bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Biologic Properties and Ultra‐Microstructure in the Senescence of Endothelial Cell Induced by AngiotensinII
Authors: Bai Xiaojuan , Shan Haiyan , Chen Xiangmei 
Published: June 2021

ObjectiveThe aim of this study was to investigate the biologic effects and morphology senescence of human umbilical vein endothelial cell (HUVEC) induced by angiotensinII and its possible action mechanism.MethodsHUVEC were cultured in vitro and intervened by AngII. The cell living rate was measured by MTT assay; SA‐β‐gal stain and cell cycle analysis were used to identify cell aging status; the early stage apoptosis was detected by flow cytometry with Annexin V‐FITC/PI double staining, morphologic changes and percentage of apoptosis were assayed with acridine orange fluorescence staining and were observed under fluorescent microscope, transmission electron microscopy was used to evaluate the ultra‐microstructure of HUVEC.ResultsInduction of AngII promoted a growth arrest with phenotypic characteristics of cell senescence, such as enlarged cell shapes, increased senescence‐associated β‐galactosidase (SA‐β‐gal) staining and depressed cell proliferation. Apoptotic number and percentage was increased in senescent cell, which a small subset of the senescent cells showed aberrant morphology such as remarkable nuclear fragmentation or multiple micronuclei.ConclusionsOur results suggest cell apoptosis is possibly an important factor for the process of pathologic and physiologic endothelial cells senescence and vascular aging.
Transcriptional regulation in the MAPT H1/H2 Haplotype Block
Authors: Rhodes C. Harker , Haroutunian V , Katsel P , Tsongalis G J , Lefferts J A , Sibille E , Lathrop M J , Fiering S N 
Published: June 2021

The protective effect of the MAPT H2 haplotype against neurodegenerative diseases suggested that a genome wide screen comparing normal control individuals with and without an H2‐containing chromosome might provide insights into the molecular biology of those diseases.RNA from the anterior cingulate and amygdala from 18 individuals with no neurologic disease was examined by U133 expression array analysis. Four of the subjects were heterozygous for the MAPT H1/H2 haplotype, one subject was an H2 homozygote, and the remaining 13 were H1 homozygotes. Comparison of H1 homozygotes and H2 carriers revealed 5 transcripts which were expressed at significantly higher levels in H2‐carriers. This result was confirmed by a quantitative PCR assay of one of the transcripts and by U133 array analysis of RNA from multiple brain areas in an independent cohort. All five transcripts map to the 1 Mb inversion containing the MAPT locus. None of the transcripts comes from a named gene or contains a known miRNA.The coordinated up‐regulation of these transcripts suggests a global difference in transcriptional regulation in the H1 and H2 chromosomes. This hypothesis is in contrast to, but is not mutually exclusive with, the usual explanation that the protective effect of the H2 haplotype is due to specific functional polymorphisms.
GABAergic circuits in the hippocampus
Authors: Klausberger Thomas 
Published: June 2021

In the hippocampal CA1 area a relative homogenous group of pyramidal cells is accompanied by 21 different classes of GABAergic interneurons. Different interneurons make synapses with distinct compartments of pyramidal cells, but why is such a large diversity of distinct interneurons required?We recorded from identified interneurons in vivo and showed that interneurons targeting different domains of pyramidal cells exhibited distinct firing patterns. The spike timing of parvalbumin expressing basket and axo‐axonic cells indicates roles in synchronizing the sub‐threshold oscillations and output of pyramidal cells, respectively. The spike timing of bistratified and O‐LM cells, which innervate dendrites of pyramidal cells in the stratum radiatum and lacunosum‐moleculare, respectively, suggests their role in phasing EPSPs and de‐inactivating voltage‐gated ion channels of pyramidal cells; bistratified cells also synchronise oblique pyramidal cells dendrites to field gamma oscillations. We discovered GABAergic neurons projecting to subiculum, retrosplenial cortex and medial septum, possibly supporting synchronisation across brain areas. Another class of interneuron expresses neuronal nitric oxide synthase and provides slowly‐rising GABAergic inhibition in pyramidal cells.Our results indicate that different GABAergic interneuron release GABA at distinct times to different domains of pyramidal cells explaining the need of diverse classes of interneurons.
A replicase sliding clamp bound to DNA and a novel chemical compound that inhibits DNA replication
Authors: O'Donnell Mike , Georgescu Roxana E , Kuriyan John , Kong Xiang‐Peng 
Published: June 2021

Cellular replicases utilize a sliding clamp that holds the replicase to DNA for high processivity. We have solved the structure of the E. coli β clamp in complex with a peptide of the Pol III replicase and compare it to the structures of β–δ and Pol IV‐β. In overview, all proteins that bind the clamp utilize a common protein binding pocket, although they also bind unique positions on β. Using this information we have devised a high‐throughput assay to screen chemical libraries for small organic chemicals that disrupt interaction between β and Pol III. We have obtained several compounds, some of which inhibit E. coli cell growth. The clamp binding pocket is conserved and some chemical compounds also inhibit gram positive clamp‐DNA polymerase interactions.We have also solved the structure of β clamp bound to primed DNA. The duplex is highly tilted within the ring. This may facilitate switching of DNA among different polymerases bound to one clamp. Surprisingly, template ssDNA fits into the protein binding pocket of β. In fact, a polymerase peptide competes the ssDNA off of β, implying that ssDNA binding to β serves a “placeholder” function that locks the clamp to the primed site after clamp loading. This interaction is severed upon association of Pol III to β, and this frees the clamp for rapid sliding on dsDNA during fast processive replication.(Funded by the NIH).
The level of nitrogen supply modulates the expression of arginine metabolizing enzymes in Caco‐2/TC7 intestinal cells.
Authors: Ventura Gabrielle , Moinard Christophe , Carrière Véronique , Chambaz Jean , Cynober Luc , De Bandt Jean‐Pascal 
Published: June 2021

Portal arginine (Arg) plays a key role in liver ureagenesis. Intestine ability to control Arg availability from the diet may depend on a modulation of arginase, glutaminase, ornithine aminotransferase (OAT), argininosuccinate lyase (ASL) and synthetase (ASS) expression, according to protein supply. The aim of this work was to study the influence of amino acid (AA) supply on the intestinal expression of these enzymes.Caco‐2/TC7 cells were exposed for 3 days to different media containing AA at 0 (0), 1 (1X), 2 (2X) or 4 (4X) fold plasma post‐prandial concentrations. Enzymes mRNA were quantified using real time PCR. 18S rRNA was used as an internal control. Results (mean ± SEM) were analyzed by linear regression.There was an inverse relationship between AA supply and glutaminase (p= 0,028), OAT (p= 0,035), ASS (p= 0,028) and arginase (p= 0,058) expressions.This study shows for the first time a direct effect of AA supply on transcriptional regulation of arginine metabolizing enzymes. Such a regulation would enable in situations of low protein supply to increase Arg catabolism and to decrease hepatic AA oxidation thus promoting nitrogen sparing.
Changes in skeletal muscle protein synthesis in trained adults during recovery from endurance exercise
Authors: Vislocky Lisa , Gaine P. Courtney , Pikosky Matthew , Bolster Douglas , Ferrando Arny , Wolfe Robert , Rodriguez Nancy 
Published: June 2021

Endurance exercise stimulates mixed muscle protein synthesis (MPS) for several hours post‐exercise. However, the time course of this response is not well characterized.PURPOSE: The purpose of this study was to characterize changes in MPS in trained persons following an endurance run.METHODS: Following an overnight fast, participants (n=14; men=9, women=5) underwent muscle biopsies at 30, 190, and 210 min after an endurance exercise bout (75 min, 70% VO2peak or 45 min, 65% VO2peak). MPS was determined using a primed, continuous infusion of [2H5]‐phenylalanine and muscle biopsies at 30 and 190 min (EARLY) and 190 and 210 min (LATE).RESULTS: LATE MPS values were significantly higher than EARLY values (0.237 ± 0.036 % / hr vs 0.067 ± 0.006 % / hr for LATE and EARLY, respectively; P < 0.001), with the eta squared statistic (0.048) indicating a large effect size of time. A strong positive correlation (r=0.571, P < 0.001) existed between time and skeletal muscle MPS values.CONCLUSION: These data document an increase in MPS three hours after completing an endurance exercise bout in the fasted state. Given that the source of amino acids to support the noted increase in MPS during the fasted state is endogenous proteins, studies should be conducted to evaluate the benefit of protein supplementation and feeding on post‐exercise on protein utilization during the recovery period.
Specificity of epithelial cell adhesion by Group A Streptococcus pilus proteins in the M1 serotype SF370 strain
Authors: Abbot Emily Louise , Smith Wendy D. , Pointon Jonathan A. , Chiriboga Carlos , Wilson Janet A. , Hirst Barry H. , Kehoe Michael A. 
Published: June 2021

Group A Streptococcus (GAS) express long polymeric pili which are encoded in the hypervarible FCT‐region. In the serotype M1 strain SF370 these pili are composed of a major subunit (Spy128) and two ancillary proteins (Spy130 and Spy125). Deletion mutants of each of the individual subunit genes failed to adhere to human tonsil epithelium or human keratinocytes (HaCATs), suggesting that pili are essential in promoting the initial stages of GAS infection. However, which of the subunits is functioning as the primary adhesin in this pilus‐dependent interaction remains undetermined. Each of the three pilus subunits were expressed as a recombinant protein and their ability to interact with various epithelial models was examined by immunofluorescence using subunit specific antisera. Both r130 and r125 proteins adhered to the surface of tonsil epithelium and HaCAT cells, however r128 failed to bind to either model. The ability of subunit‐specific antisera to reduce parent strain (WT) bacterial adherence to human tonsil or HaCAT cells was examined. Incubation of WT GAS with anti‐Spy125 sera inhibited the ability of GAS to adhere to either cell model. In contrast, anti‐Spy 128 and anti‐Spy 130 sera had no affect on bacterial binding. These results suggest that although both Spy130 and Spy125 proteins have the ability to bind host cells, pilus adhesion specificity resides in the Spy125 subunit.Sponsored by MRC.
Healthy School Lunch Behavior and the Invisible Hand
Authors: Wansink Brian , Just David , Payne Collin 
Published: June 2021

Restricting the use of prepaid lunch accounts use may improve food selection without eliminating free choice.In a school lunch cafeteria, 166 students were allowed to purchase their lunch in one of three ways: 1) cash only, 2) cash and an unrestricted debit card, or 3) cash and a restricted debit card that could be used to purchase items marked as healthy. Those given restricted debit cards ate significantly fewer calories than those given the unrestricted card (p=0.045), and they consumed healthy foods (78.6%, p=0.000).Because this payment procedure still allowed students to use their own cash to purchase less healthy options, it did not unduly restrict dining options or reduce cafeteria revenue.
Hierarchical contributions of IgM and complement C1q in protection against murine atherosclerosis
Authors: Lewis Myles Jonathan , Malik Talat H. , Ehrenstein Michael R. , Boyle Joseph J. , Botto Marina , Haskard Dorian O. 
Published: June 2021

Natural IgM antibodies and complement C1q have been implicated in atherogenesis through their ability to bind tissue debris such as denatured low‐density lipoprotein (LDL) and apoptotic cells. To investigate their relative contributions, mice deficient in serum IgM (sIgM), C1q or both C1q and sIgM were crossed with LDL receptor deficient mice (Ldlr−/−) and studied at 22 weeks. sIgM.Ldlr−/− mice showed increased lesion size in comparison to Ldlr−/− mice, both en face (~7‐fold, p<0.0001) and at the aortic root (~5‐fold, p<0.0001). Similar findings were noted on a high fat diet with a 98% increase en face (p=0.0009) and a 23% increase in aortic root lesion area (p=0.006) in sIgM.Ldlr−/− mice, and increased smooth muscle cell infiltration (p=0.007). En face and aortic root lesions were substantially larger in sIgM.Ldlr−/− than C1q.Ldlr−/− mice on either diet (all p<0.005), and the effects of combined C1q and serum IgM deficiency were no different to those of sIgM deficiency alone. Thus although IgM antibodies and C1q are both significantly atheroprotective, IgM makes a more prominent contribution. The major mechanism of protection by IgM therefore appears to be independent of classical pathway activation.Funded by the Wellcome Trust and British Heart Foundation
Total urinary bladder regeneration with restoration of native structure and pharmacological response
Authors: Bertram Timothy A , Christ George J , Wagner Belinda J , Jain Deepak , Aboushwareb Tamer , Ludlow John , Payne Richard , Jarajapu Yagna , Turner Chanda , Jayo Manuel J 
Published: June 2021

Neo‐bladder histology and pharmacological response were investigated in 7 dogs at 51 (n=1), 89 (n=3), and 179 (n=3) days (d) after radical cystectomy and implantation of a Neo‐Bladder Replacement Construct (Construct). Pharmacological responses of neo‐bladder tissue strips (51, 89, and 179 d; 3, 18, and 27 strips, respectively) were compared to age‐matched native bladder (n=13; 49 strips).By 51 d post‐implantation (p.i.) regenerated bladder was histologically consistent with native bladder including mucosal and serosal linings, detrusor muscle, vascular, and nerve composition. Logistic analysis revealed similar EC50 and slope factor values for contraction of bladder tissue strips derived from all groups induced by carbachol (Car) and electrical field stimulation (EFS). A statistically significant and progressive increase in the mean Emax values occurred in response to both Car and EFS over time. While contractile responses to Car and EFS increased over time, they were lower than native bladder responses at 51 and 89 d p.i. Car‐induced contractions, but not EFS‐induced contractions, were equivalent to native tissue at 179 d p.i.A Neo‐Bladder Replacement Construct is capable of regenerating urinary bladders in vivo with histological and pharmacological responses similar to native bladder by 179 d p.i. There was no evidence of abnormal cell growth, immune response, or adverse systemic effects.
Tissue Engineered Tubularized Urethra for Surgical Reconstruction: A Pre‐Clinical Study
Authors: Orabi Hazem Osman , Aboushwareb Tamer , Yoo James J. , Atala Anthony 
Published: June 2021

IntroductionVarious urethral conditions often require extensive surgery. The treatment options for patients requiring repair of a large segment of tubularized urethra are limited by the availability of autologous tissues. In this study we explored the feasibility of engineering clinically relevant long urethras for surgical reconstruction in a canine pre‐clinical model.Materials and MethodsAutologous bladder epithelial and smooth muscle cells were grown and seeded onto collagen based tubular matrices (6 cm in length). The urethral segment of approximately 6 cm in length was removed in 18 male dogs and replaced with the cell seeded collagen scaffolds in 12 animals, and without cells in 6 animals. The animals were sacrificed at 1, 3, 6 and 9 months for analyses.Results and DiscussionThe CT scan showed wide caliber urethras in the animals implanted with the cell seeded matrices. The urethral segments replaced without cells had tissue breakdown and scaffold collapse. The urethral implants seeded with cells showed normally appearing tissue without evidence of fibrosis grossly. Histologically, formation of an organized urethral tissue structure was evident in the cell seeded constructs.ConclusionsThese findings show that long segments of cell seeded tubularized scaffolds can be used to repair large urethral defects. This study demonstrates that tissue engineered tubularized long urethras may be used for surgical reconstruction in patients requiring urethral tissue repair.Please acknowledge NIDDK P01 DK65298‐01
Targeting quantum dots to surface proteins in living cells
Authors: Yoo Young‐Mi , Kim Yong‐Jung , Yoo Hyun‐Tae , Lee Uhn 
Published: June 2021

Quantum dots (QDs) are semiconductor nanoparticles that greatly expand the possibilities for fluorescence imaging of cells and living animals. Compared with small molecule dyes, the intense fluorescence emission of QDs makes it easier to track single protein molecules, they are remarkably resistant to photobleaching, their narrow emission spectrum facilitates imaging of many proteins simultaneously, and their largeThe size of this QD complex (≈50 nm) can affect membrane protein trafficking and can reduce accessibility to crowded locations in cells. Our aim was to develop a method to target QDs to cell surface proteins that eliminated the bulky antibodies and provided a stable linkage between the QD and the protein of interest.The speed, specificity, and simplicity of labeling with Qtracker should make it a common tool for the study of cell surface proteins.
An emerging role for thioester‐linked polyubiquitin chains in protein degradation
Authors: Ravid Tommer , Hochstrasser Mark 
Published: June 2021

The mechanisms of ubiquitin chain assembly are incompletely understood. The classical model proposes that ubiquitin is first added to a lysine residue within a substrate and then to the distal end of the growing ubiquitin chain. This model becomes less intuitively appealing when applied to substrates with long chains, because the newly added ubiquitin becomes structurally remote from original substrate. One hypothesis that tackles this structural dilemma proposes that ubiquitin chains are pre‐assembled on the catalytic cysteine of an E2 enzyme, prior to transfer onto a substrate. However, until recently no experimental evidence supported the existence of cysteine‐linked polyubiquitin chains in living cells.We found that Ubc7, a yeast E2 enzyme of the endoplasmic reticulum (ER) protein quality control system, is polyubiquitylated exclusively on its catalytic cysteine. The polyubiquitin polymer can then be transferred to a nearby lysine side chain. Moreover, thioester‐linked polybiquitylated Ubc7 undergoes proteasomal degradation when its levels exceed that of its binding partner Cue1, a transmembrane protein that tethers Ubc7 to the ER membrane. Unassembled, and thus mislocalized, Ubc7 is targeted to the proteasome by Ufd4, a HECT‐class E3 ligase. This ensures that no Ubc7 activity persists beyond the ER. Thus, cysteine‐linked polyubiquitin chains can function as an additional degradation signal.
site mapping and quantitation of O‐GlcNAcylated proteins in diabetic erythrocytes
Authors: Park Kyoungsook , Wang Zihao , Saudek Chtistopher D. , Hart Gerald W. 
Published: June 2021

O‐linked N‐acetylglucosamine (O‐GlcNAc) is a ubiquitous post‐translational modification found on nucleocytoplasmic proteins. This modification is very dynamic and sensitive to cellular nutrients and stress. Cycling of O‐GlcNAc on many proteins can modulate diverse events in cells. Elevated global O‐GlcNAcylation is associated with development of many diseases including neurodegenerative diseases, cancer and diabetes. In diabetic patients, prolonged exposure to high glucose may result in global changes of O‐GlcNAcylation and altered occupancy of O‐GlcNAc on certain proteins.We examined the changes of O‐GlcNAcylation in diabetic red blood cells and site‐mapped O‐GlcNAc on proteins using semi‐quantitative mass spectrometry method. We modified beta‐elimination/Michael addition detection (BEMAD)/mass spectrometry method by adding enrichment steps for O‐GlcNAcylated peptides. This step improved the efficiency of O‐GlcNAc detection. We have identified multiple O‐GlcNAc sites on erythrocytic proteins such as catalase and carbonic anhydrase I and observed site‐specific differential occupancy of O‐GlcNAc. Significant changes of O‐GlcNAcylation of certain proteins/sites will be used for further study to develop a diagnostic tool for diabetes.
Mechanism of redox‐ and CO‐sensing by the heme protein, CooA: Cryoradiolysis studies
Authors: Clark Robert W. , Kalafut Deanna F. 
Published: June 2021

Gas‐sensing heme proteins participate in a variety of signal‐transduction mechanisms across a broad range of biological systems. This class of proteins functions by undergoing an allosteric conformational change in response to the binding of a small gas molecule to a heme group. CooA, which is a redox‐ and CO‐sensing transcription factor that is found in several bacteria, regulates gene expression that enables growth on CO as a sole energy source. In the current study, we have employed the technique of gamma‐irradiation / cryoreduction spectroscopy (cryoradiolysis) to investigate facile changes that occur to the heme coordination structure of CooA during activation. Specifically, we conducted cryoradiolysis experiments to elucidate the chemical steps of an unusual ligand switch that occurs rapidly when the CooA hemes are reduced from the Fe(III) to Fe(II) state. Presented are our protocols for the preparation of samples suitable for cryoradiolysis experiments, as well as results we have obtained that provide evidence for the isolation of a highly‐reactive Fe(II)‐Cys/Pro species upon gamma irradiation at 77 K. Finally, we present initial results of annealing experiments that seek to identify the chemical details of the redox‐mediated ligand switching mechanism utilized by CooA from Rhodospirillum rubrum.The authors wish to thank Valparaiso University, the Hope College Chemistry Department, and the Riechel Fund for financial support.
Purification of Strep(II)‐tagged proteins using new prepacked columns
Authors: Heijbel Anna , Bergh Ann , Carlsson Marianne , Hjellstrom‐Nilsson Ulrika , Karlsson Anneli , Nilsson Martina 
Published: June 2021

The Strep(II)‐tag is an ideal tag due to the small size (8 aa, 1 kDa), often making removal of the tag unnecessary. The prepacked StrepTrap™ HP 1 ml & 5 ml columns have been specially developed for high specificity towards the Strep(II)‐tag facilitating fast and convenient purification. Purification is done under physiological conditions and mild elution preserves the activity of the target protein. The 34 um bead size of the resin allows elution in narrow peaks, minimizing the need for further concentration steps.In this work dual‐tagged fluorescent protein, (His)6‐mCherry‐Strep(II) expressed in E. coli, was purified in a purification procedure including two affinity columns, StrepTrap HP followed by HisTrap™ HP. The second affinity column was necessary for removal of a truncated variant of the target protein. The multi‐step purification was performed fully automatically using ÄKTAxpress™. Another dual‐tagged protein expressed in insect cells was purified to high purity in a single step using StrepTrap HP. The final purity was above 95% according to SDS‐PAGE. Finally, six repeated purifications of a Strep(II)‐tagged protein expressed in E. coli were performed on the same StrepTrap HP column with 0.5 M NaOH regeneration between the runs. The final purity and yield of the target protein remained almost unchanged, no tendency of decreasing values, demonstrating the high reproducibility.