridges have been documented to increase the thermostability of proteins, but little attention has been devoted to their effect on catalytic kinetics. We previously attempted to improve the stability of Escherichia coli AppA2 phytase through creation of nine de novo disulfide bridge mutants. However, only Q134C/A202C marginally increased the stability, while seven mutants showed decreased catalytic efficiency. Moreover, at 37°C, ss2‐ (C133/C408S), ss3‐ (C178S/C188) and A21C/W40C maintained the same thermostability as WT while showing decreased activity with altered pH profile and optimal temperature. We explored the mechanism of these effects through additional mutations in one mutant, T33C/L170C. Both T33C/L170C/L217M, with a mutation to counteract atom displacement by the de novo disulfide bridge, and T33C/L170C/A25insG, with a mutation to counteract decreased atom motility, had a higher (P ≤ 0.05) Vmax, kcat and kcat/Km than T33C/L170C at 37°C. These results suggest that de novo disulfide bridges in AppA2 phytase decreased catalytic efficiency partially through alterations in atom motility and displacement of atom coordinates. Research supported by Cornell CAT grant.
Functional analysis the role of the Kaposi sarcoma‐associated virus vBcl‐2 protein in cellular apoptosis (1010.13)
Authors: Yasi Emily , Caruso Brittany , Walker Stephanie , Roecklein‐Canfield Jennifer
Published: June 2021

Kaposi’s sarcoma‐associated herpesvirus (KSHV) is a transforming virus associated with various cancers, the most common being the skin cancer Kaposi’s sarcoma. KSHV viral proteins are known to disrupt many normal cellular pathways in the human host. One specific target is the apoptotic pathway, or the programed cell death process. The cellular Bcl‐2 family proteins, critical components of apoptotic signaling, converge at the mitochondria. This broad family of proteins is comprised of both pro‐ and anti‐apoptotic proteins, sharing homology in four conserved trans‐membrane domains, BH1‐BH4. KSHV encodes a protein, vBcl2, with functional and sequential homology to the anti‐apoptotic Bcl‐2 protein. This gene is expressed in the early stages of the viral lytic cycle and has been shown to affect the progression of apoptotic signaling in order to allow for proper viral replication and virion production. We analyzed the interaction between KSHV’s vBcl‐2 protein and a human protein, BIK, a member of the BH3 only family of pro‐apoptotic proteins. BIK has significant antitumor activity, mediated by its BH3 domain. Mutant constructs of both the viral vBcl‐2 protein and the human BIK protein were screened in a yeast two‐hybrid protein interaction assay to determine the critical residues required for interaction. Using fluorescent microscopy we determined the cellular localization of both wild type and mutant BIK proteins with respect to the vBcl2 protein. Understanding more fully the interaction between vBcl‐2 and BIK can illuminate the role vBcl‐2 and the BH3 only family of proteins play in the viral pathway to cancer, providing new information about the process of tumor development.
Hyperglycemia induces apoptotic signaling in first trimester cytotrophoblast cells (1010.6)
Authors: Leonard Dean , Horvat Darijana , Beeram Madhava , Allen Steven , Meininger Cynthia , Zawieja David , Kuehl Thomas , Uddin Mohammad
Published: June 2021

Objective: Diabetes mellitus is a risk factor for preeclampsia (preE). The mechanism contributing to this effect is not well known. It is evident that dysfunction of cytotrophoblast cells (CTBs) plays a role in the pathogenesis of preE. We have reported that hyperglycemia downregulates factors for invasion and induces an anti‐angiogenic milieu in CTBs. This study assessed the apoptotic signaling in hyperglycemia‐induced CTBs dysfunction. Methods: Human CTBs were treated with 100, 150, 200, 300 or 400 mg/dL glucose for 48h. Some cells were pretreated with p38 inhibitor (SB203580) or peroxisome proliferator‐activated receptor gamma (PPARγ) ligand (rosiglitazone). Thereafter, cell lysates were utilized to measure Bax, Bcl2, Cox‐2 and PPARγ expression and p38 MAPK phosphorylation by western blot. Comparisons were performed using ANOVA with Duncan’s post hoc test. Results: The expression of Bax (2.5 fold), Bcl2 (1.8 fold), Cox‐2 (2.1 fold) and PPARγ (1.8 fold) and p38 MAPK phosphorylation (1.7 fold) were up‐regulated (p<0.05) in CTBs treated with 蠅150 mg/dL glucose compared to basal (100 mg/dL). SB203580 or rosiglitazone pretreatment attenuated glucose‐induced stress and apoptotic signaling. Conclusions: Exposure of CTBs to hyperglycemia induces apoptotic signaling. Attenuation of this signaling by SB203580 or rosiglitazone pretreatment suggests involvement of p38 and PPARγ in this signaling cascade.

Grant Funding Source: N/A
Y14 positively regulates TNF‐α induced NF‐κB transcriptional activity via interacting RIP1 and TRADD beyond an exon junction complex protein (1012.8)
Authors: Matsuda Tadashi , Togi Sumihito , Kon Shigeyuki , Sekine Yuichi , Muromoto Ryuta
Published: June 2021

Although Y14 is known to be a component of the exon junction complex, we previously reported that Y14 regulates IL‐6‒induced STAT3 activation. In this study, we showed that endogenous Y14 positively regulated TNF‐α‒induced IL‐6 expression in HeLa cells. Small interfering RNA‒mediated Y14‐knockdown reduced TNF‐α‒induced and NF‐κB‒mediated transcriptional activity, phosphorylation/degradation of IκBα, and nuclear localization of NF‐κB/p65. As in the case of IL‐6 stimuli, Y14 enhanced TNF‐α‒induced STAT3 phosphorylation, which is important for its nuclear retention. However, our manipulation of Y14 expression indicated that it is involved in TNF‐α‒induced IL‐6 expression via both STAT3‐dependent and ‐independent mechanisms. We screened signaling molecules in the TNF‐α‒NF‐κB pathway and found that Y14 endogenously associated with receptor‐interacting protein 1 (RIP1) and TNFR‐associated death domain (TRADD). Overexpression of RIP1, but not TRADD, restored TNF‐α‒induced NF‐κB activation in Y14‐knockdown cells, and Y14 overexpression restored TNF‐α‒induced NF‐κB activation in TRADD‐knockdown cells, but not in RIP1‐knockdown cells, indicating that Y14 lies downstream of TRADD and upstream of RIP1. Of importance, Y14 significantly enhanced the binding between RIP1 and TRADD, and this is a possible new mechanism for Y14‐mediated modification of TNF‐α signals. Although Y14 associates with MAGOH in the exon junction complex, Y14’s actions in the TNF‐α‒NF‐κB pathway are unlikely to require MAGOH. Therefore, Y14 positively regulates signals for TNF‐α‒induced IL‐6 production at multiple steps beyond an exon junction complex protein