bluuebunny/crossref_metadata_2025 huggingface dataset:
Neuregulin decreases protein degradation in skeletal muscle
Authors: Hellyer Nathan J , Mantilla Carlos B , Argadine Heather M , Sieck Gary C
Published: June 2021

Neuregulin (NRG) is a member of the epidermal growth factor family, with 3 types varying in their structure and cellular expression but not in their receptor‐binding domain. NRG‐2 and NRG‐3 are primarily membrane bound, while NRG‐1 can be secreted. NRG‐1 is present in phrenic motor neurons. We previously reported that exogenous NRG containing only the receptor‐binding domain, increases protein synthesis in rat diaphragm. This effect was significantly inhibited by LY294002 indicating a PI 3‐kinase dependent mechanism. We also reported that NRG increases Akt phosphorylation. Interestingly, Akt phosphorylation is positively correlated with skeletal muscle growth and inversely correlated with atrophy. In this study, we hypothesized that NRG reduces protein degradation in rat diaphragm. Diaphragms were harvested from 4‐week old rats and incubated in the presence or absence of exogenous NRG. Protein degradation was measured by quantifying muscle tyrosine release via a fluorimetric assay. We found that NRG treatment resulted in a 50% reduction in tyrosine release from diaphragm preparations, indicating a reduction in basal protein degradation rates. These results suggest that phrenic motoneuron‐derived NRG‐1 regulates muscle protein synthesis and degradation rates, thereby playing a role in matching muscle fiber and motoneuron properties within motor units.
Aldosterone increases oxidant stress to inhibit soluble Guanylyl Cyclase activity in vascular smooth muscle cells
Authors: Maron Bradley Allen , Zhang Ying‐yi , Tang Shiow‐Shih , Loscalzo Joseph , Leopold Jane A.
Published: June 2021

Hyperldosteronism (ALDO) is associated with impaired vascular reactivity. We hypothesized that ALDO‐generated reactive oxygen species (ROS) decrease soluble guanylyl cyclase (sGC) function to inhibit vascular smooth muscle cell (SMC) relaxation. To test this hypothesis, bovine aortic SMC were exposed to ALDO (10−7 mol/L) or vehicle (V) for 24 h and ROS was measured by 2′‐7′‐dichlorofluorescein fluorescence. ALDO increased ROS by 43% (p<0.04) and 3‐nitrotyrosine immunohistochemistry revealed peroxynitrite formation was increased by 48% (p<0.04) compared to V. Apocynin (APO)(30 mol/L), a NADPH oxidase inhibitor, decreased ALDO‐induced ROS by 25% (p<0.04) and peroxynitrite by 75% (p<0.02). sGC was stimulated exogenously by iNOS activation after SMC exposure to cytokines for 24 h. Without influencing total NO2− levels, ALDO reduced iNOS‐mediated cGMP formation by 55% (ANCOVA, p<0.05) and decreased the NO2−/NO3− ratio by 32% (p<0.04); APO restored cGMP levels (ANCOVA, p<0.05) and the NO2−/NO3− ratio (p<0.01). Similarly, SMC exposure to DETA‐NONOate (DETA)(10−3 mol/L) for 24 h increased cGMP levels by 86% (p<0.02) compared to V. ALDO decreased DETA‐stimulated cGMP production by 54% (p<0.02), which was restored by 79% after APO treatment (p<0.02). These data suggest that ALDO‐induced ROS may uncouple NO bioactivity by disrupting key NO‐sGC signaling pathways needed for normal SMC vasodilatory function.
PGC‐1α plays a protective role against oxidative stress in skeletal muscle
Authors: Shi Yun , Liang Huiyun , Liu Yuhong , Ward Walter F , Van Remmen Holly
Published: June 2021

PGC‐1α is a transcription coactivator that regulates various biological processes such as mitochondria biogenesis, fatty acid oxidation, and oxidative fiber determination. It has also been shown to regulate genes of the reactive oxygen species (ROS) detoxifying systems. We hypothesize that PGC‐1α plays a protective role against oxidative stress in the highly oxidative tissue, skeletal muscle. To test this hypothesis, we will use a PGC‐1α transgenic mouse (PGC‐1α Tg+/0) that has ubiquitously elevated PGC‐1α in all tissues. We will study primary myoblasts and skeletal muscle tissue to examine the effect of increased PGC‐1α level on the ability to protect against oxidative stress in vitro and in vivo. PGC‐1α protein is overexpressed by 40% in skeletal muscle from the PGC‐1α Tg+/0mice and levels of downstream targets mtTFA, COX II, COX IVa are elevated as measured by real‐time PCR. Muscle mitochondrial ATP generation is unchanged, yet mitochondrial hydrogen peroxide (H2O2) production is significantly reduced by 50%. In a sciatic nerve cut denervation model known to increase muscle mitochondrial ROS generation, PGC‐1α transgenic mice appear to have attenuated release of H2O2. In addition, primary myoblasts exhibited increased resistance to hydrogen peroxide toxicity. In conclusion our data support a significant role for PGC‐1α in protection against oxidative stress in skeletal muscle.
Role of arginase in the pathophysiology of preeclampsia
Authors: Sankaralingam Sowndramalingam , Davidge Sandra T
Published: June 2021

Preeclampsia (PE), a hypertensive disorder of pregnancy, is characterized by vascular endothelial cell dysfunction resulting in increased levels of superoxide (O2.−) and reduced nitric oxide (NO) bioavailability. Also, increased peroxynitrite (ONOO−) has been observed in PE. Arginase competes with NO synthase (NOS) for the substrate L‐arginine. Upregulation of arginase can reduce L‐arginine availability for NOS, resulting in NOS uncoupling and more (O2.−) generation. We therefore hypothesize that upregulation of arginase occurs in PE resulting in increased O2.− and subsequently ONOO− formation. Immunohistochemical staining of arteries obtained from PE women show increased arginase expression when compared to arteries from normotensive pregnant (NP) women. In order to address mechanisms, HUVECs were treated with 2% plasma from NP and PE women for 24h. PE plasma significantly increased arginase expression (∼35%), increased O2.− (∼200%) and ONOO− (10 fold) generation. Plasma from either group did not affect eNOS expression. Inhibition of arginase by BEC reduced O2.− by 50%, P<0.05 but not ONOO− in response to PE plasma. Inhibition of NOS by L‐NAME also reduced O2.− by 50%. Therefore, in PE there is evidence of elevated arginase expression resulting in uncoupling of NOS leading to excess O2.− generation. However, inhibition of arginase did not ultimately reduce ONOO−. Funded by CIHR.
Use of a Planar Patch Electrode to Measure NBCe1‐A Function in Mammalian Cells
Authors: Kao Liyo , Abuladze Natalia , Newman Debra , Liu Weixin , Pushkin Alexander , Kurtz Ira
Published: June 2021

Standard whole cell‐patch clamping is labor‐intensive whereas automated planar patch clamp devices offer the possibility of higher throughput. Planar patch clamp electrodes are also attractive because their geometry facilitates obtaining electrical and optical recordings simultaneously. Previous studies have utilized the Port‐a‐Patch system (Nanion Technologies GmbH), to study ion channel activity however there are no studies which have demonstrated the utility of this approach with electrogenic cotransporters, which in general have a lower conductance. In this study, we utilized a modified Port‐a‐Patch system to measure the functional activity of the electrogenic sodium bicarbonate cotransporter, NBCe1‐A. HEK‐293T cells permanently expressing NBCe1‐A were generated. Cell monolayers were detached using 0.025% Trypsin/0.22 mM EDTA in PBS and were re‐suspended at a concentration of 500,000 cells/ml in 50% DMEM/PBS. The cells were kept at room temperature until measurements were made. Approximately 2,500 cells were placed on a planar electrode chip from which a single cell was automatically selected. Planar electrode chips with variable resistances were assessed. Using chips with a resistance of 2 – 3.5 MOhm, we routinely obtained GOhm seals using this approach. Whole cell currents were elicited by voltage ramps from −100 mV to +30 mV from a holding potential of −100 mV. Cell membrane capacitance for the HEK‐293T cells was 7.14 ± 0.67 pF (n = 21) and access resistance was 8.6 ± 0.5 MOhm (n =21). The DIDS‐inhibitable Na+‐and HCO3−‐dependent NBCe1‐A‐mediated average whole cell current was 29.3 ± 3.2 pA (n =21) at +30 mV. These results represent the first reported use of a planar patch‐clamp device to study ionic currents mediated by a heterologously expressed electrogenic cotransporter in mammalian cells.

Supported by the NIH
Interaction of the Na/HCO3 cotransporter SLC4A7 (NBCn1) and the N‐Methyl‐D‐Aspartate (NMDA) receptor subunit NR2A
Authors: Rajbhandari Ira , Kim Eunjin , Choi Inyeong
Published: June 2021

Intracellular pH affects neuronal excitability. We have previously demonstrated that the electroneutral Na/HCO3 cotransporter SLC4A7 (NBCn1) can interact with the PDZ protein PSD95, which is known to bind to NMDA receptors. In the present study, we examined the functional and physical association of NBCn1 and the NMDA receptor subunit NR2A. Human embryonic kidney (HEK) 293 cells were transiently transfected with NBCn1, NR1A/NR2A, and PSD95, and cell lysates were prepared. In immunoprecipitation with an antibody to PSD95, both NBCn1 and NR2A were detected in PSD95 immunoprecipitates. A parallel experiment using an antibody to NBCn1 showed the detection of PSD95 in NBCn1 immunoprecipitates. Immunofluorescence labeling of NBCn1 and NR2A showed both proteins colocalized on the plasma membranes of transfected HEK cells in the presence of PSD95. The colocalization decreased in the absence of PSD95. Two‐electrode voltage clamp was done to examine the functional effect of NBCn1/NR2A interaction on the NMDA receptors expressed in Xenopus oocytes. NBCn1 increased the peak glutamate current (at −40 mV) by 42% in the absence of CO2/HCO–3 and by 40% in the presence of CO2/HCO–3. However, the peak glutamate currents were unaffected during the pH recovery mediated by NBCn1. These data suggest that NBCn1 clusters with NMDA receptors via PSD95 and this clustering likely affects NMDA receptor expression and activity.
Signaling Mechanism Involving PPARα‐ligand‐Mediated Increase in NO Production
Authors: Newaz Mohammad , Mitchel Bryan , Oyekan Adebayo
Published: June 2021

Clofibrate (CF), a peroxisome proliferator activated receptor α (PPARα) ligand increases nitric oxide (NO) production via PPARαf{dependent and ‐independent pathways. As PKA/PKC are involved in NO production, we investigated whether PKA/PKC signaling pathways are involved in CF‐mediated increase in NO. NO production (over 30 min) was determined in proximal tubular (PT) suspensions isolated from PPARα knock‐out (KO) and wild type (WT) mice challenged with CF (2μM) using NO specific dye DAF‐FM2. WT and KO mice were also treated with CF (250 mg/kg; i.p, 7 days) and renal homogenate was used for protein expression. NO signal in the PT was significantly lower in PPARα KO mice (21¡Ó2%). CF enhanced NO production (WT: 88¡Ó12%; KO: 86¡Ó6%) that was abolished by LNAME (3 μM). PKA 14–22 (20 μM), a PKA inhibitor, reduced CF‐mediated NO production (WT: 56¡Ó5%; KO: 46¡Ó2%). Similarly, chelerythrine (3 μM), a PKC inhibitor, reduced NO production (WT: 47¡Ó4%; KO: 45¡Ó2%). CF also increased eNOS and iNOS mRNA and iNOS protein expression in the KO but not in WT mice and did not affect PKC or PKA protein. These data suggest that CF increased NO production by inducing iNOS gene via a PPARα‐independent mechanism. We conclude that although CF did not influence PKA/PKC protein expression, but PKC/PKA signaling pathways are required for CF‐mediated enhanced acute NO production.
Effects of perfusion pressure on Ca2+ sparks in rat renal afferent arterioles
Authors: Balasubramanian Lavanya , Sham J.S.K. , Yip K.‐P.
Published: June 2021

Mechanical force can be transduced into freshly isolated renal vascular smooth muscle cells (VSMCs) to trigger Ca2+ sparks. Sparks can trigger Spontaneous Transient Inward Currents (STICs), and regulate myogenic tone via membrane depolarization. Electrophysiological studies indicate the presence of STICs in isolated renal VSMCs. Hence, we sought to determine whether the occurrence of Ca2+ sparks is increased during pressure induced myogenic constriction. Line scan images were collected from perfused rat afferent arterioles at 500 Hz using laser scanning confocal microscopy. We detected spontaneous Ca2+ sparks in 40% of the VSMCs at a perfusion pressure of 80 mm Hg. These sparks were suppressed by 50 μM ryanodine and were augmented by 0.5 mM caffeine. The mean spontaneous Ca2+ spark frequencies were 0.14±0.04 sparks/s (n=23 cells) and 0.24±0.11 sparks/s (n=13 cells) at 80 mm Hg and 120 mm Hg, respectively. These suggest that the increase in frequency of sparks is probably related to pressure induced myogenic constriction. Intriguingly, the frequency of Ca2+ sparks observed in SHR afferent arteriole was double compared to that of normotensive rats at the same perfusion pressure. These observations implicate that the enhanced Ca2+ sparks frequency may contribute to the increase of renal vascular resistance in SHR.
Telemetric Signal‐Driven Servo‐Control of Renal Perfusion Pressure in Rats
Authors: XIA Min , Li Pin‐Lan , Li Ningjun
Published: June 2021

The present study used a DSI pressure telemetry device to develop a system for servo‐control of renal perfusion pressure (RPP). Digitized pressure signals from lower abdominal aorta, reflecting RPP, is obtained by a DSI system and dynamically exported into Excel worksheet every 15s. A Digital Data Exchange program continuously reads the pressure data from Excel and inputs them into LabVIEW, an instrument control program. LabVIEW compares the pressure data with preset pressure values and then drives a bi‐directional syringe pump, which controls the inflation of an inflatable vascular occluder implanted round the aorta above the renal artery. In this way, the RPP can be servo‐controlled within desired range no matter what the systemic arterial pressure (SAP) is. In neropinephrine or angiotensin II‐induced acute increases in SAP (115–145 mmHg), this system was able to control RPP at a constant range of 100 – 110 mmHg within 0.5–1 min. In high salt‐induced chronic hypertension in Dahl S rats, this system maintained RPP at 110 – 120 mmHg over 14 days, while SAP was 150 ± 5.9 mmHg. This system also has ability of simultaneity and multiplexing to control multiple animals concurrently. Our results suggest that this is an effective and reliable system to servo‐control RPP in rats, which can be established with general computer knowledge due to user‐friendly LabVIEW. (Supported by NIH grants HL70726, DK54927 and HL89563)
Physiological role of NHERF family proteins in the regulation of CFTR
Authors: Raghuram Viswanathan , Yang Yu , Yaemsiri Sirin
Published: June 2021

In epithelial cells, salt‐water balance is maintained by the expression and function of CFTR. The NHERF family PDZ proteins have been shown to interact with the c‐terminus of CFTR. To elucidate the functional role in human lung epithelia, we have used Calu‐3 cells derived from the serous cells of submucosal glands, as a model system. We have used RNA interference techniques to generate stable NHERF1 and NHERF2 knockdown (KD) cell lines. In these cells, the expression of NHERF1 and NHERF2 ranged from 10‐40% of Calu‐3NT cells (control cells). In these KD cells, steady‐state CFTR protein level was greatly reduced, whereas its apical localization was unaffected. We also found structural defects in the apical microvilli. In polarized NHERF1‐KD cells, membrane‐permeable cAMP analogs were unable to stimulate CFTR mediated short‐circuit currents at lower concentration. However, at higher concentration of analogs, their response was nearly normal despite reduced CFTR expression. Examinations of β‐adrenergic‐and adenosine‐receptor signaling pathways suggest that NHERF1, but not NHERF2 plays a unique role in organizing the signaling pathway.
Graduate courses in statistical and model‐based analysis of physiological data
Authors: Moore Leon C , Solomon Irene C , Clausen Chris
Published: June 2021

We have developed two modular graduate courses in statistical and model‐based analysis of physiological data. The motivation was to provide students with a common set of skills and tools that facilitate their research training. Each course lasts 5 weeks and has 2 hours of lecture and 2 hours of lab per week. The first course, Statistical Analysis of Physiological Data, consists of a review of statistical methods used in laboratory research. It focuses on the interaction between experimental design and statistical analysis. To pass, the students must analyze and present a set of 15 problems. The lab sessions consist of tutorials on software usage, reference database searching, graphic design and data presentation, as well as student presentations of the assigned problems. The second course, Model‐based Analysis of Physiological Data, focuses first on nonlinear curve fitting and how it is used to analyze transporter kinetics and band density on gels. The course concludes with sessions on programming in which the students are required to write a program that numerically solves a 4‐compartment model of lithium pharmacokinetics. Again, the students are given a set of problems that they must complete and present to pass the class. The courses have been well received by the students, and have markedly increased their ability to analyze data and solve quantitative problems.

Funded by NIH Grant DK066124
An integrated framework for teaching oxygen delivery
Authors: Wittmers Lorentz E. , Mohrman David E.
Published: June 2021

The historical development of the process by which oxygen makes its way from the atmosphere to the tissue has numerous elements: pressures, ventilation, dissociation curves, blood flows, O2 contents, curve shifts, % saturation, metabolism, etc. Students of physiology are left to assimilate these bits and pieces into some understanding of the overall process. Our goal is to integrate the aforementioned “bits” and formulate a “bottom line“ model focusing on oxygen availability to cells as judged by the PO2 around them. Whereas the latter cannot be directly measured, it can be inferred from changes in the venous PO2. Our approach includes the constraint of conservation of mass to the overall oxygen delivery, as well as addressing the interactions of cardiovascular, respiratory, and metabolic physiology. Another major component in our approach is to combine the individual influences of PCO2, pH, temperature, 2,3‐DPG, and CO on the oxyhemoglobin dissociation curve. This allows the development of a “normal operating curve” that helps predict the loss of oxygen from the blood as it passes through the tissue. The model presented here makes it possible for the student to grasp the consequences of commonly encountered pathophysiologies of oxygen availability. It provides a convenient framework for discussing physiologic responses to problems such as hypoventilation, shock, and CO poisoning.
Evaluation of the morphological aspect of portal hepatic vein branches in the hepatic hillum
Authors: MATUSZ Petru L. , HORDOVAN Eniko‐Christine , PUSZTAI Agneta Maria , SZTIKA Dorina
Published: June 2021

According to Terminologia Anatomica, the portal hepatic vein (PHV) trunk bifurcates into the right branch and the left branch in the vicinity of the hepatic hillum. The caudate branches start from the transverse portion of the left branch. We studied the branching modalities of the PHV trunk and the morphological aspect of its branches at hillum level on 100 hepatic corrosion casts. Besides the homologated type (modal type, present in 97% cases), we found major branching variants of the PHV trunk in 3% cases. These variations are represented by: branching of the PHV trunk into the left branch and the posterior branch (1%), the anterior branch and the posterior branch (1%), and trifurcation into the left branch, the anterior branch and the posterior branch (1%). In case of bifurcation into a left branch and a posterior branch, the anterior branch of the PHV originates into the transverse portion of the left branch. In case of bifurcation into an anterior branch and a posterior branch, the left branch originates from the initial part of the anterior branch. In case of trifurcation into a left branch, an anterior branch and a posterior branch, the right branch is absent as a morphologic entity. These major morphological variations of branching of the PHV trunk are due to a vascular gliding phenomenon. Knowing these morphological aspects will facilitate the evaluation of the origin of caudate branches of the portal hepatic vein. (Supported by CNMP 4.1‐092/2007)
Posterior cranial fossa dimension and descent of cerebellar tonsils after arachnoid cysto‐peritoneal shunting
Authors: Osuagwu Ferdnand C , Salamon Noriko , Lazareff Jorge
Published: June 2021

Background Descent of cerebellar tonsils (CT) has been reported following shunting of supratentorial (ST) arachnoid cyst. The phenomenon has been attributed exclusively to a difference in pressure gradient between ST space and the posterior fossa (PF). We aimed our study at determining the association between the posterior cranial fossa volume (PCFV) in this phenomenon.

Materials and Methods We compare posterior fossa volume in two children with shunted left arachnoid cyst but Chiari like condition in one.

Results The PCFV was 137.7c.c in the chiari 1 patient compared with 145.6cc in the control group peer. The PCFV area in the chiari 1 patient was 204.9cm2 while the control had 207.9cm2.

The supratentorial volume (SV) measured 1055.1c.c in the chiari 1 patient compared with 898.2c.c in control group peer. The area of the supratentorial region was 742.9cm2 in the chiari 1 patient versus 687.7cm2 in the control. The prepontine and pre‐medullary cistern measured 4.4mm in the chiari 1 patient while control was 6.3mm and 7.7mm respectively.

The percentage ratio of the posterior cranial fossa volume to the supratentorial volume (PCFV/SV X 100) in the chiari 1 patient was 13.0 versus 16.4 in the control.

Conclusion Our findings emphasize the relevance of skull anatomy on the development of Chiari like condition.
Skeletal malformations associated with mutations causing left‐right patterning defects
Authors: Gibbs Brian Clemon , Schimel Dan , Yu Qing , Chatterjee Bishwanath , Lo Cecilia
Published: June 2021

We recovered many novel mouse mutants exhibiting structural heart defects from a prenatal fetal ultrasound screen of N‐ethyl‐N‐nitrourea (ENU) mutagenized mice. Several of these mutations were found to cause congenital heart defects associated with laterality defects that included situs inversus totalis, as well as heterotaxy. Interestingly, these mutants also were found to have a variety of skeletal anomalies. In this study, we carried out a detailed analysis of the skeletal phenotypes using Alizarin red and Alcian blue staining of skeletal preparations, and by microCT imaging. In addition, histological sections and immunostaining were used to analyze the course of bone development in these mutants. As the cilia is well described to play an essential role in the specification of laterality, and more recently, in skeletogenesis as well, it is of interest to note that several of these mutants have defects related to the cilia. Thus the further characterization of these mutant mouse models promises to yield new insights into the role of the cilia in skeletal development and remodeling.
Structural Basis of RNA Polymerase II Pausing and Arrest
Authors: Wang Dong , Bushnell David , Westover Kenneth , Kornberg Roger
Published: June 2021

All cellular RNA polymerases are capable of both forward and retrograde movement (“backtracking”) on the DNA template. Forward movement is favored by the binding of nucleoside triphosphates (NTPs), while backtracking occurs especially when the enzyme encounters an impediment, such as DNA damage. We have determined the X‐ray structures of RNA polymerase II transcribing complexes in various backtracked states. The backtracked RNA is specifically bound by the polymerase, indicative of the functional significance of the backtracked state. The site of binding the backtracked RNA clashes with the site of interaction previously reported for the transcription elongation factor TFIIS. The results are pertinent to the action of TFIIS in recovery from the backtracked state and to the mechanism of proofreading the nascent transcript.
Transcriptional regulation of the growth phase regulated porin, OpnS in the mutualistic bacterium, Xenorhabdus nematophila
Authors: van der Hoeven Ransome , Forst Steven
Published: June 2021

Porins are small water filled channels that allow nutrients to diffuse across the outer membrane of Gram negative bacteria. The goal of this study was to characterize the transcriptional regulation of a novel porin, OpnS, in Xenorhabdus nematophila. RT‐PCR and urea SDS‐PAGE analysis of outer membrane proteins revealed that opnS mRNA and OpnS protein were expressed predominantly as cells transitioned into stationary phase. The EnvZ‐OmpR two‐component signal transduction system was shown to control the transcription of opnS. Expression of opnS mRNA as well as OpnS protein levels were reduced in both mutant strains. To quantify the level of transcription, a opnS‐lacZ fusion was constructed and β‐galactosidase activity was measured in wild type, ompR− and envZ− strains. β‐gal activity was reduced by 50% and 20% in the ompR− and envZ− mutants respectively. A 2‐fold increase in β–gal activity was observed in a strain in which var1, encoding a putative global regulator of transcription, was inactivated. 5′ RACE studies of the upstream region of the opnS gene revealed the presence of two transcription start sites suggesting the existence of two sigma 70 promoters. Further, promoter deletions using opnS‐lacZ fusions identified putative repressor and activation sites to both promoters 1 and 2 of the regulatory region. Understanding the regulation and function of OpnS in the life cycle of X. nematophila are in progress.
Activity of cap‐independent translation depends on a double‐loop structure in human SP‐A 5'‐UTR variants
Authors: Wang Guirong , Guo Xiaoxuan , Kimball Scot R. , Floros Joanna
Published: June 2021

Human surfactant protein A (SP‐A), a molecule of innate immunity and surfactant‐related functions, consists of two functional genes, SP‐A1 and SP‐A2. Several SP‐A 5′UTR splice variants have been identified with differences in translation efficiency and mRNA stability. To study whether these 5′UTR variants are subject to internal ribosome entry site (IRES) activity (i.e. Cap‐independent translation activity), we generated several constructs using a bicistronic reporter vector, in which each contained one SP‐A 5′UTR variant (A′D′, ABD, AB′D′, or A′CD′) between the Renilla and firefly luciferase genes. The results indicated that variants A′D′, ABD, AB′D′ exhibit significantly higher IRES activities than the negative control (plasmid without 5′UTR insert), but the A′CD′ had no IRES activity. Deletion mutation analysis of the ABD variant revealed that at least two cis‐acting regulatory elements associate with IRES activity. An element at the end of exon A attenuated activity whereas elements in the second half of exon B and the start of exon D enhanced activity. Further analysis by predicted mRNA secondary structure and site‐directed mutation, revealed that the activity of the enhancer element depends on a double‐loop structure. Similarly, elimination of a double‐loop structure in the A′D′ variant resulted in a decrease in IRES activity, but with no impact on cap‐dependent translation activity. Supported by NIH HL‐34788
Role of HuR in the regulation of bcl‐2 mRNA stability in human HL60 leukemia cells
Authors: Spicer Eleanor K , Ishimaru Daniella , Ramalingam Sivakumar , Tholanikunnel Baby G. , Fernandes Daniel J.
Published: June 2021

High bcl‐2 expression in hematological tumors is frequently an obstacle to cancer chemotherapy. We have demonstrated that Bcl‐2 overexpression in some leukemia cells is a consequence of abnormal bcl‐2 mRNA stability. Previously, nucleolin was identified as a factor that stabilizes bcl‐2 mRNA in human HL60 leukemia cells. This interaction occurs via an AU‐rich element (ARE) in the 3’‐untranslated region of bcl‐2 mRNA. Our current study demonstrated that HuR also plays a role in the regulation of bcl‐2 mRNA stability in HL60 cells. Using EMSA we observed that HuR in HL60 cell extracts binds to the bcl‐2 mRNA ARE. Also, we found that such interactions occur in vivo. Taxol treatment induced proteolysis of cytoplasmic HuR, suggesting that modulation of HuR may play a role in the downregulation of bcl‐2 that precedes taxol‐induced apoptosis. Pull‐down assays indicate that nucleolin and HuR co‐immunoprecipitate in an RNA‐dependent manner, suggesting the two proteins are part of common bcl‐2 mRNP complexes. Moreover, nucleolin and HuR can bind independently and concurrently to bcl‐2 ARE RNA in vitro. This suggests HuR and nucleolin bind to separate, non‐overlapping sites on the bcl‐2 ARE. Sh‐RNA‐mediated knockdown of HuR in A431 epithelial cancer cells leads to reduced levels of bcl‐2 mRNA and protein. Taken together, our studies suggest that both HuR and nucleolin play a role in stabilization of bcl‐2 mRNA, which contributes to the overexpression of Bcl‐2 protein in some types of leukemia cells. Supported by NCI R01CA87553 (EKS) and Leukemia and Lymphoma Society Grant 6006‐06 (DJF).
A fluorescent micro‐assay utilizing purified microsomal epoxide hydrolase
Authors: Mandichak Tracy , Qi Hongwei , Chen Stephanie , Grimes Maggie , An Gang , Van Der Keyl Harjeet , Sweitzer Sharon , Schwartz Benjamin , Brandt Martin
Published: June 2021

A low volume (10 μL) kinetic fluorescent assay to evaluate human microsomal epoxide hydrolase (mEH) activity with the α‐cyano‐methyl ester substrate, PHOME (Analyt. Biochem. (2006) 355, 71–80), has been optimized. mEH was successfully cloned into a mammalian expression vector, expressed transiently in FreeStyle 293F cells, and purified by affinity chromatography (purity > 80%). In the presence of 5 μM PHOME, reaction rates were linearly proportional to added enzyme to 0.1 μM and the reactions were linear for up to 60 minutes with respect to product formation. The lowest discernable reaction rate of 5.5 nM/min was obtained with 10 nM mEH; with an assay signal:noise of 3:1. Optimized assay buffer contained 25 mM HEPES pH 7.0, 0.01% CHAPS, and 0.005% casein. Purified mEH activity was selectively inhibited with the addition of 50 mM ZnSO4 or with the known mEH inhibitor, Valpromide (IC50 = 1 mM and 5 μM, respectively), indicating that product formation was due solely to mEH. The potency of Valpromide was shown to give similar values in an alternative assay format using mEH enriched microsomes (IC50 = 8.0 μM). AUDA, a potent sEH inhibitor, was not an inhibitor of purified mEH. Thus, purified mEH provides a means to develop a low volume, high throughput assay to further our efforts towards developing inhibitors that can selectively discriminate sEH versus mEH activity.
Selective Diminution of Leukemic Cell Growth by shRNA‐Mediated Targeting of S‐Adenosylmethionine (SAMe) Metabolism
Authors: Attia Ramy R , Gardner Lidia , Mahrous Engy , Taxman Debra , LeGros Leighton , Ting Jenny P‐Y. , Rowe Sarah , Geller Arthur M. , Kotb Malak
Published: June 2021

Leukemic cells require significantly higher amount, than normal lymphocytes, of S‐adenosylmethionine (SAMe). We exploited these differences to modulate the kinetics of methionine adenosyltransferase (MAT) that catalyzes SAMe synthesis from ATP and methionine using shRNA technology to silence the expression of MAT regulatory subunit, consequently reducing intracellular SAMe levels and diminishing leukemic cell growth in physiologic levels of methionine, both in vitro and in vivo. These cells also showed significantly higher apoptosis than control cells even at high methionine levels. Whereas control transduced Jurkat cells showed heightened engraftment in a hyper‐immunodeficient mouse model, leukemic cells where gene expression was ablated failed to engraft for up to 5 weeks. We predict that it may be possible, via dietary control of methionine intake; to adjust methionine levels in a way that would rescue normal cells while selectively induce death and apoptosis of leukemic cells that do not express MAT regulatory subunit.
The expression of pyruvate dehydrogenase kinase 1and 3 promotes metabolic switch and drug resistance in cancer cells
Authors: Lu Chun‐Wun , Lin Shih‐Chieh , Chen Ko‐Fan , Wu Chun‐Wei , Tsai Shaw‐Jenq
Published: June 2021

Metabolic switch from oxidative phosphorylation to aerobic glycolysis is a common phenomenon in cancer cells. However, the underling mechanism is still unclear. Pyruvate dehydrogenase kinase (PDK) inactivates pyruvate dehydrogenase to block tricarboxylic acid (TCA) cycle initiation. Using a bioinformatic approach we identified candidate HRE elements in PDK 1 to 3, but not in PDK4. Therefore, we hypothesized that hypoxia may regulate the expression of PDK to modulate cellular metabolism in cancer cells. Promoter activity assay demonstrated that promoter activities of PDK1 and PDK3 were elevated by hypoxia, and it could be reverse by mutation of HRE. Hypoxia and chemical drugs, such as DFO and DMOG, increased the expressions of PDK1 and PDK3 in several cancer cell lines, such as HeLa and neuroblastoma cancer cell lines. SiRNA assay demonstrated that the hypoxia‐elevated expression of PDK3 was mediated by HIF‐1α, but not by HIF‐2α. Chromatin immunoprecipitation assay provided further evidence that HIF‐1α bound to the PDK3 promoter in vivo. It was known that hypoxia increased lactate production in cancer cells. The hypoxia‐increased lactate production was reversed by knocking down the expression of PDK1 and PDK3. Moreover, lactate production was increased in HeLa cells which transfected with PDK3 under normoxia condition. Furthermore, cells overexpressed PDK3 were more resistance to anticancer drugs. These data demonstrated that increased expression of PDK1 and PDK3 beta;Ψ HIΦ–1α may play important roles in hypoxia‐mediated metabolic switch and drug resistance.
Androgen‐dependent Activation of Human Cytomegalovirus IE promoter in Prostate Cancer Cells.
Authors: moon jong‐seok , lee min‐young , park byeong‐woo , kim kyung‐sup
Published: June 2021

Human cytomegalovirus (HCMV) is a member of the herpesvirus family, which is sexually transmitted in adults. HCMV immediate early (IE) promoter is strongly activated by androgen agonist, R1881, in androgen‐dependent prostate cancer cells, LNCaP. However, IE promoter is not activated at all by R1881 in other cancer cells, which are forced to express AR. The activation of IE promoter by R1881 coincided with the changes of PKA activity and could be abbreviated by treatment with the inhibitor of PKA or the mutation of multiple copies of CRE. These results indicate that the activation of IE promoter by androgen results from the increase of PKA activities. Treatment with R1881 in LNCaP cells strongly suppressed the net production of prostaglandin E2, but enhanced the ability to activate PKA in response to PGE2 by expressing the EP4, the receptor for PGE2. This fact suggest the possibility that EP4 expression by androgen enables the cells to react to plentiful PGE2 derived from surrounding normal tissue, then activating PKA and IE promoter. The expression of catalytic beta II subunit of PKA by R1881 treatment, in a similar pattern to changes of IE promoter activities, suggest that the regulation of the expression of PKA catalytic beta II subunit might be direct upstream factor in IE promoter regulation. Taken together, present study demonstrated that HCMV IE promoter could be sustainedly activated in androgen‐dependent prostate cancer cells, which directs the expression of IE1/2 proteins containing oncogenic properties and proposed the possibility that HCMV be involved in oncogenesis in prostate.
Gene profiling of the liver in high carbohydrate‐ and high fat diet‐fed mice unravels novel aspects of the disease
Authors: Kirpich Irina , Deaciuc Ion , Song Zhenyuan , McClain Craig
Published: June 2021

Alcoholic and non‐alcoholic fatty liver (NAFL) have similar phenotypical appearance, i. e. the presence of vacuolar fat in the hepatocyte. The most widespread diagnostic tool remains histological examination. New technologies such as gene profiling of tissues have brought new diagnostic tools for many diseases. We have performed gene profiling of NAFL of dietary origin in mice exposed to various diets and identified potential “signature genes”. Mice were exposed to two diets that produce NAFL: high carbohydrate (HCD) and high fat diet (HFD) which mimicked the composition of diets consumed by humans. At the killing, the animals displayed markers of metabolic syndrome and NAFL. Using Affymetrix and Agilent technology, we identified two major groups of genes that underwent expression changes. One group can be linked to the diet composition, called “adaptive genes”. The other, a much larger group, called “non‐adaptive genes” cannot be linked to the metabolic pathways of lipids or carbohydrates. These genes may be considered as consequential to NAFL. They unravel novel, never thought‐of aspects of the NAFL disease. Our experimental results allow a distinction to be made between NAFL of various dietary origins. Also, they may be suggestive for the clinician in an attempt to establish a diagnostic of the disease as a function of its origin.
Association of IκB‐α of polymorphism with development of asthma
Authors: Park Se‐Min , Chang Hun‐Soo , Park Choon‐Sik , Rhim Tae Youn , Jang An‐Soo , Lee Mi‐Young , Park Byung Lae , Uh Soo‐Taek , Shin Hyoung Doo , Chung Il Yup
Published: June 2021

The development of asthma is determined by the interaction between host susceptibility and a variety of environmental exposures. NF‐κ B activates many immunoregulatory genes. The I κB‐α is an endogenous inhibitor of NF‐ κB. This study examined the association between I κB‐ α gene polymorphisms and susceptibility to asthma.

The SNPs were genotyped by single base extension method in Korean population. RT‐PCR and luciferase reporter assay were performed. Iκ B‐α activity was evaluated with EMSA.

One novel SNP (+3075 G>A) was discovered, and the five reported SNPs were found in the Korean population. The frequency of rare allele (−689 A>T) was significantly lower in asthmatics than normal controls (OR = 0.516, p=0.037), especially in atopic subject (p=0.007). The mRNA level was significantly higher in B cell lines of I κB‐α −689 TT type than those of AA type (p=0.024). The luciferase activity of c.‐689 T type of I κB‐α promoter was significantly higher than that of A type (p= 0.002). A nuclear protein showed stronger DNA binding affinity with a probe containing c.‐689 A than that of T.

Our data suggest that the I κB‐α −689 A>T SNP participates in the regulation of I κB‐α expression by providing a potential binding site for a repressor, and that the I κB‐α −689 A>T SNP may predict asthma phenotypes.

Funded By: the Korea health 21 R&D project. Ministry of Health & Welfare. Republic of Korea (01‐PJ3‐PG6‐01GN04‐0003).
Partial LXR agonist reduces atherosclerosis in ApoE‐deficient mice without inducing liver steatosis and hypertriglyceridemia
Authors: Kratzer Adelheid , Buchebner Marlene , Becker Tatjana M. , Pfeifer Thomas , Uray Georg , Ebner Birgit , Rathke Nora , Ahammer Helmut , Trauner Michael , Hoefler Gerald , Levak‐Frank Sanja , Levi Moshe , Kostner Gerhard M. , Kratky Dagmar
Published: June 2021

Most LXR agonists not only activate genes relevant for the reverse cholesterol transport but also increase lipogenesis. We treated ApoE‐deficient mice with the partial steroidal LXR agonist N,N‐dimethyl‐3β‐hydroxy‐cholenamide (DMHCA). Long‐term administration of DMHCA reduced lesion formation by 20% in male and 22% in female ApoE‐null mice without inducing hypertriglyceridemia or liver steatosis. DMHCA treatment slightly reduced not only plasma total cholesterol, but also plasma triglyceride (TG) concentrations and did not increase hepatic TG levels in male nor female ApoE‐deficient mice. The gene expression levels of ATP binding cassette transporters ABCA1 and G1 and cholesterol 7α‐hydroxylase were increased, while SREBP‐1c mRNA and protein quantity was unchanged. Thus, our data provide evidence that DMHCA is a useful therapeutic agent for the treatment or prevention of atherosclerosis. Currently, we are also evaluating a) potential target genes which are differentially regulated between DMHCA and the non‐steroidal LXR agonist T0901317 and b) short‐ and long‐term treatment of DBA mice with DMHCA.

This work was supported by the Austrian Federal Ministry of Education, Science, and Culture, GOLDII, C6.
Over‐expression of monoacylglycerol lipase (MGL) in mouse small intestine results in an obese phenotype
Authors: chon su‐hyoun , Zhou Yin Xiu , Quadro Loredana , Storch Judith
Published: June 2021

We recently demonstrated that, in addition to the well known anabolic metabolism of intestinal MG, MG hydrolytic activity and expression of the MG lipase (MGL) gene were present in rodent small intestine. The function of small intestinal MGL is unknown. We therefore generated transgenic mice (iMGL mice) that over‐expressed MGL specifically in small intestine using the intestinal fatty acid binding protein promoter. Marked induction of transgenic MGL mRNA was found in transgenic animals and intestinal specific expression of the transgene was confirmed. Western analysis and MGL activity measurements were also performed. Following 3 weeks of high fat feeding (40% Kcal), body weight gain and total fat pad weight were significantly increased in iMGL mice compared to non‐transgenic littermates. DXA measurements showed that body fat mass was significantly higher in the iMGL mice whereas lean mass was not altered. Triacylglycerol (TG) content of intestinal mucosa and liver were also increased in iMGL mice. Plasma TG levels in iMGL mice were high but free fatty acid and total cholesterol levels were not changed. The metabolism of 14C oleate and 3H monoolein in small intestinal mucosa were not substantially altered, although less labeled MG remained following in vivo incubation, likely due to the action of the MGL transgene. Food intake data showed that the iMGL mice were hyperphagic. Based on this phenotype, we hypothesize a role for intestinal MGL in whole body energy balance, possibly via regulation of food intake.
Mammalian acetyl‐CoA carboxylase polymerizes in association with tubulin
Authors: Lee Weissy Michele , Brownsey Roger William
Published: June 2021

Acetyl‐CoA carboxylase (ACC) catalyses the formation of malonyl‐CoA, an essential substrate for fatty acid synthase and a potent inhibitor of fatty acid oxidation. Control of ACC activity has important implications for overall energy metabolism and involves complex regulation via gene expression, allosteric mechanisms and multi‐site phosphorylation. A classic characteristic of ACC is allosteric activation by carboxylic acids, leading to the formation of filamentous polymers (up to 5–10‐MDa). Here, we test the hypothesis that ACC polymerization involves additional proteins. ACC polymers were purified by size fractionation and subjected to tandem mass spectrometry to identify associated proteins. Major ACC polymer‐associated proteins included tubulin and fatty acid synthase. The association between ACC and tubulin was validated by co‐immunoprecipitation and confocal microscopy. In reconstitution studies, purified tubulin and/or GTP had no direct effect on catalytic properties of ACC. However, based on immunocytochemical analysis, association with tubulin may be sensitive to the phosphorylation state of ACC. In conclusion, intracellular control of ACC involves complex protein‐protein interactions. These studies were supported by the Canadian Institutes for Health Research and by Scholarships from UBC and the Natural Sciences and Engineering Research Council (WML).
Parameters Affecting Cell Binding to Lectin Beads in Carbohydrate‐Based Drug Development
Authors: Zem G. C. , Goldstein L. , Lim J. , Petrosyan S. , Tanaka S. , Kravtsova A. , Allen J. , Guzelyan K. , German F. , Dayan M. , Mejia D. , Heravi M. , Ebrahimi R. , Davoodi H. , Rajaei A. , Nguyen Q. , Gonzales F. , Basmadjian S. , Jahanbakhsh A. , Zhamkochyan S. , McNee Z. , Keihani K. , Herr C. , Estrada J. , Hatanian F. , Babakhanian M. , Abedian H. , Benshian J. , Oppenheimer S. B.
Published: June 2021

Using a model system of washed yeast (Saccharomyces cerevisiae) that possess mannose‐rich cell surfaces and washed concanavalin A derivatized agarose beads that preferentially bind to glycans containing mannose/glucose residues, we tested 4 concentrations of alpha methyl mannose and the same 4 concentrations of D‐melezitose (0.025 mM – 25 mM) and a 50/50 mixture of these 2 sugars at the same final concentrations, over a 30 minute time course, assaying how much yeast remained bound to the beads at 10 minute intervals with and without sugar at each sugar concentration tested. In 15,379 replicate experiments, averaging about 300 trials for each sugar at each time tested, alpha methyl mannose and D‐melezitose caused dissociation of yeast from the Con A beads in a concentration dependent manner with an almost linear time course with little dissociation occurring immediately and incrementally increased dissociation occurring after 10,20 and 30 minutes. The highest sugar concentration (25 mM) tested here was most effective in causing dissociation and the 50/50 mixture of sugars was less effective than the single sugars in causing dissociation at the lower concentrations (less than 25 mM). The results suggest that careful attention should be paid to sugar concentration and incubation times in experiments using lectin derivatized beads in molecular purification protocols and in development of carbohydrate‐based drugs and diagnostic tests (Supported by NIH NIGMS SCORE (S06‐48680, NIH MBRS RISE, NIH MARC and the Joseph Drown Foundation).
Regulation of the rat intestinal phosphate transporter NaPi‐2b by dietary phosphate
Authors: Giral Hector , Breusegem Sophia , Barry Nicholas , Sutherland Eileen , Brodsky Kelley , Wilson Paul , Wang Xiaoxin , Jiang Tao , Levi Moshe
Published: June 2021

Elevated serum phosphate (Pi) remains a clinical challenge in patients with chronic kidney diseases. Pi homeostasis is determined by intestinal absorption and renal excretion. Intestinal Pi absorption is mediated by sodium‐dependent Pi uptake across the apical membrane of enterocytes by the NaPi‐2b transporter. Studies in mice have shown expression of NaPi‐2b mainly in the ileum. In contrast, in rats absorption of Pi occurs in the duodenum and jejunum, but not in ileum, a pattern similar to humans. Protein studies in rats have been not possible because of a lack of suitable antibodies. We report here the use of a specific antibody used successfully in rats for the study of the regulation of NaPi‐2b. We found that NaPi‐2b transporter mRNA and protein is mainly expressed in jejunum, with lower levels in duodenum and negligible amounts in ileum. NaPi‐2b expression as well as Na‐dependent Pi uptake was increased in rats adapted to a chronic low Pi diet (0.1% Pi) both in duodenum and jejunum. All these results were confirmed by immunofluorescence microscopy in intestinal sections. In addition, we studied mRNA expression of the type III NaPi transporters: Pit‐1 is highly expressed in all three segments and Pit‐1 mRNA is regulated by dietary Pi. In conclusion, regulation of intestinal Pi transport and NaPi‐2b by dietary Pi in rats is concentrated at duodenum and jejunum sections. This research was supported by NIH and AHA.