bluuebunny/crossref_metadata_2025_split huggingface dataset:
The transcription factor Erg regulates angiogenesis and endothelial apoptosis
Authors: RANDI ANNA MARIA , Birdsey Graeme , Dryden Nicola , Amsellem Valerie , Gebhardt Frank , Haskard Dorian , Dejana Elisabetta , Mason Justin Charles 
Published: June 2021

Tight regulation of the balance between apoptosis and survival is essential in angiogenesis. The Ets transcription factor Erg is required for angiogenesis. Inhibition of Erg expression in human umbilical vein endothelial cells (HUVEC) using antisense resulted in detachment of cell‐cell contacts. A key regulator of junction stability in EC is VE‐cadherin. Using chromatin immunoprecipitation (ChIP), we found that Erg binds the VE‐cadherin promoter in HUVEC; moreover, inhibition of Erg expression in HUVEC resulted in a significant decrease in VE‐cadherin levels. Both Erg and VE‐cadherin provide survival signals to EC; apoptosis induced by inhibition of Erg was partly rescued by over‐expression of VE‐cadherin‐GFP, suggesting that VE‐cadherin is involved in the Erg‐dependent survival signals. Additional survival signals mediated by Erg may be via the anti‐apoptotic molecule Bcl‐2, since Erg binds the Bcl‐2 promoter and inhibition of Erg expression in HUVEC results in decreased Bcl‐2 levels. To validate the role of Erg in angiogenesis in vivo, we used Erg siRNA in a Matrigel plug model. Matrigel, mixed with bFGF and either Erg or control siRNA, was injected subcutaneously in mice. Erg inhibition resulted in a significant decrease in vascularization of the plugs, as measured by H&E and CD34 staining. These results identify a new pathway regulating angiogenesis via Erg, VE‐cadherin and endothelial survival.Research supported by the British Heart Foundation
Pharmacokinetic studies of four novel soluble epoxide hydrolase (sEH) inhibitors and anti‐inflammatory efficacy of the most promising one t‐AUCB
Authors: Liu Junyan , Tsai Hsing‐Ju , Hwang Sung Hee , Jones Paul D. , Morisseau Christophe , Hammock Bruce D. 
Published: June 2021

Earlier sEH inhibitors such as AUDA are effective anti‐hypertensive and anti‐inflammatory agents in multiple animal models. However, their poor metabolic stability and water solubility make them difficult to use pharmacologically. For obtaining a compound easy to use in vivo, we present the PK profiles of four conformation‐restricted sEH inhibitors and anti‐inflammatory efficacy of the most promising one.Better PK parameters (higher Cmax, longer t1/2, and greater AUC) were obtained from the four tested inhibitors than AUDA with t‐AUCB showing the best result. Oral bioavailability of t‐AUCB (0.1 mg kg−1) was 75±12% (n=4). Furthermore, when mice were administrated t‐AUCB in drinking water at 4 mg L−1, a fairly stable blood concentration of ~70 nM t‐AUCB was maintained since one day administration, and was totally cleared from blood after 3 days of drug withdrawal. Finally, oral gavage of 0.5 and 1.0 mg kg−1 t‐AUCB were effectively anti‐inflammatory evidenced by reversing LPS‐induced hypotension in a dose‐dependent manner, and significantly shifting the ratio of lipid epoxides to corresponding diols in plasma. t‐AUCB appears around 10 times more orally active than AUDA in this model. Thus t‐AUCB is promising for further in vivo studies related to the biology of sEH and its potential therapeutic applications.
Expansion of the Eicosanoid Profiling Methodology by the Addition of w‐3 Fatty Acid Metabolites
Authors: Georgi Katrin , Hwang Sung‐Hee , Nording Malin , Yang Jun , Zivkovic Angela , Pedersen Theresa , Newman John W , Hammock Bruce D 
Published: June 2021

Eicosanoids, a large group of arachidonic acid (AA) metabolites, play an important role in several physiological processes such as inflammation or hypertension. They are produced via three different pathways by cytochrome P450, lipoxygenase and cyclooxygenase enzymes. It has been established that these enzymes metabolize not only AA but also other poly unsaturated fatty acids (PUFAs), i.e. eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA).Recent studies indicate that w‐3 fatty acids and their metabolites play an important protective role in multiple inflammatory conditions including IgAN. We are expanding our analytical method by adding separate platforms for lipid amides and leukotrienes but also by adding more targets to the oxylipin method. For example we incorporated metabolites of the three most important w‐3 fatty acids (EPA, DHA and a‐linolenic acid) in our existing eicosanoid profiling methodology. Thus, we developed a precise and sensitive method using LC‐MS/MS to identify and quantitate 88 oxylipin metabolites in plasma samples. This work was supported by NIEHS SBRP Grant P42 ES04699.
Using Low‐Carbohydrate Diet to Teach Metabolic Pathways to Undergraduates and Medical Students
Authors: Pogozelski Wendy K. , Makowske Mary , Feinman Richard D. 
Published: June 2021

The need to understand metabolic pathways has never been greater, given recent increases in obesity‐related illnesses. We previously described how discussion of low‐carbohydrate (low‐CHO) diets can assist undergraduates1 and medical students2,3in learning metabolism. Here we describe further benefits of this approach.The medical establishment largely holds to low‐fat diets as a treatment for obesity, diabetes and metabolic syndrome but the method is under attack by a minority documenting benefit of CHO restriction as a preferred approach. We use this controversy to engage student interest and to provide context.The underlying principle of low‐CHO diets lies in control of insulin and relief of insulin resistance. The role of insulin ties together many disparate pathways. Discussion of metabolic syndrome and its dependence on insulin response reinforces the approach. The method has been a highly effective tool for maintaining student interest in metabolism.
Neuropathological phenotype of a new type of lissencephaly associated with mutations of TUBA1A
Authors: Fallet‐Bianco Catherine , Loeuillet Laurence , Loget Philippe , Chapon Françoise , Poirier Karine , Chelly Jamel , Francis Fiona 
Published: June 2021

In 2007, Keays et al identified in an ENU‐induced mutant mouse, a mutation in Tuba1a, a gene coding for alpha 1 tubulin protein, associated with behavioral disorders and a disturbance of the cortical cytoarchitectony. Subsequently, mutations of TUBA1A were found in children with mental retardation and lissencephaly with abnormalities of the corpus callosum and cerebellum, on MRI. Recently, mutations of TUBA1A were found in 4 fetuses, with a prenatal diagnosis of cerebral dysgenesis leading to a medical termination of pregnancy according to French laws.These fetuses, not mutated for LIS1, DCX and ARX, were studied at 23, 25, 26 and 35 gestational weeks respectively. The neuropathological study demonstrated a phenotype including abnormalities affecting constantly the neocortex, hippocampus, corpus callosum, cerebellum and brainstem, and inconstantly, basal ganglia, olfactory bulbs and subventricular germinal zones. This phenotype was different from that of LIS1, DCX, ARX lissencephalies, and in agreement with the pattern of expression of TubA1A studied in the mouse embryo. Compared with children mutated for TUBA1A, fetal cases are located at the most severe end of the spectrum, suggesting that prenatally diagnosed cases are probably the most severe forms of TUBA1A lissencephaly.
HEPATITIS C VIRUS: Effect Of NS5A On The NS5B in vitro Replication Reaction
Authors: Quezada Elizabeth M , Kane Caroline M 
Published: June 2021

The Hepatitis C virus replicates its viral RNA template by a process that involves a protein complex containing the RNA dependent RNA polymerase (RdRp) NS5B, the helicase NS3, the membrane inserted protein NS4, and the phospho‐protein NS5A along with a number of host proteins. In vitro studies of the replication reaction have shown that the rate of nucleotide incorporation of NS5B is slow when compared to other DNA dependent RNA polymerases, suggesting that other factors such as host or non structural proteins, membrane structures, cis‐elements in the template or a combination of these factors are required to achieve higher rates. To understand how NS5A stimulates NS5B, we have developed an in vitro assay to study the effect of NS5A in the replication reaction.The NS5A recombinant protein was expressed and highly purified from bacterial cells. Using gel filtration, two isoforms of different molecular weight were isolated. The multimeric form stimulates the in vitro NS5B activity in a concentration and in an order of addition dependent manner. The monomeric isomer also will be tested. A pre‐incubation step with NS5B is required for the enhancement effect of NS5A on the replication reaction. Using the most terminal 98 nucleotides of the viral 3’UTR genome, NS5A stimulates the replication reaction three fold. Further studies using templates containing structural elements of the 3’UTR of the positive strand and negative strand are being tested. Stimulation is also seen with a synthetic RNA template. With this latter template, individual steps in replication can be studied; and preliminary data suggests that the activation by NS5A seems to occur at the elongation phase. In addition, the pause profile changes when NS5A is present in the reaction. We will explore in detail these features of the stimulation mechanism as well as analyze the affinity of the template to the complex NS5A‐NS5B.
Substrate specificity and conformational preferences of prolyl 4‐hydroxylase
Authors: Gorres Kelly L , Raines Ronald T 
Published: June 2021

Prolyl 4‐hydroxylase (P4H) catalyzes the post‐translational hydroxylation of proline residues in procollagen strands. P4H specifically forms 4(R)‐hydroxyproline (Hyp) from proline residues in Pro–Gly sequences of polypeptides. The presence of Hyp is crucial for the folding, secretion, and stability of the collagen triple helix under physiological conditions.The current study examines the substrate recognition and specificity of P4H. We have synthesized peptide substrate analogs containing 4(R)‐fluoro‐L‐proline (Flp), 4(S)‐fluoro‐L‐proline (flp), and 4‐thio‐L‐proline (Thp). The peptide containing Flp was not found to interact with P4H, whereas the flp‐ and Thp‐containing peptides were determined to be substrates of P4H, forming products containing 4‐keto‐L‐proline and 4(R)‐sulfoxoproline. These results suggest that the cis/trans conformation of the substrate is a factor in recognition by P4H, and provide evidence for a role of conformational changes in collagen biosynthesis.This work is supported by Grant AR44276 (NIH). KLG was supported by the NIH Chemistry–Biology Interface training program.
Novel method for fast immunodetection using vacuum filtration
Authors: Gutierrez Sara , Rapiejko Peter , Emerick Marc , Sharma Ajay 
Published: June 2021

Western blotting is a well establish technique used for the routine detection of specific proteins from complex mixtures. The immunodetection step of western blotting is a long and tedious process, involving multiple changes of solutions, and is usually extended over several hours to overnight. This paper describes an innovative, vacuum‐based immunodetection approach, which allows the detection of the protein in less than 45 minutes. The system can be used for rapid antibody optimization studies, antibody screening, simultaneous detection of different proteins in multiples blots, or routine western blot analysis. Vacuum filtration enables efficient blocking and washing of the membrane, speeds up the overall process and make the system easy to use. Blocking of the membrane occurs rapidly in the presence of low concentration of non fat dry milk or other common blocking reagents. Since incubation steps are typically performed in ten minutes, users can sequentially process several blots in a day. The system in compatible with PVDF or nitrocellulose membranes and detection can be accomplished by any chemiluminescence or fluorescence detection method. The SNAP id™ system is a novel immunodetection system that uses the basic steps of the standard immunodetection protocol, is easy to use and offers a substantial reduction in processing time.This research has been totally funded by Millipore Corporation.
Influence of Complex Nucleotide Substance from baking yeasts on selective cells cytotoxicity.
Authors: Orlova Elena V. , Orlova Valentina 
Published: June 2021

It was shown that Complex Nucleotide Substance (CNS) from Saccharomyces cerevisiae been added in final concentration 0.2 ‐1.0 mg/ml to HeLa, U937, MCF7 and L929 cells culture gave cytotoxic effect in all these cell types but not on human leucocytes.CNS also activated caspase‐3 in dose‐dependent manner in all the above cancer cell lines.Under cell‐cytometry investigations CNS increased apoptotic cells quantity and decreased the number of G2/M of all cancer cells lines.Thus, we can conclude that CNS is a selective apoptotic agent and it could give good results in cancer therapy.
EVI5 is a cytokinesis‐associated protein with dynamic subcellular localization
Authors: Faitar Silviu Leonard , Dabbeekeh Jeremy TS , Cowell John K 
Published: June 2021

The EVI5 gene was originally identified in a screen for integration sites of ectopic viruses that gave rise to tumors in mice. The product of the gene is an 810 amino acids long ubiquitously expressed protein. It carries two functional domains, a Tre2/Bub2/Cdc16 (TBC) consensus sequence in the N‐terminal region, which is known to have a GTPase activating function, and an extensive coiled coil in the C‐terminal region. The goal of the present study was to elucidate the cellular localization and function of the EVI5 protein. A highly specific antibody against the C‐terminal section of the protein was used in an immunocytochemical analysis, and demonstrated that EVI5 is a nuclear protein, that is also present in the pericentriolar region of interphase cells. EVI5 is localized to the mitotic spindle during chromosome migration, and is also found in the midzone and midbody in late mitosis and during cytokinesis. Knockdown of EVI5 using siRNA results in a high incidence of binucleate cells with evidence for incomplete septation. The role of EVI5 in cytokinesis is further supported through the demonstration of its interaction with members of the chromosome passenger complex (INCENP, aurora B kinase and survivin). Taken together, these results suggest that EVI5 is involved in the completion of cytokinesis.This work was supported by NINDS grant NS35791 from the National Institutes of Health.
Influence of vitamin D on androgen receptor‐mediated signaling in LNCaP prostate cancer cells
Authors: Rybarczyk Megan Michelle , Russell Nicholas , Mordan Lawrence , Welsh JoEllen , Tenniswood Martin 
Published: June 2021

The efficacy of the anti‐androgen Casodex relies on the interaction with the androgen receptor (AR). This study is designed to elucidate the interactions between AR signaling and the vitamin D receptor (VDR) signaling pathways. Using the LNCaP prostate cancer cell line as a model of early stage prostate cancer, we have shown that Casodex induces apoptosis in a time dependent manner which is co‐incident with the loss of the AR from the nucleus, in agreement with previous studies. While 1,25(OH)2D3 induces apoptosis in this cell line in the absence of exogenous testosterone, in the presence of testosterone, treatment with 1,25(OH)2D3 induces cell cycle arrest, but even at doses as high as 100 nM there is no evidence of apoptosis. Furthermore, in the presence of testosterone, 1,25(OH)2D3 blocks the Casodex‐mediated degradation of the AR and abrogates the induction of apoptosis by Casodex. These data suggest that there is significant cross talk between the AR‐mediated signaling and VDR‐mediated signaling that influences the susceptibility of the cells to anti‐androgen induced cell death. Changes in the expression of several p73 isoforms, monitored by Western blotting, correlate with the sensitivity of the cells to Casodex, suggesting that the responsiveness of tumors to anti‐androgens may be inversely related to vitamin D status.(Supported by USPHS RO1 CA101114‐04 and the Coleman Foundation)
Kinetics of EGF receptor dimer formation
Authors: Yang Katherine S , Pike Linda J 
Published: June 2021

The EGF receptor is thought to exist in the membrane as a monomer that dimerizes in response to ligand binding. Studies on the relationship between ligand binding and dimer formation have been hampered by the lack of an assay that can follow receptor dimerization in living cells in real time. Here we report the use of a split‐luciferase enzyme complementation assay to study the kinetics of EGF receptor dimerization in intact cells. For the assay, the EGF receptor was C‐terminally fused to the N‐terminal or C‐terminal half of firefly luciferase and both constructs transfected into CHO cells. In the absence of ligand, significant basal light production was observed. Addition of EGF led to a rapid decrease in light production during the first two minutes, followed by a rebound in light production over the ensuing 3 to 5 min. The data suggest that under resting conditions, the C‐terminal tails of the EGF receptor are in proximity to each other, possibly in pre‐formed dimers, and that ligand binding induces a conformational change that disrupts these interactions but ultimately allows the formation of an alternative oligomeric state of the receptor.This work is supported by NIH grant GM64491to LJP.
Protein kinase C ζ‐dependent LKB1 phosphorylation at serine 428 induces LKB1 nuclear export and apoptosis in endothelial cells
Authors: Song Ping , Xie Zhonglin , Wu Yong , Xu Jian , Dong Yunzhou , Zou Ming‐Hui 
Published: June 2021

LKB1 is a serine‐threonine protein kinase that, when inhibited, may result in unregulated cell growth and tumor formation. However, the mechanisms underlying regulation of LKB1 are unknown. The aim of the present study was to define the upstream signaling molecules responsible for peroxynitrite (ONOO‐)‐induced LKB1 phosphorylation. Exposure of cultured human umbilical vein endothelial cells (HUVECs) to pathological concentrations of ONOO‐ significantly increased the phosphorylation of LKB1 at Ser428 and protein kinase C [zeta] (PKC[zeta]) at Thr410. These effects were accompanied by, increased activity of the lipid phosphatase PTEN, decreased activity and phosphorylation (Ser473) of Akt, and induction of apoptosis. In LKB1‐deficient HeLa S3 cells or in HeLa S3 cells transfected with kinase‐dead or inactive LKB1, ONOO‐ enhanced Akt‐Ser473 phosphorylation. Overexpression of wild‐type LKB1 in these cells mimicked the effects of ONOO‐ in HUVECs. Further analysis revealed that PKC[zeta] directly phosphorylated LKB1 at Ser428 in vitro and in HUVECs, resulting in increased PTEN phosphorylation at Ser380/Thr382/383. ONOO‐ also enhanced PKC[zeta] nuclear import and LKB1 nuclear export. We conclude that PKC[zeta] mediates LKB1‐dependent Akt inhibition in response to ONOO‐, resulting in endothelial apoptosis.This work was supported by NIH grants (HL079584, HL07439, and HL080499).
Identification of a New Target in the Interactions between Staphylococcal Enterotoxin B with the Renal Proximal Tubule Epithelial Cells
Authors: Hammamieh Rasha , Sumaida Dena , Miller Stacy , Lin Yixin , Morris Sam , Jett Marti 
Published: June 2021

Staphylococcal eneterotoxin B (SEB) a monovalent T cell mitogen and inducer of T suppressor cells, is known to form complexes with the major histocompatibility complex class II and T‐cell receptor in a manner that is quite different from the way the natural ligands bind to the same receptors and direct normal cellular responses it bypasses the normal route of antigen processing by binding as an intact protein to the complex formed by the MHC class II receptor on the antigen‐presenting cell and the T cell receptor.It is estimated that within 90 min. post‐exposure to SEB, >75% of the toxin is cleared to the renal tubule epithelial cells. We hypothesize that SEB binds to these cells using a mechanism that does not include MHCII.Binding assays carried out in vitro in proximal tubule epithelial cells suggests that glycosphingolipids and lipid rafts play a major role in SEB interactions with these cells.CD1d is an MHC class I homologous protein that is present in the plasma membrane lipid rafts and is proposed to be involved in presenting the antigen to the NKT cells.We have studied SEB interactions with the proximal tubule epithelial cells using imaging and direct protein‐protein interaction studies and showed that SEB can interact with CD1d on the proximal tubule epithelial cells.
Altered Behavior in the Novelty‐Induced Hypophagia Test in FoxG1+/− Mice
Authors: Flores Natasha Mahealani , Beech Robert 
Published: June 2021

For this study we used mice with a heterozygous deletion of the FoxG1 gene to examine the relationship between adult neurogenesis and symptoms of depression as modeled by the Novelty‐Induced Hypophagia test. The mice were given a 0.2 mg/ml dosage of fluoxetine through drinking water for 3 weeks. Testing occurred in both the home cage and novel cage. We did not find an effect of antidepressant treatment on behavior in either the FoxG1+/− mice or wild type mice. Compared to wild type controls the FoxG1+/− mice displayed transgenic mice had decreased consumption of milk in the home cage as well as increased latency to feed. These are similar to two features of depression in humans, increased anxiety and decreased response to rewarding stimuli. Thus our results are consistent with the hypothesis that decreased neurogenesis is responsible for some symptoms of depression found in humans.This study was funded by Yale University's Summer Undergraduate Research Fellowship, The Leadership Alliance and Chaminade University of Honolulu.
A career management program for basic science junior faculty
Authors: Sukalski Katherine A , Fox LaVonne , Moulton Patricia , Flower Ann M , Burns Elizabeth E , McHugo Jeanie , Larson Annette 
Published: June 2021

A Career Management Program was implemented for new tenure track faculty (basic science and educator scholars) in a small community‐based medical school. The program was designed based on best practices in the literature to be a resource for faculty, complementing the mentoring provided by department chairs. Interactive panel presentations were led by senior faculty from basic science and allied health departments focusing on research, teaching, service, and strategic career planning. The basic science scholars also met as a cohort, with an experienced basic scientist to discuss specific issues raised in previous panel discussions. The program successfully completed year one with eleven junior faculty (five basic science) participating on a regular basis. The session evaluations resulted in 90% or higher for the value of the sessions and usefulness/transferability of the information to the participant's position and job responsibilities. This program demonstrates that a career management program can combine faculty from multiple tracks as long as they have opportunities to address career‐specific issues.Sponsored by the North Dakota Women's Health CORE, A National Center of Excellence in Women's Health Region VIII Demonstration Project, Elizabeth Burns, PI.