bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hepatic carbohydrate and lipid metabolism are altered in rats fed creatine‐supplemented diets (LB151)
Authors: da Silva Robin , Kelly Karen , Jacobs Rene 
Published: June 2021

Non‐alcoholic fatty liver disease (NAFLD) encompasses a wide spectrum of liver damage. Insulin resistance often accompanies NAFLD and these phenomena are hallmarks of the metabolic syndrome. Recently, we reported that creatine supplementation prevents hepatic steatosis, lipid peroxidation and insulin resistance in rats fed a high‐fat diet. In order to examine potential mechanisms underlying these observations, we used McArdle RH‐7777 rat hepatoma cells treated with oleic acid as a model of hepatocyte lipid accumulation. We found that cells cultures with creatine had a dose dependent reduction in cellular TG accumulation. Using radiolabeled tracers we have demonstrated that incubation of hepatoma cells with creatine increases fatty acid oxidation and decreases both fatty acid and TG synthesis. In‐line with increased fatty acid oxidation, analysis of mRNA from hepatoma cells treated with creatine had increased expression of PPARα and its targets CPT1a and LCAD. We have also found that creatine treated hepatoma cells have increased expression of the PEPCK and pyruvate kinase. Our preliminary data suggests that rats fed a creatine‐supplemented high‐fat diet have significantly improved glucose tolerance compared to high‐fat diet fed control animals. Together these data suggest that dietary creatine influences carbohydrate metabolism as well as lipid metabolism.This research is funded by the CIHRGrant Funding Source: Supported by CIHR
Mitochondrial integrity is impaired in MELAS patients (LB164)
Authors: Joseph Rosette , Green Alexander , Cameron Jessie , Hood David 
Published: June 2021

Mitochondrial diseases are disorders that can be caused by either mitochondrial or nuclear DNA mutations. One of the most prominently studied mitochondrial diseases is mitochondrial myopathy, encephalopathy, lactic acidosis and stroke‐like episodes (MELAS), caused by a mutation in the tRNA‐leucine gene within mtDNA. While the symptoms of this disease have been studied, the molecular manifestations of this mutation remain to be clearly understood. Since alterations in organelle function underlie the pathophysiology of various mitochondrial diseases, we explored mitochondrial morphology, cytochrome c oxidase activity, reactive oxygen species (ROS) production and mitochondrial membrane potential in patient (n=3) and control fibroblast cell lines (n=3). Cells transfected with Mito ds‐Red and visualized using confocal microscopy revealed an increase in mitochondrial fragmentation in cells from MELAS patients, compared to control, coincident with reduced cytochrome c oxidase activity, higher ROS production and a greater membrane potential. The increase in organelle fragmentation observed in MELAS cells led us to investigate the proteins which regulate mitochondrial fission (Fis1 and Drp1) and fusion (Opa1). Our data suggest that the increase in organelle fragmentation was due to a tendency for a higher expression of Fis1 in MELAS cells, suggesting that mtDNA mutations have consequences for the expression of nuclear‐encoded fission proteins, resulting in altered organelle morphology. Future work will investigate the use of fission protein inhibitors to ameliorate mitochondrial function in MELAS cells.Grant Funding Source: Supported by Mitacs and Panacea Global
Characterization of mitochondrial Lace1 ATPase (LB168)
Authors: Stiburek Lukas , Cesnekova Jana , Houstek Josef , Zeman Jiri 
Published: June 2021

Lace1 (Lactation elevated 1) is a human homolog of yeast Afg1 (ATPase family gene 1) ATPase with sequence identity ranging from 41,5 % H. sapiens vs. S. cerevisiae to 88,9 % H. sapiens vs. M. musculus. The protein consists of ATP/GTP binding P‐loop motif and a common five domain structure. The aim of our work was to perform detailed cell biological characterization of human Lace1 utilizing stable shRNA‐based RNA interference approach, proteomics and expression analyses using human embryonic kidney cell line (HEK293). We found that human Lace1 is a mitochondrially localized integral membrane protein with an apparent molecular weight of 50 kDa. The prepared stable shRNA LACE1 knockdown HEK293 cell line showed markedly reduced Lace1 protein levels (<10% of controls). SDS‐PAGE western blotting demonstrated markedly elevated levels of subunit SDHA of respiratory complex II, Cox2, Cox3, Cox4, Cox5a and Cox6aL subunits of respiratory complex IV, and ATP d subunit of complex V. On the other hand, the amount of the antiapoptotic factor Bcl‐2 was found to be decreased in these cells. Furthermore, slightly elevated levels of the tumor suppressor p53 were found in LACE1 knockdown cells. Quantitative 2D‐PAGE analysis coupled to mass spectrometric identification (MS) showed elevated accumulation of chaperonine 10 and ATPase F1‐alpha. Our work have thus far identified Lace1 as a mitochondrial factor playing a role in protein turnover of subunits of the oxidative phosphorylation system, possibly confirming its predicted function as an adaptor protein of mitochondrial proteases. Future experiments will involve short term downregulation of Lace1 using Stealth siRNA approach as well as co‐immunoprecipitation analyses coupled to MS identification with P‐loop mutant Lace1 protein.Grant Funding Source: Grant Agency of the Czech Republic Project GACR 13‐072235 and institutional projects UNCE 204011 and RVOVFN6465.
Histidine methylation of yeast ribosomal protein Rpl3p is required for proper 60S subunit assembly (LB185)
Authors: Al‐Hadid Qais , Roy Kevin , Chanfreau Guillaume , Clarke Steven 
Published: June 2021

Histidine protein methylation is an unusual posttranslational modification. In the yeast Saccharomyces cerevisiae, the large ribosomal subunit protein Rpl3p is methylated at histidine‐243, a residue that contacts the 25S rRNA near the P‐site. Rpl3p methylation is dependent upon Hpm1p, a candidate seven beta strand methyltransferase. In this study, we characterized Hpm1p activity in vitro and in vivo. Amino acid analysis of cell lysates and subcellular fractions reveals that Hpm1p is responsible for all detectable histidine protein methylation. The modification is found in a polypeptide corresponding to the size of Rpl3p in ribosomes and in a nuclei‐containing organelle fraction, but was not detected in proteins of the ribosome‐free cytosol fraction. In vitro assays demonstrate that Hpm1p is a methyltransferase that catalyzes the modification of ribosome‐associated but not free Rpl3p, suggesting that its activity depends on interactions of Rpl3p with other ribosomal components. hpm1 null mutant cells are defective in early rRNA processing resulting in a deficiency of 60S subunits and decreased translational activity in minimal media. Finally, cells lacking Hpm1p are resistant to cycloheximide, indicative of ribosome structure abnormalities. We propose that Hpm1p plays a role in orchestrating the assembly of biogenesis factors during early ribosome assembly.Grant Funding Source: NIH
Phosphorylation regulates myo‐inositol 3‐phosphate synthase: A novel regulatory mechanism of inositol biosynthesis (LB190)
Authors: Deranieh Rania , He Quan , Caruso Joseph , Greenberg Miriam 
Published: June 2021

Myo‐inositol phosphate synthase (MIPS) plays a crucial role in inositol homeostasis. Transcription of the coding gene INO1 is highly regulated. However, regulation of the enzyme is not well defined. We previously showed that MIPS is indirectly inhibited by valproate (VPA), suggesting that the enzyme is post‐translationally regulated. Using 32Pi ‐ labeling and phosphoamino acid analysis, we show that yeast MIPS is a phosphoprotein. Mass spectrometry analysis identified five phosphosites, three of which are conserved in the human MIPS. Analysis of phosphorylation‐deficient and phosphomimetic site‐mutants indicated that the three conserved sites in yeast (S184, S296, and S374) and human (S177, S279, and S357) affect MIPS activity. Both S296A and S296D yeast mutants and S177A and S177D human mutants exhibited decreased enzymatic activity, suggesting that a serine residue is critical at that location. The phosphomimetic mutations S184D (human S279D) and S374D (human S357D) but not the phospho‐deficient mutations decreased activity, suggesting that phosphorylation of these two sites is inhibitory. The double mutation S184A/S374 caused an increase in MIPS activity, conferred a growth advantage, and partially rescued sensitivity to VPA. Our findings identify a novel mechanism of regulation of inositol synthesis by phosphorylation of MIPSGrant Funding Source: Supported by National Institutes of Health Grant DK 081367, and Wayne State University Graduate Research Fellowship and Graduate Research Enhancement Funds
Histone H3 lysine 4 methyltransferase subunits regulate cytokinesis (LB191)
Authors: Bailey Jeffrey , Fields Alexander , Cheng Kaijian , Lee Albert , Wagenaar Eric , Lagrois Remy , Schmidt Bailey , Xia Bin , Ma Dzwokai 
Published: June 2021

Cytokinesis, the physical separation of two dividing cells, occurs at the midbody and affects the growth and development of all organisms. As the final step of cytokinesis, abscission requires membrane fission and cytoskeletal rearrangement at a specific midbody region called the secondary ingression. Here, we report that histone H3 lysine 4 methytransferase (H3K4MT) subunits reside at the midbody. Knockdown of individual H3K4MT subunits increases the incidence of multinucleated cells, slows cell proliferation, and impairs cytokinesis at the abscission step. Further investigation shows that the abscission delay is due to slower formation of secondary ingressions in the knockdown cells. Importantly, the midbody microtubules in H3K4MT knockdown cells display enhanced resistance to depolymerization by nocodazole and the abscission defect in these cells can be partially rescued by drug‐induced microtubule disassembly. We propose that a midbody‐localized H3K4MT complex or subcomplex promotes abscission, at least partially, by facilitating midbody microtubule disassembly.Grant Funding Source: 5R21‐MH086853‐02
Increased rate of endocytosis in calreticulin knockout MEF cells is mediated via Caevolin‐1 overexpression (LB195)
Authors: Viswanathan Divya , Pillai Dhanya , Massaeli Hamid , Mesaeli Nasrin 
Published: June 2021

Calreticulin (CRT) is a highly expressed protein in mammalian cells, with both calcium binding and chaperone activity. As an endoplasmic reticulum chaperone, CRT is involved in quality control process during the folding and maturation of proteins. Previous reports illustrated that loss of CRT function (crt‐/‐) results in onset of ER stress, elevated ubiquitin‐proteasome activity, and increased resistance to apoptosis. We recently observed a significant increase in connexin 43 expression accompanied with a significant decrease in its function and accumulation of connexin 43 in intracellular vesicles. Thus we hypothesized that loss of CRT function increases rate of endocytosis. To test this hypothesis we examined rate of endocytosis using Wheat Germ Agglutining (WGA). Our data showed that in crt‐/‐ WGA was completely endocytosed within 30 minute incubation while in wild‐type cells WGA was localized both on cell membrane and intracellular vesicles at the same time. Furthermore, inhibiting proteasome with MG132 reduced endocytosis rate in crt‐/‐ cells. Western blot analysis illustrated a significant increase in caveloin‐1 protein level in crt‐/‐ cells. Quantitative real time PCR showed 3 fold increase in expression of caveolin‐1 in crt‐/‐ compared to wt cells. Furthermore, ubiquitin pull down experiment showed significantly higher caveolin‐1 ubiquitination in crt‐/‐ compared to wt cells. Our data show for the first time involvement of CRT in endocytotic pathways. Furthermore, we showed that knock out of CRT gene results in enhanced endocytosis that is dependent on the ubiquitin‐proteasome activity.Grant Funding Source: Qatar National Research Foundation
Human cytokines activate JAK‐STAT signaling pathways in porcine ocular tissue (LB214)
Authors: Fasler‐Kan Elizaveta , Barteneva Natasha , Nurzhanova Asil , Wunderlich Kerstin , Meyer Peter 
Published: June 2021

Introduction The pig has been used extensively in experimental studies of human eye diseases. Pig retina shares many morphological features with the human eye. The use of porcine ocular cells (POC) as putative xenotransplants appears theoretically possible. We investigated cytokine‐induced changes of the JAK/STAT signaling pathways in POC in response to human cytokines.Methods POC were used to study the nuclear translocation of STAT proteins by gel‐shift assay, immunohistochemistry, confocal laser microscopy, flow and imaging cytometry.Results JAK/STAT signaling pathways could be activated in porcine pigmented epithelial ciliary body cells, pigmented iris epithelial cells and in lens epithelial cells in response to porcine and human cytokines. All POC showed very strong STAT1 activation upon stimulation with porcine interferon‐gamma. POC also respond to human cytokines; IFN‐alpha caused strong activation of STAT1 in all assays whereas activation of STAT3 was less strong in EMSA, but strong in flow cytometry and immunofluorescence. Human recombinant IL‐6 activated STAT3 and human IL‐4 activated STAT6. With the help of immunofluorescence assay and flow cytometry we observed nuclear localization of STAT proteins after activation of POC with cytokines and interferons. Human IFN‐α had an inhibitory effect on POC in proliferation assays.Conclusion Our data demonstrate the upregulation of the members of the JAK‐STAT signaling pathway in POC. Human cytokines and interferon activate JAK‐STAT signalling pathway in POC. We hypothesize that direct stimulation of the JAK‐STAT pathway in POC in response to human cytokines will lead to complications or failure, if pig‐to‐human ocular tissue xenotransplantation were to be carried out. For successful xenotransplantation among other obstacles there must be new approaches developed to regulate signalling pathways.Grant Funding Source: University of Basel
Optimization of Chemical Inhibition Conditions for NMD Studies in p53‐Positive Cancer Cell Lines (LB234)
Authors: Soares Robert , Connelly Zachary , Vazquez Oscar , Carastro L. Michael 
Published: June 2021

We report on the treatment of p53‐positive cancer cell lines with chemical inhibitors of transcription, translation and PI‐3 kinase‐like kinases (PIKKs). Our goal was to determine optimal concentrations of, and exposure times for, chemical inhibitor treatments. These experiments were prerequisite for our studies of the role nonsense‐mediated mRNA decay (NMD) plays in p53 expression. Cultured p53‐positive cancer cells were treated with varied concentrations of cycloheximide, caffeine, wortmannin, or actinomycin D to determine optimal concentrations. Cells were also treated with constant inhibitor concentrations in time‐course experiments to determine optimal duration of exposure. Treated cell samples were divided equally for protein and total RNA isolation. Protein samples were subjected to SDS‐PAGE and western analyses for p53 and beta‐actin. RNA samples were used to synthesize cDNA, which was subjected to TaqMan quantitative RT‐PCR (qRT‐PCR) analysis for p53 and beta‐actin. Western and qRT‐PCR data are consistent with optimal chemical inhibitor concentrations being substantially higher for HepG2 cells compared to MCF‐7 and HeLa. However, chemical inhibitor exposure times required to achieve optimal effect was not widely varied between cell lines studied. Through our determination of optimal conditions for chemical inhibition of transcription, translation and PIKK activity in p53‐positive cell lines, we conclude that cell lines studied require widely different concentrations of, but not different exposure times to, chemical inhibitors. Therefore, we determined optimal concentrations and exposure times for chemical inhibition in our studies of the role of NMD in p53 expression.Grant Funding Source: University of Tampa Chemistry, Biochemistry & Physics Departmental Student Research Support Fund
High‐throughput screening for inhibitors of iron transport in E. coli (LB238)
Authors: Hanson Mathew , Newton Salete , Klebba Philip 
Published: June 2021

Iron acquisition is a component of the pathogenesis of Gram‐negative bacteria, therefore as a form of 'nutritional immunity' host organisms sequester iron. To obtain iron bacteria secrete siderophores that scavenge iron. The E. coli outer membrane protein FepA actively transports the siderophore ferric enterobactin (FeEnt) into the periplasm. We observe this uptake reaction by fluorescently labeling FepA in live bacteria, monitoring quenching that occurs upon binding of FeEnt, and then fluorescence recovery during transport. We developed methods to screen for iron transport inhibitors using a cell‐based high‐throughput screening platform (HTS). This novel assay measures the fluorescence of bacterial cells before and after addition of FeEnt, and monitors its transport. Using this microtiter assay we differentiate between three possible events: no binding of FeEnt to FepA, binding without transport, and binding with transport into the periplasm. After optimizing the assay parameters (cell density, FeEnt concentration, and measurement timing) we found a Z' factor of ~0.7. This test allows for evaluation of libraries of compounds in 96‐well or 384‐well formats using live bacteria, in less than 1 hour.Grant Funding Source: Supported by: NIH GM53836
Metalloproteomic approach of mercury in breast milk samples of lactating women in the Amazon region, Brazil. (LB254)
Authors: de Queiroz João , dos Santos Felipe , Braga Camila , Leite Aline , Buzalaf Marília , Zara Luiz , Okoshi Marina , Padilha Cilene do , Padilha Pedro de 
Published: June 2021

The aim of this study is to present the metalloproteomic results obtained for the protein fractionation in breast milk samples of lactating women from JIRAU Hydroelectric Power Plant ‐ Madeira River/Amazon region, Brazil. The protein spots obtained after the fractionation by 2D‐PAGE were cut from the gel and analyzed by Thermal Decomposition and Amalgamation Coupled with Atomic Absorption and the protein spots, in which were detected mercury, were characterized by mass spectrometry (ESI‐MS/MS). The mercury determinations indicated that this element is linked in two protein spots with molar mass (Mm) of 10.80 (spot 1) and 15.00 kDa (spot 2) and with concentration of 4.70 and 12.10 mg of mercury per gram of protein in the protein spots, respectively. Based on these concentrations was possible to estimate one mercury atom per four protein molecules in relation to the spot 1 and one mercury atom per protein molecule in relation to the spot 2. The ESI‐MS/MS analysis allowed to characterize the spot 1 as a fragment of beta‐casein (Mm = 26.00 kDa) and the spot 2 of lysozyme (Mm = 17.70 kDa).Grant Funding Source: Financial Support: ESBR PD‐6631‐0001/2012 and Fapesp
Regulation of Snf1 kinase by a cross talk between ubiquitylation and phosphorylation (LB256)
Authors: Hsu Hsiang‐En , Liu Tzu‐Ning , Lo Yi‐Chen , Kao Cheng‐Fu 
Published: June 2021

A search for genes reported to interact with UBP8 and UBP10, coding for two highly‐conserved ubiquitin proteases required for deubiquitylation of histone H2B, revealed the yeast homologue of AMPK, SNF1. Snf1 is required when cellular glucose is limited, as it activates stress‐responsive transcription factors. We used a mutant that lacks Ubp8 and Ubp10 to investigate whether Snf1 is regulated by ubiquitylation and degradation. SNF1 mRNA levels in the mutant were no different to the wild type, but Snf1 protein levels were dramatically decreased. The decreased the level of Snf1 protein in the mutant was mediated ubiquitin proteasome pathway. Interestingly, the remaining Snf1 in the mutant was hyper‐phosphorylate when the glucose is exhausted; furthermore, we observed no change in tolerance to stress and lifespan, suggesting the sustainment of Snf1 activity. We hypothesize that Snf1 activity is enhanced in the mutant to compensate for the defect. When Snf1 activity approaches the adaptive level, the cell can overcome the stress and survive. In conclusion, our results suggest that Snf1 is protected from degradation by both Ubp8 and Ubp10. Adaptive hyper‐phosphorylated Snf1 might trigger stress‐responsive transcription and prevent Snf1 from being degraded further, thereby protecting cells against stress. We demonstrate the regulation of Snf1 by a crosstalk between phosphorylation and ubiquitylation.Grant Funding Source: Supported by National Science Council, Taiwan and Academia Sinica
Beneficial effects of Gelidium amansii ethanol extracts on the adipogenesis in 3T3‐L1 preadipocytes (LB260)
Authors: Park Hey jin , Lee Myoungsook 
Published: June 2021

Abstract We believed that the prevalence of obesity could be driven by changes of lifestyle and environments as well as the individual genetics. Gelidium amansii (GA‐3) contains a flush of plant fiber that plays a role of food digestion and excretion. Even though many reports had been shown the beneficial effect of GA‐3 ethanol extracts on the reduction of cholesterol absorption, their effect on anti‐obesity was not proved so far.To investigate the anti‐obese effects of GA‐3 ethanol extracts, biomarkers for adipogenesis (PPARλ, C/EBPα, FABP, ACC & SREBP‐1), lipolysis (HSL & AMPK) and inflammation (TNF‐a, IL‐6, IL‐10) were tested by western blotting, real time PCR and ELISA in 3T3‐L1 cells.Intracellular lipid accumulation was significantly decreased in preadipocytes differentiated in the presence of GA‐3 ethanol extracts compared to control as judged by Oil Red O staining. GA‐3 ethanol extracts reduced the lipid accumulation in corresponding to the reduction of protein and mRNA and protein level of PPARλ, C/EBPα, SREBP‐1, LPL, AQP7, and Insulin in concentration–dependent manner. However, adiponectin mRNA expression was not increased significantly. GA‐3 ethanol extracts also increased the phosphorylation of HSL and AMPK expression, which are active forms of lipolysis function on the intracellular signaling. IL‐6 mRNA expression decreased.We found that GA‐3 ethanol extracts have a anti‐obesity effects because they significantly reduced inflammation‐induced adipogenesis but activated lipolysis. These results suggested GA‐3 ethanol extracts may develop the functional food to prevent obesity in future.Grant Funding Source: Korea Inst. of Planning Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (811003‐3) and Ministry of Oceans and Fisheries