bluuebunny/crossref_metadata_2025 huggingface dataset:
Meclizine is not an inverse agonist or antagonist of human constitutive androstane receptor
Authors: Lau Aik Jiang , Yang Guixiang , Rajaraman Ganesh , Baucom Christie C. , Chang Thomas K. H.
Published: June 2021

Certain tissues express both pregnane X receptor (PXR) and constitutive androstane receptor (CAR), and various target genes are cross‐regulated by these receptors. Meclizine was reported to be an inverse agonist and antagonist of human CAR (hCAR) and an activator of human PXR (hPXR) in cell‐based reporter gene assays. Therefore, activation of hPXR by meclizine may attenuate its apparent hCAR inverse agonistic effects. To test this hypothesis, we investigated whether meclizine would still be capable of suppressing hCAR target gene expression (CYP2B6) in cultured human hepatocytes, which are known to express hCAR and hPXR. Meclizine (0.03–60 μM) did not decrease constitutive CYP2B6 mRNA expression or attenuate hCAR agonist‐mediated increase in CYP2B6 mRNA and CYP2B6‐catalyzed bupropion hydroxylation levels. These findings reflect hPXR agonism and the lack of hCAR inverse agonism and antagonism by meclizine, which were assessed by a reporter gene assay and a mammalian two‐hybrid assay in transfected HepG2 cells. Control experiment indicated that PK11195, which is a hCAR inverse agonist and antagonist, decreased constitutive and hCAR agonist‐mediated hCAR activity. In conclusion, meclizine does not act as a hCAR inverse agonist or antagonist in human hepatocytes. Therefore, it is not appropriate to use this drug as a pharmacological tool to study hCAR function in this cell type.[Supported by CIHR and MSFHR]
Insulin growth factor 2 mRNA binding protein 1 (IGF2BP1) regulates translation of the multidrug resistance protein 2 (MRP2) by binding to its 5′‐untranslated region (5′UTR)
Authors: Vore Mary , Zhao Tianyong , Zhang Yanbin , Yan Baoxiang , Thompson Sunnie R
Published: June 2021

MRP2 is a member of the ATP binding cassette superfamily (ABC) of efflux transporters and mediates the ATP‐dependent efflux of glucuronide and glutathione conjugates from liver, kidney and intestinal cells. The MRP2 5′UTR was investigated for its regulation of downstream gene expression. Both transient gene expression in HepG2 cells and in vitro translation assays of MRP2 5′UTR‐luciferase constructs showed that a 35 nt RNA motif in the MRP2 5′UTR stimulates downstream open reading frame (ORF) translation. One, 2, 3 and 5 copies of the 35 nt motif were inserted between renilla and firefly luciferase ORFs in bicistronic constructs and transfected into HepG2 cells, where analysis of gene expression showed that the 35 nt motif enhanced translation of the 3′ cistron without affecting its mRNA expression. In vitro translation assays of the bicistronic transcripts showed that the 35 nt motif increased translation of the 3′ cistron, consistent with potential Internal Ribosome Entry Site (IRES) activity of the 35 nt RNA motif. Using RNA EMSA, affinity purification and Mass Spectrometry, IGF2BP1 was identified as a protein that bound to the 35 nt RNA motif, while over‐expression of IGF2BP1 in HepG2 cells increased MRP2 translation. Thus, IGF2BP1 stimulates cap‐dependent and cap‐independent translation initiation through binding to the MRP2 5′UTR 35 nt motif.
Immunological Effects of Gulf Oil Spill: Crude Oil, COREXIT® EC9500A Dispersant and Oil/Dispersant Mixtures
Authors: Anderson Stacey E , Franko Jennifer , Lukomska Ewa , Frasch H Fred , Barbero Ana M , Munson Albert E , Meade B Jean
Published: June 2021

Workers involved in the gulf oil spill containment and cleanup efforts have reported health concerns including pulmonary distress, dermatological problems, and an increased incidence of infection. These concerns have prompted toxicological evaluation of samples recovered from the gulf oil spill including: COREXIT® EC9500A (CR), crude oil and crude oil/CR mixtures. Potential for hypersensitivity and immune suppression were evaluated in dermal exposure murine models. Dermal exposure to 50 μl of CR for 3 days induced irritation and significant increases in lymphocyte proliferation at concentrations of 10% and greater, while exposure to oil induced significant elevations in lymphocyte proliferation at concentrations of ≥50%. No alterations in serum IgE were observed after exposure to either test article. Exposure to crude oil was also found to be immunosuppressive. The spleen IgM response to sheep red blood cells was significantly inhibited when mice were exposed to 50 μl at concentrations of ≥25% crude oil for 14‐days. Increased liver and decreased spleen weights were also documented. Mixtures of crude oil and CR did not enhance either effect. Consistent with these results, CR did not enhance in vitro epidermal penetration of naphthalene. These studies suggest potential immunological effects from dermal exposure to crude oil and CR.
A novel role of macrophages in turning‐off endotoxin‐induced neutrophiliclung inflammation and thereby preventing acute lung injury associated withGram negative bacteria
Authors: Chava Koteswara Rao , Mohammad Tauseef , Mehta Dolly
Published: June 2021

Acute lung injury (ALI) associated with Gram‐negative bacterial sepsis is a life threatening neutrophilc inflammatory lung (NLI) disease. In this study, we used transgenic CD11b‐diphtheria toxin receptor (DTR) mice whereby administration of Diphtheria toxin (DT) selectively depletes macrophages/monocytes by inducing apoptosis to investigate the role of macrophage in NLI. We confirmed that DT injection significantly depleted F4/80+ cells in the BAL fluid and markedly reduced monocyte in the blood without altering other cell type. Control mice and DT mice were then exposed to nebulized LPS for an hour, after which lung inflammation was determined at 4, 24 and 48 hrs post LPS challenge. We show that WT lungs develop edema and NLI within 4 hrs after LPS challenge and which is resolved within next 24 hrs, reached near the basal levels in 48 hrs. Surprisingly, DT mice displayed delayed neutrophilic inflammation i.e. there was no lung edema and neutrophil infiltration at 4 hrs after LPS challenge and they were evident at 24 hrs, and NLI did not resolve even at 48 hrs. Further CD11 mice were subjected to LPS inhalation and then DT was given. We show that depletion of macrophages post LPS‐induced injury produce persistent NLI and lung edema. These data suggest that macrophages play a vital role in the autoregulaory loop of resolution of inflammatory process and thereby play a fundamental role in preventing the lung injury.
L‐Carnitine Attenuates Doxorubicin‐Induced hepatotoxicity Through Antioxidant Signaling
Authors: AL‐SHABANAH OTHMAN A , Sayed‐Ahmed Mohamed M
Published: June 2021

This study examined the mechanism whereby carnitine supplementation attenuates doxorubicin‐induced hepatotoxicity in rats. Male Wistar albino rats were divided into six groups: Rats in group 1 were injected I.P. with normal saline and served as control; rats in group 2, 3 and 4 were injected every other day with doxorubicin (3 mg/kg, i.p.), to obtain treatments with cumulative doses of 6, 12, and 18 mg/kg. Rats in the fifth group were injected with L‐carnitine (200 mg/kg, i.p.) for 10 consecutive plus a cumulative dose of doxorubicin (18 mg/kg). Administration of doxorubicin resulted in a significant dose‐dependent increase in serum markers of liver function, and tissue levels of thiobarbituric acid reactive substances, nitric oxide production, and protein carbonyl content. Moreover, doxorubicin significantly decreased gene expression and activity of superoxide dismutase, glutathione peroxidase, glutathione‐s‐transferase, and catalase in liver tissues. Interestingly, L‐carnitine supplementation completely reversed the biochemical and gene expression levels induced by doxorubicin to the control values. In conclusion, data from this study suggest that L‐carnitine supplementation prevents the development of DOX‐induced hepatocellular damage by decreasing oxidative stress and preserving both the activity and gene expression levels of antioxidant enzymes.
Effects of metformin on spontaneous and cholinergic contractions of the rat bladder
Authors: Peuler Jacob D. , Senn Christopher J. , Phelps Laura E.
Published: June 2021

Diabetes causes autonomic neuropathy which impairs bladder parasympathetic release of acetylcholine (ACh) during the active phase of micturation, resulting in inadequate voiding of urine. Diabetes also causes incontinence which is a problem during storage of urine in the resting bladder. Previously we reported that a high (10 millimolar) concentration of the antidiabetic drug metformin can rapidly enhance rat bladder contractile responses to ACh (FASEB J. 22:916.6, 2008).We tested whether long‐term (4‐hour) pretreatment with a lower, more therapeutically‐relevant concentration of metformin (100 micromolar) would do the same. First, after 4 hours the 100 micromolar metformin exerted contrasting effects on cholinergic (ACh‐induced) vs non‐cholinergic (e.g. K‐induced) contractions of isolated rat bladder tissues (p < 0.05), tending to enhance the former (by 31–51%) and inhibit the latter (by 9–18%). Second, during the pretreatment period the 100 micromolar metformin interacted with pretreatment time (p < 0.05) to specifically increase amplitudes of resting spontaneous contractile activity (after 2–4 hours) without altering the midpoint level of resting contractile tone or the frequency of the spontaneous activity. These findings may be relevant to potential effects of metformin on the abovementioned clinical problems in diabetic patients. Support: Midwestern University.
Re‐examination of role of α2‐adrenergic receptors in pithed rat pressor responses
Authors: Docherty James R
Published: June 2021

Yohimbine has been employed in a number of studies to identify α2‐adrenergic components to nerve and agonist induced pressor responses in the pithed rat. However, yohimbine has moderate affinity for α1D‐receptors and these have also been reported to mediate pressor nerve responses in the pithed rat. We have reexamined the actions of yohimbine in the pithed rat. Pressor nerve responses to 1 Hz stimulation were markedly inhibited by the α1A‐receptor antagonist RS 100329 (0.1 mg/kg) and by the α1D‐receptor antagonist BMY 7378 (0.1 mg/kg). Yohimbine (0.1 mg/kg) significantly increased, but yohimbine (1 mg/kg) significantly reduced, pressor nerve responses. Following BMY 7378 (0.1mg/kg), yohimbine (1 mg/kg) did not produce any further inhibition of pressor nerve responses. The α2A‐adrenoceptor antagonist BRL 44408 (1mg/kg) did not inhibit pressor nerve responses. Yohimbine or BRL 44408 (both 1mg/kg) significantly shifted, but BMY 7378 (1mg/kg) failed to affect, the pressor potency of the α2‐adrenergic agonist xylazine. Hence, responses to exogenous agonists in the pithed rat involve α2‐receptors but there is no clear evidence for the involvement of α2‐receptors in pressor nerve responses.
Regulation of endothelial permeability by protein S‐nitrosation and de‐nitrosation
Authors: Mujica Patricio E. , González Francisco G. , Durán Walter N.
Published: June 2021

Venular hyperpermeability to macromolecules is a hallmark of inflammation. Platelet‐activating factor (PAF) is a pro‐inflammatory agent that increases permeability by stimulating endothelial nitric oxide synthase (eNOS) activity and nitric oxide (NO) production, which can either activate soluble guanylyl cyclase (sGC) and/or induce protein S‐nitrosation. Because the thioredoxin (Trx)/thioredoxin reductase (TrxR) system can reverse S‐nitrosation, we studied the significance of S‐nitrosation and the Trx/TrxR system on endothelial permeability in human microvascular endothelial cells (HMVEC) and in hamster cheek pouch microvascular endothelial cells (HCPMEC). We evaluated PAF‐induced protein S‐nitrosation using immunofluorescence, Trx/TrxR expression using immunofluorescence and western‐blot, and the efficacy of TrxR inhibitors on PAF‐induced hyperpermeability. PAF produces maximum protein S‐nitrosation at 1 and 15 min of stimulation. The Trx/TrxR system is expressed in HMVEC and HCPMEC. Inhibition of Trx/TrxR increased both basal and PAF‐stimulated permeability. Our results suggest an important role for the regulatory enzymes that control the levels of S‐nitrosation in the regulation of microvascular permeability. (Supported by NIH grants 5RO1 HL088479 and 5RO1 HL070634).
Activation of Ca‐dependent Potassium Channels Improves Impaired EDHF Signaling in Uterine Arteries during Diabetic Pregnancy
Authors: Gokina Natalia I , Bonev Adrian , Gokin Alexander , Phillips Julie
Published: June 2021

Diabetic pregnancy is associated with adverse fetal complications in part due to impaired uteroplacental blood flow. In this study we define: (1) diabetes‐induced changes in uterine arterial responses to ACh and NS309; (2) the effect of NS309 on EDHF‐mediated dilatation; and (3) the dependence of this effect on changes in endothelial cell (EC) [Ca2+]i.

Diabetes was induced in rats by i.p. injection of streptozotocin (STZ) on day 2 of pregnancy. NS309 and ACh were tested in uterine arteries pre‐constricted with phenylephrine (PE). A subset of the vessels was pre‐treated with 1 μM NS309 before PE and ACh application. [Ca2+]i responses were studied using fura‐2. MP was recorded with glass microelectrodes. Apamin and charybdotoxin (CTX) were tested in single SMCs.

NS309‐induced dilatation, hyperpolarization and [Ca2+]i were reduced in diabetes. SMC K+ currents were insensitive to apamin and CTX. Vasodilation to ACh was impaired in diabetes (22 ± 8 % vs. 78 ± 6 % in controls at 1 μM ACh), but was markedly enhanced with 1 μM NS309 (75 ± 6 %). NS309 produced no significant changes in ACh‐induced EC [Ca2+]i responses.

Impaired EDHF‐dependent dilatation in diabetes can be markedly enhanced by activation of endothelial SKCa/IKCa channels without changes in EC [Ca2+]i. Openers of these channels may serve as a pharmacotherapy for improvement of maternal uteroplacental blood flow in diabetic pregnancies.

Supported by NIH HL088245
DIVERGENCE BETWEEN ARTERIAL PERFUSION AND FATIGUE RESISTANCE IN SKELETAL MUSCLE IN THE METABOLIC SYNDROME
Authors: Frisbee Jefferson C. , Goodwill Adam G. , Butcher Joshua T. , Olfert I. Mark
Published: June 2021

The obese Zucker rat (OZR) exhibits many vascular alterations, although the integrated impact on active hyperemia remains unclear. Pressor responses and skeletal muscle perfusion were assessed in lean Zucker rats (LZR) and OZR during adrenergic stimulation (phenylephrine), challenge with thromboxane (U46619) and endothelium‐dependent dilation (methacholine). OZR were hypertensive vs. LZR, but this was abolished by adrenoreceptor blockade (phentolamine); pressor responses to U46619 were similar and were abolished by the PGH2/TxA2 receptor antagonist, SQ‐29548. Depressor responses to methacholine were impaired in OZR, but improved by the antioxidant TEMPOL. Active hyperemia to gastrocnemius muscle was restrained by adrenergic constriction in OZR, although this diminished with increased VO2. O2 extraction, reduced in OZR across levels of metabolic demand, was improved by TEMPOL or SQ‐29548; phentolamine did not impact extraction and neither TEMPOL nor SQ‐29548 improved perfusion. While VO2 and muscle performance were reduced in OZR, treatment with all three agents improved outcomes; individual agents were less effective. These data suggest that contributions of vascular dysfunction to perfusion, VO2 and muscle performance are spatially distinct, with adrenergic constriction impacting proximal resistance and endothelial dysfunction impacting distal exchange. (NIH DK64668, RR2865AR)
Does G‐protein Coupled Receptor Activation Enhance Cerebral Arterial Mechanosensitivity
Authors: Anfinogenova Yana , Welsh Suzanne E Brett , Welsh Donald G
Published: June 2021

This study determined whether the mechano‐sensitivity of a TRPC current in cerebral arterial smooth muscle could be enhanced by G‐protein coupled receptors (GPCRs) in a manner that facilitates myogenic tone development. An initial RT‐PCR analysis revealed the expression of TRPC1, 3, 6 & 7 mRNA in smooth muscle cells isolated from anterior cerebral arteries. Whole cell patch clamp electrophysiology subsequently confirmed the presence of a doubly rectifying current that displayed properties characteristic of a heteromultimeric TRPC3/6/7 channel. In particular, this non‐selective cation current was blocked by micromolar trivalents (Gd3+ and Ln3+) but insensitive to Cl− inhibitors (DIDS, niflumic acid, tamoxifen and flufenamic acid) and amiloride. GPCR agonists (UTP and U46619) augmented the activity of the TRPC‐like current as did a mechano‐stimulus like hyposmotic challenge (300 to 200 mOsm). While GPCR agonists can directly modulate TRPC‐like currents, this study found no evidence to suggest that they facilitate mechano‐sensitivity. The preceding findings were consistent with functional work which revealed that while GPCR agonists cause constriction, they do not enhance the myogenic reactivity of cerebral arteries. In closing, our findings suggest, in contrast to previous HEK expression studies, that GCPR activation does not enhance the mechano‐sensitivity of TRPC‐like currents nor the myogenic sensitivity of intact cerebral arteries. Supported by NSERC.
The effects of melatonin and/or treadmill exercise on ischemic/reperfusion brain damage in focal cerebral ischemic rats
Authors: Lee Minkyung , Lee Seunghoon , Shin Jinhee , Hong Yonggeun
Published: June 2021

Acute ischemic stroke results from sudden decrease or loss of blood supply to an area of the brain, resulting in a coinciding loss of neurological function. The aim of this study was carried out to examine the effect of melatonin and/or treadmill exercise on improvement of neurological functions in focal cerebral ischemic rats. Following the MCAo, the 8 week old Sprague‐Dawley (SD) rats were treated twice daily with melatonin (10 mg/kg) at 7 and 19 hrs, trained twice daily with treadmill exercise (20 m/min, 6 days/week) at 17 and 22 hrs and control group animals were provided with the same intervention. After 1 week of MCAo, the rat brains were collected for gene analysis and histological measurement. Histological data showed the significant reduction in the loss of neural cells on the primary motor cortex in melatonin treated rats. The gene expression showed the upregulation of HIF‐1α. Likewise, the neuronal cell and oligodendrocyte progenitor cell specific marker, MAP2 and NG2 were significantly upregulated in melatonin or treadmill exercise MCAo rats (p<.01). However, melatonin with treadmill exercise combined rat was not significantly changed. Based on the present study, melatonin or treadmill exercise might be the possible therapeutic approaches. Then, excessive intervention might be lead to negative effect on acute ischemic stroke.
Genetic basis of impaired myogenic response and cerebral blood flow autoregulation FHH rats
Authors: Roman Richard J. , Williams Jan M. , Reddy Pabbidi M. , Burke Marilyn , Juncos Julio , Renic Maria , Harder David
Published: June 2021

This study examined the effect of transfer of a small region from chromosome 1 (Chr 1) of the Brown Norway (BN) rat (FHH.1BN AR+ strain) into the Fawn Hooded Hypertensive (FHH) rat genetic background on autoregulation of cerebral blood flow (CBF) and the myogenic response of isolated perfused middle cerebral arteries (MCA). FHH rats exhibited poor autoregulation measured by laser Doppler flowmetry and CBF rose by 50% when perfusion pressure was increased from 100 to 150 mmHg. Autoregulation of CBF was completely restored in the AR+ strain. The diameter of isolated middle cerebral artery (MCA) of FHH rats increased by 10% when transmural pressure was increased from 40 to 140 mmHg. In contrast, the diameter of the MCA fell from 127±16 to 65±14 μm in the AR+ strain when pressure was increased over this range. FHH rats exhibited a much more prolonged hyperemia and larger infarct than that seen in the AR+ strain following ischemia/reperfusion. These results indicate that a gene that plays a critical role in the myogenic response of cerebral arteries lies within this critical 2.6 Mb region containing just 11 genes and that transfer of this region restores autoregulation of CBF and reduces infarct size following ischemia/reperfusion injury. The impaired myogenic response is likely due to hyperpolarization of VSM cells due to increased BK channel activity. NIH HL36279, DK79306, HL59996.
Functional evidence of TRPV4‐mediated Ca2+ signals in cortical astrocytes
Authors: Dunn Kathryn M , Bonev Adrian D , Nelson Mark T
Published: June 2021

A rise in intracellular calcium ([Ca2+]i) in astrocytes is required to communicate the energy demands of neurons to the cerebral microcirculation to precisely match local cerebral blood flow to neuronal metabolism. This “neurovascular coupling” is essential for neuronal viability and homeostasis. A moderate rise in [Ca2+]i in the perivascular “endfoot” terminals of astrocytes in response to neuronal activation produces vasodilation of the adjacent parenchymal arteriole, whereas high endfoot [Ca2+]i can produce vasoconstriction. Thus, precise regulation of astrocytic [Ca2+]i is necessary for proper function of the cerebral microcirculation. Transient receptor potential vanilloid 4 (TRPV4) channels are Ca2+‐permeant plasma membrane cation channels that participate in intracellular Ca2+ signaling in a variety of cell types. While there is some immunohistochemical evidence for TRPV4 expression, there is no evidence of functional TRPV4 channels in astrocytes. Here we provide evidence of TRPV4‐mediated Ca2+ oscillations in endfeet of cortical astrocytes obtained by multiphoton confocal microscopy in mouse brain slices. Activation of TRPV4 channels with the selective agonist GSK1016790A concentration‐dependently increased the amplitude of Ca2+ oscillations in astrocytic endfeet. Blocking neuron‐to‐astrocyte communication did not inhibit this effect, suggesting a direct effect of TRPV4 activation on astrocytic endfoot Ca2+. Additionally, the effect of TRPV4 activation on endfoot Ca2+ oscillations was blocked by the selective TRPV4 antagonist HC067047. These results provide evidence of functional TRPV4 channels in cortical astrocytes, and suggest that TRPV4 may play a role in regulating local cerebral blood flow through effects on astrocytic endfoot Ca2+. This study was supported by National Heart, Lung, and Blood Institute grant HL44455.
Age related effects on pathophysiology of chronic cerebral hypoperfusion
Authors: Wappler Edina Amalia , Nemeth Istvan B , Bari Ferenc , Nagy Zoltan
Published: June 2021

We investigated the effects of age on cerebral hypopoperfusion in 6‐, and 18 months old male Wistar rats after 3 months of bilateral common carotid artery occlusion (BCCAO). Brain pathology was investigated on coronal sections using Nissle staining (cell number and satellitosis) and immunohisotology for microtubule‐associated protein 2 (MAP‐2), synaptophysin, and growth associated protein 43 (GAP‐43). Increased satellitosis was found in the hippocampus and the frontal and parietal cortices with decreased cell number in the same regions in young animals. Decreased dendritic and synaptic density was observed in every investigated region in the young hypoperfused brain; however, some groups of neurons had higher synaptophysin expression than others. Old control animals had similar changes to young hypoperfused animals, but the, old hypoperfused rats had more serious changes that those of young BCCAO animals. In summary, cerebral hypoperfusion in young animals induces several changes in the brain that are similar to that of non‐BCCAO old rats. Additionally, hypoperfusion at a later age causes the same pattern of changes but of more serious nature than in young BCCAO rats. We conclude that cerebral hypoperfusion seems to be an important factor which accelerates the deterioration associated with brain aging.
The estrogen metabolite, 2‐MeOH, induces arterial dilation by increasing BK channel expression
Authors: Jordan Constance Renee , Hill Brent J.F.
Published: June 2021

Abnormal coronary blood flow is associated with inhibition of Ca2+‐activated K+ (BK) channels and/or stimulation of voltage‐gated Ca2+ channels. Our lab previously demonstrated that the estrogen metabolite, 2‐methoxyestradiol (2‐MeOH) inhibits Ca2+‐dependent contractions. The objective of this study was to determine if 2‐MeOH can induce vasodilation via BK channels. This study used right coronary arteries from hearts obtained from female pigs. The arteries were cut into 4 mm rings to measure isometric tension in response to a depolarizing KCl solution. The rings were exposed to 2‐MeOH (10 nM to 10 μM) or its vehicle (ethanol) in the presence and absence of the BK blocker, tetraethylammonium (TEA, 1 mM). Physiological [2‐MeOH] increased the KCl‐induced contraction in the presence of TEA by 2‐fold. Coronary arteries were also cut into longitudinal strips to determine BK protein expression using Western blots. The strips were incubated (37°C at 5% CO2) for 24 hrs in 2‐MeOH (10 nM to 1 μM) or ethanol. Densitometric analyses of the blots indicate that 2‐MeOH increased BK channel expression greater than 2‐fold. Data from our study indicate that physiological concentrations of 2‐MeOH induce vasodilation by increasing the protein expression of the BK channel. Support: NCRR of the NIH, Grant #P20 RR‐16460.
Cell Division Cycle 7 is Essential for the Initiation of Transforming Growth Factor‐β‐induced Smooth Muscle Differentiation
Authors: Chen Shiyou , Shi Ning
Published: June 2021

The objective of this study was to determine if cell division cycle 7 (Cdc7) plays a role in smooth muscle cell (SMC) differentiation. Cdc7 stimulates cell proliferation by facilitating the initiation of DNA replication. We found that Cdc7 is also important for the initiation of SMC differentiation. TGF‐β induced an increased Cdc7 expression and phosphorylation in the initial phase of SMC differentiation. Cdc7 specific inhibitor or shRNA suppressed TGF‐β‐induced expression of SMC early marker genes α‐SMA, SM22α and calponin. Cdc7 overexpression, on the other hand, enhanced the marker expression in a dose‐dependent manner. Cdc7 function in SMC markers was independent of DBF4, the catalytic subunit of Cdc7 critical for cell proliferation, suggesting that Cdc7 mediates SMC differentiation through a mechanism distinct from that of cell proliferation. Cdc7 appeared to be involved in activating SMC gene transcription. ShRNA knockdown of Cdc7 inhibited TGF‐β‐induced SMC marker promoter activity, while overexpression of Cdc7 activated the promoter activities. Importantly, CArG mutation diminished Cdc7‐induced activation of α‐SMA and SM22α promoters, suggesting that Cdc7‐induced SMC differentiation is SRF‐dependent. Taken together, our data demonstrate a novel role for Cdc7 in regulating the early phase of TGF‐β‐induced SMC differentiation. (Funding source: NIH R01HL093429)
Characterization of a Novel in Vitro Model for Smooth Muscle Differentiation
Authors: Chen Shiyou , Guo Xia
Published: June 2021

The objective of this study was to characterize a novel in vitro model for SMC differentiation from mesenchymal stem cells (huMSCs) derived from human embryonic stem cells. We found that huMSCs were differentiated into SMC phenotype by transforming growth factor‐β (TGF‐β) and retinoid acid (RA) in a dose‐ and time‐dependent manner, as demonstrated by the expression of SMC specific genes including smooth muscle α‐actin, SM22α, calponin, and smooth muscle myosin heavy chain. Both TGF‐β and RA induced huMSC to become an elongated, spindle‐shaped contractile morphology. Importantly, the SMCs differentiated from huMSC contracted in response to the stimulation of muscarinic agonist carbachol and KCl. Additional studies demonstrated that TGF‐β‐induced SMC differentiation from huMSC was SRF‐dependent because CArG box mutation diminished α‐SMA and SM22α promoter activities. Taken together, this simple and efficient cell model system is likely to serve as a valuable tool in understanding molecular mechanisms of human SMC differentiation during vascular development. It may also become a potential cell source for therapeutic application to treat vascular diseases. (Funding source: NIH R01HL093429)
Interaction of RAGE with focal adhesion molecules during alveolar epithelial‐mesenchymal transition
Authors: Ehrhardt Carsten , Medina Carlos , Buckley Stephen Timothy
Published: June 2021

Ezrin/radixin/moesin (ERM) proteins provide a linkage between F‐actin and membrane proteins. RAGE also associates with components of the cytoskeleton. This study tested the hypothesis that RAGE‐ERM interaction plays a role in regulating EMT‐associated structural changes in alveolar epithelial cells.

A549 cells were stimulated with TGF‐β1 for 72 h. The interaction of ERM proteins and F‐actin was investigated by immunofluorescence microscopy (IFM) and Western blot (WB). RAGE and sRAGE expression and localisation was determined by WB and IFM. MMP‐9 activity was evaluated using gelatin zymography. Co‐immunoprecipitation studies and IFM was performed to assess the association of ERM and RAGE, and ERM and CD44.

Exposure to TGF‐β1 gave rise to re‐distribution of ERM to the cell periphery and co‐localisation with EMT‐associated actin stress fibres. Concurrently, ERM underwent phosphorylation. Expression of RAGE was diminished, coupled with release of its soluble isoform (sRAGE) in a MMP‐9 dependent manner. IFM and co‐IP identified strong association between ERM and RAGE under basal conditions, which was disrupted when challenged with TGF‐β1, with pERM forming a complex with membrane‐linked CD44.

These data indicates that disruption of the ERM‐RAGE complex may be a crucial step in actin microfilament re‐arrangement during cytokine‐induced EMT of human alveolar epithelial cells.
Biomechanical analysis of the human head‐neck‐shoulder complex: Asymmetry as a natural experiment
Authors: Osborn Michelle L. , Homberger Dominique G.
Published: June 2021

The unique human mastoid processes and clavicles are a consequence of the shoulder girdle being suspended from the skull. When one arm is used, the upper trapezius and sternocleidomastoid muscles contract to counterbalance the added weight on that shoulder. Core muscles acting on the atlanto‐occipital and cervical joints synchronously contract to prevent retroflexion of the head. Our hypothesis was tested by a natural experiment based on the asymmetry of biomechanically relevant paired skeletal features in right‐handed males. Our biomechanical free‐body diagram analyses show that asymmetrical muscle forces act on the mastoid processes, skull, and clavicles when one arm is favored and predict that these forces, over time, stimulate different bone growth at their attachment sites. Quantitatively, our data show that at least 70% of the individuals had a wider right and a longer left mastoid process; a higher and/or more robust right superior nuchal line; a broader left scapula; and/or a greater right humerus diameter. Qualitatively, however, each individual displayed a unique combination of at least two of these features indicative of right‐handedness. Individual differences are probably caused by long‐term force regimes due to idiosyncratic movements and athletic or professional activities.

Funding: LSU Robert C. West Fund to MLO; LSU Foundation to DGH

Grant Funding Source: LSU Foundation
Characterization of a trinitrobenzene sulphonic acid (TNBS)‐induced mouse model of Periodontitis
Authors: San Miguel Symone M. , Tillberg Connie M. , Opperman Lynne A. , Svoboda Kathy K.H.
Published: June 2021

The 2,4,6‐trinitrobenzenesulfonic acid (TNBS)‐murine model has been used for immunopathogenesis of inflammatory bowel disease. The use of TNBS induces severe colonic inflammation when administered intrarectally in mice. The colitis which results from this procedure also presents clinical and histopathological findings common in periodontitis. This study will characterize parameters needed for successful induction of TNBS‐colitis as well as monitoring periodontal disease levels among 20 adult CD1 mice. The experimental groups were given 25 μL gingival injections of TNBS and the sham groups were given 25 μL saline solution (0.9% NaCl) in each quadrant of both maxillary and mandibular arches. Each group received 50 μL of TNBS or saline solution orally thrice weekly and was given both a gel diet in addition to solid mouse pellets. Oral and systemic tissue samples were collected at 2, 4, 6, 10 weeks. Radiographic examinations of both arches were taken to supplement histologic evaluations of changes in tooth supporting structures. Soft tissue samples were obtained from the large intestines and analyzed for the extent of systemic inflammation caused by TNBS. Colitis was present from second week on, with TNBS treatment. Radiographic and histochemical analyses revealed some changes in the alveolar bone pattern. Morphologic changes were noted in the colonic mucosa with extensive inflammatory infiltrates and some ulceration. This novel approach can help elucidate the relationship between alveolar bone loss and systemic manifestation common to gingival and periodontal disease for future therapeutic purposes.

Grant Funding Source: PerioSciences, LLC
Histological and lectin histochemical characterization of the efferent ductules in dromedary camels (Camelus dromedarius)
Authors: Hafez Shireen Abdelgawad , El‐Shafey Samir , Caceci Thomas
Published: June 2021

To study the microscopic anatomy of the ductuli efferentes in the dromedary camel, tissue sections were stained with H&E and a combination of Masson's trichrome and Verhoeff's stains. Efferent ductules showed variations in diameter. Two morphologically distinct types of epithelium were detected, both including ciliated and non‐ciliated cells. Close to the rete testis, the lining epithelium was a low pseudostratified columnar. Close to the epididymal duct the epithelium varied in height from place to place similar in appearance to the initial portion of the epididymal duct; however, the nuclei maintained their specific pattern pertaining to the efferent ductules and not to the initial part of the epididymal duct. Each tubule was surrounded by a delicate circular layer of smooth muscle cells with loose connective tissue interspersed between the tubules. Histochemical characteristics of epithelial cells were investigated using an array of six lectins: Con‐A, DBA, MAA, SBA, SNA‐1, and WGA. Specific sugar residues of MAA and SNA‐1 were not detected. Carbohydrate binding of Con‐A and WGA was readily detected. Binding of SBA and DBA was mild. Regional variations in binding of these lectins are discussed and they suggest a high degree of functional compartmentalization of the epithelium of the efferent ductules.

Grant Funding Source: Professional Development
T7 DNA Polymerase Rescues the DNA Loading Defect of the A257T Linker Region Mutant of T7 Helicase‐Primase Protein
Authors: Patel Gayatri , Johnson Daniel S , Pandey Manjula , Yu Xiong , Egelman Edward H , Wang Michelle D , Patel Smita
Published: June 2021

Bacteriophage T7 gp4 is a bifunctional primase‐helicase that oligomerizes into ring‐shaped hexamers around DNA in the presence of dTTP. The helicase and primase activities in T7 gp4 reside in two separate domains that are connected by a linker region. The linker region plays an important role in oligomerization and point mutations in the linker region have deleterious effects on the helicase function. In this study, we have investigated the biochemical and structural properties of linker region mutant A257T, which is analogous to the A359T mutant of the human mitochondrial DNA helicase protein Twinkle that is linked to human diseases. We show that A257T gp4 is highly defective in unwinding duplex DNA when measured by ensemble assays. Interestingly, we found that in single molecule unwinding assays, A257T gp4 was almost as active as WT gp4. We show that although A257T gp4 is normal in forming rings in the presence of dTTP, it is critically defective in loading onto the DNA. We discovered that T7 DNA polymerase rescued the DNA loading defect of A257T gp4. We propose that the inability to load on the DNA at efficient rates explains why A257T gp4 does not support T7 phage replication.
Lysine rich C‐terminal domain of KU protein from Mycobacterium smegmatis facilitates DNA end‐joining and regulates DNA interaction
Authors: Kushwaha Ambuj Kumar , Grove Anne
Published: June 2021

KU protein binds to DNA double strand break ends and is required for the non‐homologous end joining (NHEJ) pathway of DNA repair. It is evolutionarily conserved from yeast to humans and a homologue is also encoded by some eubacteria, including Mycobacteria. KU protein from M. smegmatis has a basic C‐terminal tail with PAKK repeats. Here we show that M. smegmatis KU preferentially binds to longer DNA irrespective of the kind of ends (blunt or cohesive) and overhangs (3′ or 5′) as shown by electrophoretic mobility shift assay and competition assay. Further, KU exhibits some sequence preference as indicated by EMSAs. A mutant of KU protein lacking C‐terminal PAKK repeats (TKU) shows enhanced binding affinity to 37 bp linear DNA, Kd = 11.0±1.1 nM as compared to KU (barely detectable complex) and assembles on DNA as a dimer as indicated by its stoichiometric ratio estimated for 37 bp linear DNA. TKU prefers linear DNA over supercoiled DNA as opposed to KU which prefers supercoiled DNA over linear DNA. TKU inhibits end‐joining and protects DNA ends against exonucleolytic cleavage as opposed to KU, which promotes end‐joining and does not inhibit exonucleolytic cleavage. These data indicate that the C‐terminal PAKK repeats of the KU protein facilitates DNA end‐joining and has a regulatory role in DNA interactions.
Metacaspase gene function in the mushroom fungus Schizophyllum commune
Authors: Hanley Matthew , Horton J Stephen
Published: June 2021

The overall goal of this project is to investigate the role of putative metacaspase genes in the fungus Schizophyllum commune by means of gene knockout experiments. We have utilized a strain of S. commune which has had its Ku80 gene knocked out, rendering it unable to integrate DNA via the non‐homologous end joining pathway. This forces transformants to integrate homologously, as required for gene knockout. The gene Scp1 encodes a likely metacaspase, proteins suspected to have activity similar to members of the caspase protein family. A non‐functional version of Scp1 was previously generated by inserting a gene that confers the ability to synthesize tryptophan (Trp1) into the Scp1 coding region. This knockout construct was transformed into a Trp‐, ΔKu80 strain of Schizophyllum commune. It was observed that while some S. commune colonies grew at the rate expected of a typical strain, others took about ten days to become macroscopically visible. It is anticipated that we will be able to confirm the presence of Scp1 knockouts via PCR, and then examine these strains for perturbations in mushroom development. This process of generating knockout constructs and transforming them into S. commune will be carried out for other genes, including the other members of the metacaspase family. Support from a Davenport Summer Research Fellowship is gratefully acknowledged.
Dynamic imaging of cancer invasion, plasticity, and resistance
Authors: Friedl Peter
Published: June 2021

Molecular programs controlling metastatic cancer progression are diverse in different cancers as well as within the microenvironment of a single lesion. Multiphoton microscopy (MPM) has become the method of choice for investigating cell structure and function in tissues and organs, including the invasion and progression of cancer lesions. Using a novel approach of infrared‐excited (IR‐)MPM at wavelengths above 1080 nm that enhances deep tissue microscopy in orthotopic fibrosarcoma and melanoma xenografts, we here show deep collective invasion of hundreds of connected cells. These multicellular masses proliferate and simultaneously move with velocities of up to 200 μm per day along pre‐existing blood vessels but not tumor‐induced neovessels and proliferate (“invasive growth”). Collective invasion strands further maintain invasion and robust survival during otherwise regression‐inducing experimental radiation therapy. Both, primary growth and survival and increased resistance to experimental therapy were ablated by interfering with beta1/beta3 integrins, implicating integrin‐mediated signals as microenvironmental denoinators of response to therapy. In conclusion, intravital deep tumor imaging by IR‐MPM identifies the tumor‐vessel interface as preferred niche of invasive growth, radioresistance and enhance preclinical anti‐cancer therapy.
Protein folding shapes the adaptive value of genetic variation
Authors: Jarosz Daniel F. , Lindquist Susan
Published: June 2021

How can species remain unaltered for long periods yet also undergo rapid diversification? By linking environmental stress to protein homeostasis, the protein chaperone Hsp90 might provide a molecular explanation for both phenomena. This hypothesis has been controversial because the nature, scope, and adaptive value of Hsp90‐contingent traits are uncertain. Using ecologically and genetically diverse yeasts, we find that such traits are common and frequently beneficial. Most have a clear underlying genetic basis in coding or regulatory sequences that are diverse in character. The expression of genetic variation is affected in a broadly similar way by environmental stress and Hsp90 inhibition. Strikingly, both manipulations improve correlations between genotype and phenotype. Our data suggest that this system has influenced the evolution of current genomes by playing a crucial role in determining the adaptive value of genetic variation
A pH‐dependent Switch Regulates Chaperone Activity
Authors: Rajagopal Ponni , Jehle Stefan , Dove Katja , Pierini Chris , Delbecq Scott , Klevit Rachel E
Published: June 2021

Hypoxic conditions often found in cancerous and ischemic cells induce the over‐expression of a non‐ATP dependent stress chaperone, αB‐Crystallin (αB). Investigation of αB's response to hypoxia can help develop therapeutic strategies to combat disease. αB is a polydisperse oligomer, ~ 580 kDa in size and composed of ~28 subunits. We have used solution‐state and solid‐state NMR, SAXS, EM data, and computational methodologies to determine a structural model of the oligomer. NMR data show that there is an increased exposure of substrate binding sites in αB at lower pH (acidification is a consequence of hypoxia) presumably to confer protection to proteins and enhance cellular survival. αB has a conserved α‐crystallin domain flanked by variable N‐ and C‐ terminal regions. The α‐crystallin domain forms a homodimeric (20 kDa) building block of the oligomer and the inter‐subunit interactions of the N‐ and C‐termini leads to higher order oligomers. The truncated α‐crystallin domain has proved to be a good functional mimic of the oligomer and we are using the domain to identify the basis of the pH‐dependent behavior. We have identified critical residues that are responsible for the sharp pH‐dependent switch by determining pKa values and using mutational analysis. Mutations that abrogate this pH‐dependent switch have an adverse effect on the chaperone activity of αB.

Funding from NIH‐1R01EY017370 is acknowledged.
Chronic exposure of clonal pancreatic β‐cells (INS‐1 832/13) to acetoacetate inhibits glucose‐induced insulin secretion
Authors: Burritt Nathan E. , Ferrante Thomas C. , Corkey Barbara E. , Deeney Jude T.
Published: June 2021

Objective

Type 2 diabetes (T2D) occurs when pancreatic β‐cells fail to compensate for insulin resistance. Insulin resistance can be caused by a sustained elevation in insulin secretion, which may develop through a mechanism independent of the well‐established pathway described for fuel‐stimulated secretion. We hypothesize that nutrient availability can alter the redox state (NAD(P)H/NAD(P) ratio) leading to increases in reactive oxygen (or nitrogen) species (ROS) ultimately affecting insulin secretion.

Methods

To test this hypothesis we have incubated clonal pancreatic β‐cells (INS‐1 832/13) in increasing concentrations of acetoacetate (Acoc) (oxidizer) (1–20 mM) or in varying ratios of Acoc and β‐hydroxybutyrate (reducer) (βOHB) for 18–48 hrs in order to modify the REDOX capacity of the cell. Insulin secretion (HTRF), O2 consumption (multiwell plate analyzer), REDOX (autofluorescence), and intracellular lipid accumulation (Nile red) were measured.

Results

Acoc exposure decreased insulin secretion in a concentration dependent manner with a maximal inhibition of 60% occurring at 5 mM. REDOX and O2 consumption were not significantly altered after exposure to 5 mM Acoc while lipid accumulation was increased 2‐fold.

Conclusion

Chronic exposure of INS‐1 cells with Acoc inhibits insulin secretion without affecting glucose‐induced REDOX changes or respiration. This research was supported with funds from the NIH.
Regucalcin mediated up‐regulation of Wnt/β‐catenin signaling
Authors: DAR MOHD JAMAL , MONGA SP
Published: June 2021

Senescence marker protein‐30 (SMP30), also known as regucalcin (RGN), functions as a lactonase. The penultimate step in vitamin‐C biosynthesis in nonprimate mammals is catalyzed by this enzyme. RGN also regulates the transcriptional process by binding protein and DNA in the nucleus. There is growing evidence that regucalcin plays a multifunctional role as a regulatory protein in Ca2+dependent and independent signaling processes in many tissues especially liver. Recently, we identified RGN as a novel target of β‐catenin in the liver establishing an important role of β‐catenin in vitamin‐C homeostasis and cell survival. We also identified β‐catenin‐RGN association in liver cells. In the current study we investigated whether regucalcin itself has any impact on the Wnt/β‐catenin signaling. GFP‐coupled full‐length (FL), and amino (N)‐, carboxy (C)‐ or middle domain‐deletion mutants of RGN were generated in the laboratory. These constructs were transiently overexpressed in the HEK293 cells and human hepatoma cells (HepG2) cells. We observed RGN‐GFP to be prominently located in the nucleus of cells after transfection, which was blocked with staurosporine, an inhibitor of protein kinase C. Both human and mouse FL‐RGN showed a 2–3‐fold increase in β‐catenin‐dependent transcriptional activity as measured by TOPflash reporter assay over the empty GFP‐vector‐transfected cells. Moreover, deletion of both the N‐ & C‐ but not the middle domain‐deletion mutants of regucalcin abrogated TOPflash activity after transfection. Thus, we have identified a novel role of regucalcin in stimulating the Wnt pathway through direct interaction with nuclear β‐catenin for which amino‐ and carboxy‐ terminals of RGN are of relevance.
Diet Induced Obesity Disrupts Hepatic Redox Homeostasis Leading to Oxidative Stress in Mice: Protective Effect of Natural Compounds
Authors: Sarna Lindsei K , Wang Pengqi , Wu Nan , Siow Yaw L , Karmin O
Published: June 2021

Oxidative stress is associated with obesity, and while observed in several tissues, its occurrence in the liver during diet induced obesity (DIO) remains controversial. Therefore, our objectives were to investigate the effect of DIO on hepatic oxidative stress and to identify natural compounds capable of protecting against hepatic oxidative injury. Mice were fed either a control (10% kcals fat) or DIO (60% kcals fat) diet for 12 weeks. Compared with control mice, DIO mice weighed significantly more after the 12 week feeding period. In association with weight gain, DIO mice showed significant increases in serum malondialdehyde (MDA), a marker of lipid peroxidation, as well as a severe reduction in total serum antioxidants. Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), indices of liver injury, were markedly higher in DIO mice. Hepatic MDA levels were also significantly higher in these mice, indicating a disruption in redox balance in this organ. In accordance, hepatic NADPH oxidase‐mediated superoxide (O2−) production was strikingly increased in DIO mice while the activity of the O2− detoxifying enzyme superoxide dismutase was impaired. Our preliminary results also indicate that certain natural compounds are hepatoprotective in DIO which may be mediated by their antioxidant effects in the liver. This study was supported by NSERC and MHRC.