bluuebunny/crossref_metadata_2025 huggingface dataset:
Participation of Locus coeruleus (LC) noradrenergic neurons on breathing in female rats
Authors: de Carvalho Debora , Marques Danuzia A , Szawka Raphael E , Alselmo-Franci Janete , Bicego Kenia C , Gargaglioni Luciane H.
Published: June 2021

LC has been reported to regulate CO2‐drive to breathing in males. Since, sex hormones have a significant impact on ventilation; the present study examined the role of LC noradrenergic neurons in estradiol‐treated ovariectomized (OVX+E2) rats in response to hypercapnia. To selectively destroy LC noradrenergic neurons we administered 6‐hydroxydopamine (6‐OHDA, 8 μg in 1 μL of vehicle) bilaterally or vehicle (ascorbic acid) into LC of OVX+E2 rats. Bilateral inhibition of LC neurons with 6‐OHDA reduced resting respiratory frequency (57±3.53, vs. vehicle: 93.5±3.75 breaths/min) and the increase in ventilation produced by hypercapnia (7% CO2) (1052.5±11.5, vs. vehicle: 1625±89.7 mL/min/kg). Our results also suggest that noradrenergic LC neurons play an important modulation in the control of ventilation during resting or hypercapnic conditions in female rats. Financial support: FAPESP, CNPq and INCT of Comparative Physiology
Mu‐Opioid Receptors (MORs) in Pre‐Botzinger Complex (PBC) Are Involved in Fentanyl Switching of Pulmonary C‐Fibers (PCFs)‐Mediated Rapid Shallow Breathing (RSB) into An Apnea in Anesthetized Rats
Authors: Zhang Zhenxiong , Zhang Cancan , Xu Fadi
Published: June 2021

Our previous study showed that fentanyl (iv), a selective MOR agonist, could switch the PCFs‐mediated RSB into an apnea and that peripheral MORs are necessary for triggering this switch. Here we further tested if central MORs, especially those in the medial nucleus tractus solitarius (mNTS) receiving PCFs’ inputs and the PBC responsible for respiratory rhythmogenesis, were also involved in such switching. Right atrial injection of phenylbiguanide (3–5 μg/kg) was performed to stimulate PCFs in anesthetized rats before and after fentanyl (8 μg/kg, iv) without or with prior central administration of naloxone methiodide (M‐NLX, an opioid receptor antagonist). The central administration was achieved by injecting M‐NLX into the cisterna magna (8 μg/4μl), the mNTS or the PBC (10 mM, 15 nl in each side). Our results showed that phenylbiguanide alone shortened expiratory duration (TE) by 32 ± 6 % (RSB), while it markedly prolonged TE by 5.5‐fold (an apnea) after fentanyl. M‐NLX injected into the cisterna magna or the PBC, but not the mNTS, prevented the fentanyl switching with the TE of RSB slightly but significantly prolonged. This study, along with our previous results, suggests that although peripheral MORs are critical to trigger the fentanyl switching of PCF‐mediated RSB into an apnea, central MORs, especially those in the PBC, are necessary to establish this switching (Supported by ALA RG‐191095‐N and HL 107462).
Hypoxic vasoconstriction of rat skeletal artery mediated by NOX4 and NOS/sGC pathway
Authors: Kim Sung Joon , Han Jung-A , Seo Eun Yeoung , Kim Kyung Soo , Zhang Yin-Hua
Published: June 2021

Deep femoral arteries (DFA) of rats show hypoxic (3% PO2) vasoconstriction (HVC), which required a pretone by α‐adrenergic (PhE) stimulation. Nifedipine completely abolished HVC. HVC appeared to be endothelium‐independent. However, both NO synthase (NOS) inhibitor (L‐NAME) and soluble guanylate cyclase (sGC) inhibitor (ODQ) largely augmented the PhE‐induced contraction of DFA, upon which no further HVC was elicited. Upon the PhE‐induced pretone, strong contraction was induced by NOX4 inhibitor (DPI or plumbagin), and additional hypoxia induced relaxation instead of HVC. Pretreatment with NOX2 inhibitor (apocynin) or with ROS scavengers did not inhibit HVC. The inhibitors of mitochondrial electron transport chain (rotenone, antimycin A, NaCN) or uncoupler (CCCP) suppressed HVC as well as the PhE‐induced pretone, indicating a general inhibition of contractility. Also, RhoA‐dependent kinase inhibitors (Y27632 and fasudil) suppressed HVC along with the inhibition of pretone. Taken together, it is suggested that NOX4 might play a critical early step for the HVC; NOX4‐dependent hypoxic inhibition of NOS/sGC might have augmented the PhE‐induced contraction of DFA. The HVC of skeletal arteries might have a physiological implication for matching the imbalance between the cardiac output and the skeletal blood flow under severe exercise or emergent hypoxia combined with increased sympathetic tone. This study was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF 2011‐0017379 and 2011‐0001175).
Intermittent Hypoxia Induces Fra‐1 in Rat Carotid Body Cells Expressing Leptin
Authors: Messenger Scott , Moreau Jason M. , Ciriello John
Published: June 2021

Carotid body glomus cells have recently been found to express leptin. In this study, we investigated whether intermittent hypoxia (IH) induced immediate early gene Fra‐1 expression in carotid body cells expressing leptin. Adult male Sprague‐Dawley rats were subjected to eucapnic and isobaric IH (120s normoxia, 80s hypoxia; 6.5% O2) or normoxia for 8 h. A blood sample was immediately taken after IH for determination of plasma leptin levels and then the rats were transcardially perfused with Zamboni's fixative. The carotid bodies and the carotid arteries at their bifurcation were excised, paraffin embedded and sectioned at 6 μm for immunohistochemistry. Circulating leptin levels were increased in response to IH. Additionally, glomus cells containing leptin immunoreactivity were found to co‐express Fra‐1 in response to IH. Additionally, these glomus cells that expressed leptin, also expressed the Ob‐Rb, and an increase in pSTAT3 to IH. Taken together, these data suggest that the activation of carotid body glomus cells during IH may contribute to the elevated circulatory leptin levels and that leptin released by the glomus cells may also act in an autocrine/paracrine manner to alter the activity of carotid body glomus cells during IH. Supported by the Heart and stroke Foundation of Ontario.
VEGF up‐regulation and vascular area enlargement induced by intermittent hypoxia in the rat carotid body is not secondary to oxidative stress
Authors: Iturriaga Rodrigo , Moya Esteban A , Rio Rodrigo Del
Published: June 2021

Chronic intermittent hypoxia (CIH), a main feature of obstructive sleep apnea, potentiated the carotid body (CB) chemosensory responses to acute hypoxia, enlarges the vascular area and increased the VEGF expression in the CB. Since oxidative stress plays an essential role in the CIH‐induced CB chemosensory potentiation and by the fact that VEGF may be directly up‐regulated by oxidative stress, we hypothesized if the treatment with an antioxidant may impede the increased expression of VEGF and the enlarged vascular area in the CB of rats exposed to CIH. Thus, we tested the effects of ascorbic acid on the vascular area enlargement and the VEGF immunoreactivity (VEGF‐ir) in the CB of rats exposed to 5%O2, 12 times/hr for 8 hrs or sham condition for 21 days. We found that CIH increased VEGF‐ir and enlarged the vascular area in the CB by increasing the size of the blood vessels, although the number of blood vessels was unchanged. Ascorbic acid, which prevented the CB chemosensory potentiation to hypoxia failed to impede the vascular enlargement and the increased VEGF‐ir in the CB. Thus, our results suggest that CB vascular changes, including VEGF up‐regulation may result of the direct hypoxic stimulation during CIH exposure, and not secondary to the increased oxidative stress, which is involved in the potentiation of the CB chemosensory responses to hypoxia.

Supported by FONDECYT 1100405
Intermittent Hypoxia Elicits a Rapid Up‐Regulation of Cav3.2 T‐type Ca2+ Channels Mediated by Reactive Oxygen Species
Authors: Ahmmed Gias , Nanduri Jayasri , Khan Shakil A , Reddy Damodara , Wang Qiang , Fox Aaron , Prabhakar Nanduri R
Published: June 2021

We previously reported that intermittent hypoxia (IH) augmented Cav3.1 and Cav3.2 T‐type calcium channel subunit expression in adrenal chromaffin cells from neonatal rats. In the present study, we examined the mechanism of T‐type Ca2+ channel up‐regulation by IH in HEK cells stably expressing the Cav3.2 subunit. HEK cells were exposed to between 10–120 cycles of IH (one cycle is 30 sec at 1.5% O2 followed by 5 min at 20% O2). Whole cell patch‐clamp recordings showed that IH treatment increased peak CaV3.2 current density in a stimulation dependent manner and was not reversed for at least 16 hours following re‐oxygenation. IH also augmented plasma membrane expression of CaV3.2 protein levels, as determined by immunoblot. IH elevated ROS levels and ROS scavengers (MnTmPyP; 50μM or PEG‐catalase 500 U/ml) inhibited IH augmented Cav3.2 currents. Xanthine (250 μM)/xanthine oxidase (50 mU/ml) which generate ROS exogenously, mimicked the effects of IH. NADPH oxidase (NOX) are the major source of ROS, mediating cellular responses to IH. NOX activity and protein levels were up‐regulated by IH in HEK cells. The NOX inhibitors apocyanin (500 μM) and AEBSF (100μM), as well as silencing of NOX2, prevented IH induced up‐regulation of CaV3.2 current. These results demonstrate that in HEK cells expressing Cav3.2, ROS generated by NOX mediates IH‐increased CaV3.2 T‐type Ca2+ channel expression. (Supported by NIH‐HL‐90554, HL‐86493).
Modulation of voltage‐gated potassium channels by sodium channel SCN1b subunits
Authors: Nguyen Hai Minh , Calhoun Jeffrey Dennis , Goldin Alan L. , Isom Lori L. , Chandy George K.
Published: June 2021

Although voltage‐gated Nav1 sodium channels and Kv1 potassium channels are both known for their tandem function in action potential firing and have ubiquitous tissue expressions, to date, no functional association between members of the Nav1.x channel family and the Kv1 channel family have been reported. Here we report that SCN1b modulates the Kv1‐subfamily of K+ channels, each in a unique fashion, when co‐expressed in Xenopus oocytes or mammalian cells (Table). Biochemical experiments show that SCN1b is physically coupled to Kv1 channels. Using chimeras of SCN1b and the myelin Po protein, we demonstrate that the external domain of SCN1b is essential for channel modulation. Two known epilepsy‐causing mutations in the Ig‐domain of SCN1b, R85C and C121W, disrupt Kv1 channel‐modulation. Thus, sodium channel beta subunits may regulate action potential firing and propagation in normal and diseased conditions by modulating the function of both Nav and Kv channels.
Electrophysiological analysis of the spinal network function in the in situ adult interferon regulatory factor 8 (IRF8) knockout mice
Authors: Yazawa Itaru , Yoshida Yuko , Yoshimi Ryusuke , O'Donovan Michael , Ozato Keiko
Published: June 2021

IRF8 is a transcription factor expressed mainly in myeloid cells, such as macrophages and microglia (macrophage‐like cells in the CNS). It provides innate resistance to infectious pathogens. IRF8 also directs the development of myeloid cells. Thus, IRF8−/− mice develop a leukemia‐like disease, where granulocytes (neutrophils) are systemically increased, even in the spinal cord. IRF8−/− macrophages and microglia are defective in functions, including cytokine production.

We electrophysiologically investigated whether the alterations arising from the absence of IRF8 influence the spinal network function of the adult IRF8−/− and wild type mice (P11 to 12 wks) with the in situ whole and hindlimb preparation. Neuronal discharge was obtained from the phrenic and bilateral peripheral motor nerve. In both mice, locomotor‐like discharge and episodes were induced by the modulated sympathetic tone and by the drug (serotonin, N‐methyl‐D,L‐aspartate, and noradrenalin). Comparisons of episodes, induced by the modulated sympathetic tone and/or by the drug, demonstrated that the lumbosacral cord of IRF8−/− mice was functionally immature. Our results suggest the possibility that IRF8 plays a role in normal development of the spinal network function, possibly through the regulation of myeloid cells in the CNS.

Supported by NINDS and NICHD, National Institutes of Health.
Exploration of pressure‐natriuresis mechanisms using a lumped, two‐nephron whole‐kidney model
Authors: Moss Robert , Grosse Thibault , Thomas S. Randall
Published: June 2021

We present a two‐nephron whole‐kidney model of salt and water balance, which represents the short and long nephron populations and the associated cortical and medullary vasculature. The aim is to provide a context with sufficient complexity to explore and compare proposed mechanisms and features of pressure‐natriuresis, including: (1) the effects of blood pressure (BP) on proximal tubule sodium transport; (2) the sensitivity of medullary blood flow (MBF) to renal perfusion pressure and consequences for natriuresis and diuresis; (3) perturbations of TGF and myogenic effects on glomerular filtration rate (GFR). Without addressing detailed kinetic transport mechanisms in different cell‐types along each nephron segment, the model nonetheless contains sufficient detail of segment‐specific transport to serve as a basis for simulating the effects of the principal regulators of renal function (hormones, local paracrine effects, sympathetic nerve activity). The model can be adapted to different species (total number of nephrons, relative numbers and lengths of short and long nephrons, etc.), and will be used to replace the simpler kidney module of the classic Guyton model of blood pressure regulation. The model will be provided as an open source program with a flexible user interface for visualization and exploration of user‐defined scenarios.
Regulation of c‐myb by NF‐kB family members during human induced pluripotent stem cell differentiation into hematopoietic precursors
Authors: Fleury Michelle , Lynch Molly , Kowalsky Dan , Leonard Jessica , Toth Charles
Published: June 2021

c‐Myb is a transcription factor and proto‐oncogene that is important for hematopoietic cell development. This gene is predominately expressed in progenitor cells of adult bone marrow and down regulated during differentiation. Through gene knockout experimentation, the importance for c‐myb in developing cells has been determined. Null mutants show lethality in certain blood cell lines, strengthening the ideas that c‐myb plays an important role in cell line differentiation. Specifically, in murine embryonic cells, the absence of c‐myb results in the disruption of specific blood cell types. Previous studies have shown that NF‐κB family members are involved in regulating the activation of c‐myb by binding to Rel‐Related Proteins Binding Elements (RRBE) in the first intron. This leads to the hypothesis that NF‐kB regulates expression of c‐myb during development of hematopoietic cells. We have set up an in vitro differentiation protocol for differentiating induced pluripotent stem cells into blood cell precursors via a hemangioblast intermediate using defined cytokine and growth factors. Activation of c‐myb and NF‐kB was analyzed using qPCR to analyze the kinetics of c‐myb expression. Work is continuing using mobility shift assays and chromatin immunoprecipitations to determine the localization of NF‐kB to the c‐myb locus. Research support provided by a Providence College Committee on Aid to Faculty Research Grant.
Regulation of c‐myb by NF‐kB family members during murine erythroleukemia differentiation
Authors: Kowalsky Dan , Lynch Molly , Fleury Michelle , Leonard Jessica , Toth Charles
Published: June 2021

c‐Myb is a transcription factor and proto‐oncogene that is important for hematopoietic cell differentiation. The regulation of the c‐Myb protein and its down stream targets is well studied while the regulation of expression of the c‐myb gene is not well understood. c‐myb expression is controlled by a conditional block in transcriptional elongation in the first intron of the gene. Previous studies have shown that NF‐kB family members are involved in regulating the activation of c‐myb by binding to Rel‐Related Proteins Binding Elements (RRBE) in the first intron. This leads to the hypothesis that NF‐kB regulates expression of c‐myb during development of hematopoietic cells. Here we show using chromatin immunoprecipitations (ChIPs) and real‐time PCR in murine erythroleukemia cells that the down regulation of c‐myb expression via hexamethylene bisacetamide‐induced differentiation can be over‐come by activation of adenyl cyclase by forskolin. ChIP results demonstrate a correlation between occupancy of the RRBE sites by the p65 subunit of NF‐kB with elevated c‐myb mRNA levels. This study demonstrates for the first time a correlation between NF‐kB occupancy of RRBE sites and c‐myb expression in vivo in hematopoietic cells. Research support provided by a Providence College Committee on Aid to Faculty Research Grant.
The characteristics and roles of TamR, a MarR family transcriptional regulator from Streptomyces coelicolor
Authors: Huang Hao , Grove Anne
Published: June 2021

Multiple antibiotic resistance regulator (MarR) family transcriptional regulators usually play their regulatory role by responding to specific ligands. In this study, TamR (trans‐aconitate methyltransferase regulator), a member of the MarR family transcriptional regulators from Streptomyces coelicolor was investigated in order to understand its characteristics and its roles. After the tamR gene was cloned from the genomic DNA of S. coelicolor and introduced into E. coli, TamR was overexpressed and purified to apparent homogeneity. Gel filtration, cross‐linking, EMSA, fluorescence quenching and other experiments were used to investigate its characteristics and roles. The results show that TamR exists as dimer. A divergently oriented adjacent gene tam, which encodes trans‐aconitate methyltransferase is regulated by TamR. TamR binds with high affinity to the intergenic region between the tamR and tam genes, a genomic region that covers the promoter region of both genes and forms two different DNA‐protein complexes. Trans‐aconitic acid can attenuate the binding of TamR with DNA. In addition, other chemical compounds related to the citrate isomerization step in citric acid cycle, which include cis‐aconitic acid, citric acid and isocitrate, can also attenuate the interaction of TamR with DNA. A proposed model of TamR regulation is presented in order to explain the possible roles of TamR in S. coelicolor.
Telomere dysfunction as a tool to identify novel DNA damage factors
Authors: Bartocci Cristina , Yates John , Denchi Eros Lazzerini
Published: June 2021

The DNA damage response (DDR) pathway plays an essential tumor suppressive role ensuring the integrity of genomic information. Two major limitations have hindered the research in this field: i) the unpredictable location of DNA damage sites induced by conventional DNA damage‐inducing agents, ii) the lack of a technique for the isolation of proteins associated with a given DNA substrate. I have overcome these limitations by taking advantage of: i) telomere dysfunction as a tool to induce DNA damage to specific genomic loci, ii) a novel technique developed to isolate proteins associated with specific chromatin loci termed Proteomics of Isolated Chromatin segments (PICh). I performed the PICh purification on Terf2F/F Rosa26‐Cre‐ERT2 mouse embryonic fibroblasts (MEFs), in which deletion of both alleles of Terf2 gives rise to a DDR at chromosome ends. The purification identified 95 proteins uniquely associated with dysfunctional telomeres, among which I selected a subset of candidates for confirmation of their localization to damaged telomeres and for their characterization as potential novel DDR factors.

Research Support Source ADI
Identification of Pro‐Metastatic Signatures in Vesicles Released from Breast Cancer Primary Tumors
Authors: Pinto Samantha , Duelli Dominik M.
Published: June 2021

Breast cancer is one of the deadliest cancers in the world. Effective metastasis of the primary tumor to the bone, brain, lungs, and other organs are the causes for morbidity of breast cancer patients. Therefore, there is a necessity to detect metastasis early for successful therapy. One approach for detecting metastases early is to identify ways in which metastatic breast cancers modify the blood by releasing particles from the cancer cells. Here we are testing the idea that metastatic breast cancer cells export particles that are different from nonmetastatic cells. To do so, we raised antibodies against particles exported from breast cancer cells. We then tested whether these antibodies could distinguish between low and high metastatic breast cancer cell lines, and breast cancer cells that have a propensity to metastasize to different organs. We found that antibodies detected CD81 and integrin alpha‐3 beta‐1 at much higher abundance in particles exported from low metastatic cells than high metastatic cells. This result supports the idea that CD81 and alpha‐3 beta‐1 integrin may be metastatic suppressors. In addition, we identified several other antibodies that bind differently to particles exported from the brain, bone, and lung specific organotropic breast cancer cells. Our findings suggest that particles exported from breast cancer cells change during metastatic progression, and raise the possibility that antibodies detecting such differences might be of prognostic value. Future work will identify the antigens for these antibodies, and test whether they can indeed distinguish organotropic metastases of breast cancer in blood plasma.
Evaluation of the effects of linolenic acid on chronological aging and mitochondrial function of yeast
Authors: Quintero Maricela Fernández , Aguilar-Toral Roxana , Campos-Arroyo Ana Gabriela , Calderón-Cortés Elizabeth , Covián-Nares José Fernando , Cortés-Rojo Christian
Published: June 2021

The importance of the unsaturation degree of fatty acids (FA) from mitochondrial membranes in aging has been exposed by an inverse correlation between the aging rate of several species and the unsaturation degree of its membrane FA and peroxidation sensitivity. Although S. cerevisiae has been accepted as a model for the study of the molecular mechanism involved in aging, it can yield limited information about the role of FA in aging because of its membrane FA composition, which consists of FA resistant to peroxidation. To address this issue, we have studied the effects of linolenic acid (C18:3) in yeast chronological aging and mitochondria function. C18:3 decreased in > 50% the cell viability at 6 days of cultivation while almost completely abolished viability at 28 days. This was consistent with a decrease of > 60% in the activity of complexes II and III in C18:3 cells of any age. No mitochondrial membrane potential and loss of respiratory capacity were observed in 28‐days cells with C18:3. These results suggest that native fatty acids of yeast extend its longevity by preserving mitochondrial functions related with energy metabolism. This work was supported by a CONACYT grant (130638 to CCR)
Role of Hyaluronidases in Prostate Cancer Metastasis
Authors: Labrada Lyana , Gurski Lisa , van Golen Kenneth
Published: June 2021

Prostate cancer (PCa) is the second most common type of cancer in American males. If caught early, there are several effective therapies to treat this disease. However, once the cancer metastasizes to the bone, there is no cure. Therefore, there is a defined clinical need to identify drug targets to treat or prevent bone metastatic PCa. Here, we study the expression of the hyaluronidases, Hyal1 and Hyal2, in relation to their role in PCa metastasis. Hyaluronidases degrade hyaluronan (HA), a glycosaminoglycan present in the extracellular matrix. By degrading HA, these enzymes may facilitate invasion by clearing a path for cancer cells to spread, thereby facilitating metastasis to the bone. Using western blotting, Q‐PCR and substrate gel electrophoresis, we measured the expression and activity of these enzymes in PCa cell lines of different metastatic abilities. By looking at differences across the cell lines, we hope to identify whether increased expression and/or activity of any of the hyaluronidases correlates with increased metastatic capability of the PCa cells. Western blotting showed similar expression of hyaluronidases throughout PCa cell lines while Q‐PCR showed increased Hyal1 expression in C4‐2 cells. A hyaluronidase activity assay using substrate gel electrophoresis showed increased hyaluronidase activity in more aggressive cell lines. Ongoing investigations use the hyaluronidase inhibitors disodium cromoglycate, L‐ascorbic acid 6‐palmitoyl, and octyl gallate to test the effects of hyaluronidase inhibition on PCa cell invasion in a 3D HA hydrogel. This project is funded by the Milton Stetson Award.
Decreased Phosphorylation of Histone H3 serine 10 by Genistein is Associated with the Transcriptional Upregulation of ATF3 in DLD‐1 Colon Cancer Cells
Authors: Zhang Yukun , Strakovsky Rita S. , Chen Hong , Pan Yuan-Xiang
Published: June 2021

Activating transcription factor 3 (ATF3) is transcriptionally regulated by a variety of bioactive compounds during carcinogenesis. The present study investigated the mechanisms behind the regulation of ATF3 gene expression by genistein (GEN), a soy isoflavone, in DLD‐1 colon cancer cells. We examined the effect of GEN on histone modifications and the binding of transcription factors within the ATF3 promoter using Chromatin immunoprecipitation (ChIP). GEN upregulated ATF3 mRNA expression and the extra‐cellular signal regulated kinase (ERK) in a time‐dependent manner. Inhibition of ERK phosphorylation by U0126 did not abolish GEN‐induced ATF3 upregulation. However, GEN repressed the phosphorylation of Jun‐N‐terminal kinase (JNK). Inhibiting the phosphorylation of JNK (pJNK) by SP600125 resulted in increased ATF3 mRNA, indicating that JNK negatively regulated its expression. GEN reduced the phosphorylation of histone H3 at serine 10 (pH3S10), and the JNK inhibitor experiment confirmed that this was due to the reduction of pJNK. The GEN‐induced increase of ATF3 promoter activity was associated with decreased pH3S10 and increased ATF2 binding at the promoter. Our results indicate that GEN reduces pH3S10 at the promoter of an actively transcribed gene, providing a promising direction for investigating GEN‐mediated gene regulation and its role in colon carcinogenesis.
The Characterization and Identification of Ceramide‐1‐Phosphate Binding Proteins
Authors: Ward Katherine E , Stahelin Robert V
Published: June 2021

The activation of cytosolic phospholipase A2 α (cPLA2α) has been implicated in atherosclerosis and cerebral ischemia in previous animal models. The sphingolipid metabolite ceramide‐1‐phosphate (C1P) is known to specifically regulate the activation of cPLA2α and has been shown to increase enzymatic activity 5–10 fold through increased membrane residence. We have recently determined the amino acids that are responsible for C1P binding to cPLA2α in vitro and in a cellular system. In addition to heart disease, the conversation of ceramide to C1P has been implicated in cancer and phagocytosis signaling, suggesting that there are unidentified proteins that are regulated by C1P binding in membranes. Using the novel cPLA2α consensus sequence to search the proteome, we have predicted and are currently investigating new C1P binding proteins. Through the characterization of the C1P binding site, we have constructed a novel model for C1P recognition in membranes that deviates from the traditional lipid binding mechanism. This molecular characterization provides great insight into the molecular basis of C1P recognition and selectivity in biological membranes and will significantly increase the molecular basis of C1P binding by proteins. The understanding of this binding site in particular would lay the groundwork for a promising therapy for heart disease.

AHA 11PRE7640028 (KEW)

NIH T32GM075762 (KEW)
Expression of ABCB1 in the human adrenocortical cell line H295R
Authors: Powers Jennifer Lynn , Kim Jia , Oso David , Przeluska Joanna , Jenkins Philip , Walden Seth
Published: June 2021

The ATP‐binding cassette (ABC) transporter, ABCB1 (MDR1), has been shown to be involved in transport of xenobiotics as well as certain steroids. Its highest expression is in intestines, liver, kidney, adrenal, and capillary endothelial cells of the blood‐brain barrier. Adrenocorticotropic hormone (ACTH), which increases steroid production, causes an increase in expression of mdr1b in mice. Treatment with 2‐aminoglutethimide, an inhibitor of the enzyme that converts cholesterol to pregnenolone, abolishes this ACTH‐stimulated increase. This suggests a feed‐forward regulation for this steroid transporter. We wished to examine this possibility in the human adrenocortical cell line, H295R. These cells respond to both angiotensin II (AII) and ACTH, stimulating secretion of different steroids. H295R cells were treated with varying concentrations of AII for various times. RNA was isolated and treated with DNase, then cDNA was synthesized. Quantitative RT‐PCR was done using Roche's Universal Probe software to design primer and hydrolysis probe combinations. Data suggests that AII causes a decrease in ABCB1 expression. This suggests there may be species and tissue differences in control of ABCB1 expression. Since ABCB1 gene expression in many cell lines has been shown to be increased by activation of PXR/SXR nuclear receptors, we examined the H295R cell line for the presence of this and other nuclear receptors, such as CAR, with methods described above and also with a panel of nuclear receptor genes. The PXR/SXR is expressed at very low levels in this cell line, again suggesting tissue differences in control of ABCB1 expression.
Oleic acid attenuates trans‐10, cis‐12 (10,12) conjugated linoleic acid (CLA)‐mediated inflammatory signaling in primary human adipocytes
Authors: Reardon Meaghan Ann , Gobern Semone , Martinez Kristina , Chung Chia Chi , Reid Tanya , McIntosh Michael
Published: June 2021

CLA is a popular weight loss supplement consisting of 10,12 and cis‐9, trans‐11 (9,11) isomers that reduces adiposity. However, the consumption of CLA, particularly the 10,12 isomer, is associated with adverse side effects including chronic inflammation and insulin resistance. We hypothesized that these side effects of 10,12 CLA were linked to reduced activity of lipogenic enzymes including stearoyl‐CoA desaturase (SCD)‐1, a delta‐9 desaturase essential for the production of monounsaturated fatty acid (MUFA)s that are required for phospholipid and neutral lipid synthesis. Furthermore, blocking SCD‐1 increases saturated fatty acids like palmitate and stearate, which increase inflammation and insulin resistance. Thus, cultures of human adipocytes were treated with 50 uM 10,12 CLA and 30–300 uM oleic acid for 18–48 h. Consistent with our hypothesis, oleic acid reduced CLA‐mediated inflammatory gene expression in a dose‐dependent manner. However, oleic acid treatment did not attenuate CLA‐mediated insulin resistance. Notably, supplementation with stearic acid, a substrate for SCD‐1, or 9,11 CLA did not prevent inflammatory gene expression in 10,12 CLA‐treated cultures. Collectively, these data show that oleic acid attenuates CLA‐mediated inflammatory signaling, possibly by alleviating stress caused by SCD‐1 inhibition. Supported by NIH NIDDK‐ODS 5R01 DK063070‐09 and the UNCG URA program.
Functional characterization of melanoma upregulated long noncoding RNA SPRY4‐IT1
Authors: Perera Ranjan Joseph , Zhao Wei , Mazar Joseph
Published: June 2021

The elevated expression of SPRY4‐IT1 in melanoma cells compared to melanocytes, its accumulation in cell cytoplasm, and its effects on cell dynamics, including increased rate of invasion, proliferation and motility, suggest that the higher expression of SPRY4‐IT1 may have an important role in the molecular etiology of human cancers. To identify the molecular mechanism of SPRY4‐ IT1, we have engineered human melanocytes to ectopically express SPRY4‐IT1 and engineered cells were assayed to identify genes and proteins expression changes compared to vector only cells. The results indicate that the modulation of a sub‐group of anti‐apoptotic and cell cycle genes. We have also observed that SPRY4‐ IT1 is associated with the appearance of multinucleated giant cells and increases cell proliferation and invasion in engineered melanocytes. RNA‐FISH probes that were designed to various regions (5′ and 3′) of SPRY4‐IT1 demonstrate the existence of a 5′ excision event during the RNA processing and 5′RACE and Northern‐blot analysis confirmed these results. The characterization of RNA from monosome and polysome fractions followed by qRT‐PCR and Northern blots demonstrates that SPRY4‐IT1 is predominantly transported in to the polysome fractions. SPRY4‐IT1 molecular function, transcriptional regulation, cellular compartmentalization, and its interaction with other RNAs and proteins will be discussed.
Gene expression levels of the three hemoglobins of the clam Lucina pectinata differ between different tissues
Authors: Montes Ingrid Marie , Lopez-Garriga Juan , Cadilla Carmen Lydia
Published: June 2021

The clam Lucina pectinata has three different hemoglobins (Hb): hemoglobin I (HbI), a sulfide‐reactive protein and two oxygen‐reactive proteins, hemoglobin II (HbII) and hemoglobin III (HbIII). In order to better understand the physiological role of these hemoproteins, we evaluated their gene expression at the mRNA level by real time PCR. The Hb mRNAs expression levels were compared to that of the histone H3 mRNA and the 18S rRNA. Total RNA was isolated by the TRIzol method in order to obtain higher RNA yields than that obtained using column‐based methods. RNAs were further purified by n‐butanol and diethyl ether extractions in order to achieve better A260/A230 ratios and higher amplification efficiencies during one step real time RT‐PCR. The histone H3 gene was expressed in similar levels in most tissues except in visceral mass, while the 18S rRNA gene levels were more uniform throughout all tissues. Thus, we compared the three hemoglobin mRNA levels to that of the 18SrRNA using the clam's foot tissue as calibrator. The Hb mRNAs were most highly expressed in ctenidia, whereas foot and muscle had lower levels of expression. In ctenidia, oxygen‐reactive Hb mRNAs were expressed at higher levels than HbI mRNA, as reflected at the protein level in previous studies. In contrast, the visceral mass mRNA expression of HbI was higher than for HbII and HbIII. This research was supported by NIH grants 8G12‐MD007600 and 5R25GM088023.
Development of an assay to discover novel cytosolic factors for cholera toxin retro‐translocation
Authors: Moore Paul Christopher , Tsai Billy
Published: June 2021

Cholera toxin (CT) is transported from the plasma membrane of host cells to the endoplasmic reticulum (ER) where the CTA1 subunit retro‐translocates to the cytosol to induce toxicity. In the ER, the toxin co‐opts machinery that disposes misfolded proteins to the cytosol for ubiquitin‐dependent proteasomal destruction. However, once extracted into the cytosol the toxin evades proteasomal degradation, refolds, and becomes catalytically active. To elucidate this mechanism we developed an in vitro assay that permits release of CTA1 from intact ER membranes upon addition of cytosolic extract. The toxin must be delivered to the ER and processed normally into CTA1 for release. As expected, treatment with the proteasome inhibitor epoxomicin has no affect on toxin release. Furthermore, heat inactivation and energy depletion of the extract indicate that a proteinaceous, energy‐dependent activity exists. Preliminary fractionation of the cytosol indicates that this activity does not co‐fractionate with p97, Hsp90 and Hsp70; primary candidates for the retro‐translocation factor. Concurrent experiments with TCRα show that the assay can be applied to a variety of ERAD substrates. Funding: Burroughs Wellcome & NIH.
Expression of constitutively active Jak2 in the hypothalamus leads to weight gain that correlates with increased SOCS3 levels
Authors: Ryder Daniel J , Nabilsi Nancy , Sayeski Peter , Scarpace Philip
Published: June 2021

The leptin pathway is of great clinical interest for weight loss. However, intricate feedback signals obscure viable strategies to affect leptin‐mediated weight loss. We aim to understand this pathway and determine if a constitutively active positive regulator of leptin signaling, Jak2 V617F, alters weight gain and energy intake.

Hypothalamic injection of pLentiV617F and pLenti‐GFP was carried out. Rats were given high fat diet and monitored for food intake/weight changes. Oxygen consumption and fat pad analysis was conducted. After tissue extraction QPCR was used to verify expression of transgenes and evaluate levels of endogenous Jak2 transcript and immunoblot to measure Jak2 and SOCS3 protein.

Body weight, intake and food efficiency for the V617F rats was up. Concordantly, respiration and voluntary wheel running activity was down. Transgene expression was elevated, as expected, along with leptin and hypothalamic SOCS3 protein in V617F‐injected rats.

V617F is indicated to cause caloric increase and metabolic decrease. SOCS3 was up, indicating induction of an inhibitory pathway. We suspect this obese phenotype is caused by an inhibition of Jak2 signaling by SOCS3 in the hypothalamus. This delicate balance between Jak2 expression and metabolic/energy intake regulation and stands as a cautionary tale for pharmacologists who choose to alter these proteins by pharmacological means.
Human Leukotactin‐1/CCL15 up‐regulates expression of cell adhesion molecules and stimulates monocyte adhesion to endothelial cells via JAK2/STAT3 signaling pathway
Authors: Park Keun Hyung , Lee Tae Hoon , Kim Chan Woo , Kim Jiyoung
Published: June 2021

Leukotactin‐1 (Lkn‐1)/CCL15 is a potent chemo‐attractant for leukocytes and endothelial cell and is implicated in inflammatory diseases. Lkn‐1/CCL‐15 enhances cell migration and activation of neutrophils and endothelial cells mainly through CCR1 signal pathway. We found that Lkn‐1/CCL15 significantly increased mRNA and protein expression of ICAM‐1 and VCAM‐1 compared with those in non‐stimulated endothelial cells. We showed that Lkn‐1/CCL15 increased cell adhesion of human U937 monocytoid cells to the endothelial cell. Furthermore, we provide evidence that Janus kinase (JAK) and STAT pathway plays a role in Lkn‐ 1/CCL15‐induced gene expression of cell adhesion molecules in endothelial cells. We observed that phosphorylation of JAK and STAT3 was stimulated after Lkn‐1/CCL15 treatment of HUVECs. Pretreatment of these cells with inhibitors for JAK, PI3K, and AKT prevented the Lkn‐1/CCL15‐induced expression of ICAM‐1, suggesting the involvement of JAK pathway in ICAM‐1 gene activation by Lkn‐1/CCL15. The results suggest that Lkn‐1/CCL15 and its signaling pathway may be a potential target for treating inflammatory diseases involving up‐regulation of cell adhesion molecules. .
14‐3‐3ε Induces Epithelial‐Mesenchymal Transition and Suppresses E‐cadherin Expression via Inducing Zeb‐1 in Hepatocellular Carcinoma
Authors: LIU TZUAN , JAN YEE-JEE , LIANG SHU-MAN , KO BOR-SHENG , LIOU JUN-YANG
Published: June 2021

14‐3‐3 proteins are adaptors for assembly multiprotein complexes to regulate a variety of signal pathways. 14‐3‐3 proteins composed of seven isoforms which play crucial roles in regulating multiple cellular processes including the cell cycle regulation, DNA repair, apoptosis, cell adhesion and motility. Our earlier study indicated that increased expression of 14‐3‐3ε is associated with higher rate of tumor metastasis and worse overall survival in hepatocellular carcinoma (HCC). To further investigate the molecular mechanism how 14‐3‐3ε regulate tumor progression, we established 14‐3‐3ε overexpressing HCC stable cells. We found that overexpression of 14‐3‐3ε significantly promoted cell migration, epithelial‐mesenchymal transition (EMT), and transcriptionally repressed E‐cadherin expression in HCC cells. Results from analysis of 14‐3‐3ε/E‐cadherin expression with clinicopathologic characteristics indicates that combination of elevated 14‐3‐3ε expression with reduced E‐cadherin expression is correlated with higher incidence of HCC metastasis and poor 5‐year overall survival. Moreover, overexpression of 14‐3‐3ε significantly induced Snail and Zeb‐1 expression, however, the effect of 14‐3‐3ε‐reduced E‐cadherin was specifically restored by knockdown of Zeb‐1 but not Snail with their specific siRNA. Taken together, 14‐3‐3ε may act as an important regulator to modulate tumor metastasis by promoting EMT as well as migration, and serve as a potentially prognostic biomarker and therapeutic target for HCC.
Elucidation of CXCR4‐CXCL12 mediated angiogenesis mechanism in endometriosis
Authors: Echevarría Martha , Ruiz Abigail , Baez Perla , Colón Maricarmen , Monteiro Janice , Flores Idhaliz
Published: June 2021

Endometriosis is defined as the presence of the endometrial tissue outside of the uterine cavity, primarily on the pelvic peritoneum, and ovaries. The main symptoms are chronic pelvic pain, pain during intercourse, and infertility. High CXCR4 mRNA levels were reported in endometriotic lesions in a rat model of endometriosis and protein expression is higher in human endometriosis tissues compared to control endometrium. CXCR4 is a G‐protein coupled receptor, activated by its specific ligand, chemokine stromal cell‐derived factor (CXCL12). CXCR4 is predominantly expressed by endometrial epithelial cells (EEC) and its ligand, CXCL12, by endometrial stromal cells. The CXCR4‐ CXCL12 axis is a potent inducer of angiogenesis, migration/invasion and cell proliferation. It has been shown that the CXCR4‐CXCL12 axis can induce angiogenesis by inducing expression of vascular endothelial growth factor (VEGF). We hypothesize that CXCL12 can induce the expression of VEGF and its receptors, (VEGFR1 and VEGFR2) in EEC. The aim of this study is to determine if CXCL12 induce the expression of the angiogenic factors, VEGF and its receptors, in EEC. Cells were incubated with hrCXCL12α, and the expression of VEGF and its receptors was assessed by ELISA and Western blot, respectively. Preliminary results showed that EEC only expresses VEGFR1. We expect to determine the effects of CXCL12 in the expression of the angiogenic factors studied in EEC and endometriotic epithelial cells. Our findings may have implications for future therapeutical strategies that may target the angiogenic components of endometriosis. R25GM096955, 1R25‐GM082406
New IMAC media enabling purification of histidine‐tagged proteins directly from eukaryotic cell culture supernatants
Authors: Granér Therese , Algotsson Mattias , Andersson Lars C , Björner Maria , Carlsson Marianne , Glad Gunnar , Hedlund Helena , Hörnsten Lena , Le Grevès Pierre , Lindgren Helena , Lindqvist Staffan , Öberg Katarina
Published: June 2021

Immobilized metal affinity chromatography (IMAC) purification of secreted histidine‐tagged proteins in eukaryotic cell culture supernatants is often problematic. Incompatibility with the cell culture media appears as stripping of the immobilized metal ions required for binding of target proteins. Due to low target protein concentration in cell culture supernatants, large sample volumes are often needed, aggravating the stripping effect.

In this study, purifications of histidine‐tagged proteins were performed using two novel Sepharose™ based IMAC media; Ni Sepharose excel and magnetic His Mag Sepharose excel. Both media have a new type of chelating ligand with exceptionally strong binding of nickel ions. Data showing successful purification of histidine‐tagged proteins from CHO cell culture supernatants will be presented.

Furthermore, the characteristics of new media enabled purification of target protein from insect cell culture supernatants. The purification was easily scaled up from 20 μl His Mag Sepharose excel beads to 1 mL pre‐packed columns.
Nuclear Bmp4 interacts with the SCF E3 ubiquitin ligase complex and inhibits the cell cycle
Authors: Rogers Aubrey C. , Nichols Brandt A. , Loos Trina J. , Bridgewater Laura C.
Published: June 2021

A variant of BMP‐4, called nBmp4, is translated from a downstream alternative start codon and localized to the nucleus. A yeast two‐hybrid screen identified the RING finger protein Regulator of Cullins 1 (ROC1) as a binding partner of nBmp4. ROC1 is a component of several different SCF E3 ubiquitin ligase complexes, and co‐immunoprecipitation experiments revealed that nBmp4 also interacts with several other SCF E3 ligase components including ROC2, Cullins 1, 2, 3, 4A, and 5, and F‐box proteins Skp2 and beta‐TrCP1. Overexpression of nBmp4 in HEK293T cells in culture caused cells to accumulate in G0/G1 phase of the cell cycle (as measured by DNA content) and increased the number of small cells in the population (as measured by forward scatter in a flow cytometer), suggesting that nBmp4 inhibits the cell cycle and may lead to cell death. Two of the known targets of ubiquitination by ROC1‐containing SCF E3 ligase complexes are the cell cycle inhibitors p21 and p27, and targeted inactivation of ROC1 causes p27 to accumulate, inhibiting embryonic cell division. To determine whether nBmp4 similarly causes p27 and/or p21 to accumulate by interfering with ROC1 function, nBmp4 was overexpressed in cultured cells and protein isolates were analyzed by immunoblotting. Neither p27 nor p21 levels were elevated, suggesting that nBmp4 inhibits the cell cycle by a different mechanism. Funding was provided by the NIH #AR048839.
Sargassum micracanthum, brown algae, inhibitslipid accumulation and reactive oxygen species (ROS) production during differentiation of 3T3‐L1preadipocytes
Authors: Lee Ok-Hwan , Yoon Bo-Ra , Lee Young-Jun , Kim Bo-Hee , Choi Hyeon-Son , Lee Boo-Yong
Published: June 2021

Obesity is characterized by an increase in the number and size of adipocytes differentiated from precursor cells, preadipocytes. Recently, increased ROS production in 3T3‐L1 adipocyte facilitates adipocyte differentiation and fat development. This study was to investigate whether reduced ROS production by Sargassum micracanthum extract could protect the development of obesity through inhibition of adipogenesis. The extent of differentiation reflected by amount of lipid accumulation and ROS production was determined by Oil red O staining and NBT assay. Treatment of Sargassum micracanthum extract significantly inhibited ROS production and adipocyte differentiation that is depend on NADPH oxidase (NOX4), a major ROS generator, and peroxisome proliferator‐activated receptor gamma (PPARγ) and CCAAT/enhancer‐binding protein alpha (C/EBPα), a key adipogenic transcription factor. These results indicate that Sargassum micracanthum extract can inhibit adipogenesis through a reduced ROS level that involves down‐regulation of NOX4 expression or via modulation of adipogenic transcription factor.