bluuebunny/crossref_metadata_2025_split huggingface dataset:
ATP release induced by electrical stimulation increases glucose uptake via AKT in skeletal muscle
Authors: Osorio‐Fuentealba Cesar , Altamirano Francisco , Espinosa Alejandra , Jaimovich Enrique 
Published: June 2021

Insulin activates downstream pathways in skeletal muscle including PI3K and Akt. On the other hand, ATP is released by contractile activity in skeletal muscle cells and extracellular ATP is able to act as an autocrine or paracrine factor in muscle cells.We determined the effects of extracellular ATP in Akt signaling and glucose uptake. Adult muscle fibers and rat myotubes were exposed to electric field tetanic stimulation (400 pulses of 1 ms at 45 Hz), 100 nM insulin or 100 ìM ATP. Electrical stimulation induced ATP release and Akt phosphorylation in myotubes. We measured uptake of the fluorescent hexoses 2‐NBDG and 6‐NBDG at room temperature using confocal microscopy. The hexose dyes were excited at 488 nm and images were collected at 505–550 nm. Fluorescent images were analyzed and glucose uptake was normalized to the basal condition. Tetanic electrical stimulation, insulin and ATP were able to induce glucose uptake in both myotubes and adult fibers. ATP, before insulin addition, increased glucose uptake induced by insulin. 10 μM each Akt VIII inhibitor and LY 294002 inhibited the increase in glucose uptake in myotubes.Results suggest that electrical stimulation potentiates insulin signaling in skeletal muscle via ATP, which may play an important role as a mediator of glucose uptake during muscle activity.FONDAP 15010006, FONDECYT 11090301, AT‐24100067
Energy metabolic block induces accelerated senescence in human cancer cells
Authors: Krynetskiy Evgeny 
Published: June 2021

Glycolytic pathway is an essential source of energy in cancer cells; therefore, it can be selectively targeted by anticancer pharmacotherapy. The pivotal enzyme in this pathway is glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) which executes both enzymatic and regulatory functions. We evaluated the role of GAPDH in the control of energy homeostasis in isogenic (p53+/+ and p53−/−) human carcinoma cells by using RNA interference, site mutagenesis, and confocal microscopy. Knockdown of GAPDH induced accelerated senescence of cancer cells via p53‐regulated pathway, in the absence of DNA damage. Depletion of GAPDH by short interfering RNA decreased protein level, GAPDH enzymatic activity, and ATP. In response to reduced ATP, AMPK pathway was activated leading to p53 stabilization, and cell proliferation arrest. GAPDH‐depleted cells acquired senescence phenotype revealed by changes in morphology and senescence‐associated markers. Rescue experiments aimed to reinstate energy metabolism (by adding the downstream metabolite pyruvate), or GAPDH level partially restored ATP level but not cell proliferation suggesting that energy crisis by GAPDH depletion may not be the only cause of accelerated senescence. These results indicate that accelerated senescence induced by GAPDH depletion may provide a strategy to control proliferation of cancer cells.This work was supported in part by NCI grant R01 CA104729.
Differential proteomic analysis of the brain synaptic plasma membrane proteins from DHA‐deficient and adequate mice using 16O/18O labeling
Authors: Sidhu Vishaldeep K , Huang Bill , Kim Hee‐Yong 
Published: June 2021

Polyunsaturated fatty acids, especially docosahexenoic acid (DHA, 22:6n‐3) plays an important role in normal brain development in mammals. DHA promotes hippocampal neurite growth and synaptogenesis while DHA‐ deficiency causes impairments in hippocampus‐dependent learning and memory. In this study, we compared the synaptic plasma membrane (SPM) proteins obtained from DHA‐adequate and deficient mice using 16O/18O labeling to provide insights into the role of DHA in synaptic protein expression and function. Synaptosomal preparations from DHA‐deficient and adequate mice cortex were subjected to osmotic shock to obtain SPM. The SPM fraction was analysed by SDS‐PAGE coupled to nanoLC‐MS/MS. For quantification, we differentially labeled the peptide samples from DHA‐adequate and deficient mice using O16/O18 labeling technique. The heavy to light ratios (18O/16O) were calculated using Mascot Distiller. This strategy allowed us to detect more than 400 proteins from the SPM fraction including various receptors, kinases and transporter proteins. The 18O/16O ratios revealed differential expression of several pre‐ and postsynaptic proteins involved in neurotransmission. Some of these proteins were validated by western blot analysis. Altered levels of synaptic proteins important for synaptic transmission suggest a role of DHA in proper brain development and function.National Institutes of Health, MD.
Molecular bases of disease expression of syndromic visual dystrophies by RPGR‐dependent and differential subcellular sorting and processing of RPGRIP1
Authors: Ferreira Paulo A , Cho Kyoungin , Yi Haiqing , Guruju Mallikarjuna 
Published: June 2021

RPGR and RPGRIP1 play direct roles in syndromic retinal dystrophies by elusive mechanisms. RPGR interacts with the RPGRIP1 via its RHD domain, which is homologous to RCC1, a nucleotide‐exchange factor for RAN GTPase. Molecular modeling of RHD of RPGR to RCC1 shows that all disease mutations in RHD map to a distinct contact interface from that found between RCC1 and RAN GTPase. Two‐hybrid assays show that disease‐causing mutations in RHD or ORF15 domains differentially impair RPGR interaction with RPGRIP1. Cell and time‐lapse microscopy assays support that expression of RPGRIP1α1 isoform alone promotes the genesis of aggregates, whereas its co‐expression with the RPGR1–19 isoform targets RPGRIP1α1 to the Golgi. Conversely, RPGRORF15 co‐expression with RPGRIP1α1 promotes its pan intracellular dispersion and clears out pre‐existing RPGRIP1α1 deposits. Mutations singly in Rpgr1–19, RpgrORF15 or Rpgrip1, do not affect the colocalization of RPGR1–19 or RPGRORF15 with RPGRIP1α1, whereas co‐expression of similar mutations in Rpgr variants and Rpgrip1 abrogates their colocalization and association. RPGRORF15, but not RPGR1–19, prevents the C‐terminal proteolytic processing of RPGRIP1α1. Hence, Rpgr1–19 and RpgrORF15 exert distinct effects on RPGRIP1α1 sorting and processing and they are necessary but not sufficient to the coupling to, and subcellular targeting of, RPGRIP1α1.NIH support GM083165 & EY019492
Micropatterned substrates: Tools for studying cell motility and aiding rational drug design
Authors: Huda Sabil , Soh Siowling , Pilans Didzis , Mahmud Goher , Kandere‐Grzybowska Kristiana , Grzybowski Bartosz A. 
Published: June 2021

We have developed a novel class of cell biology tools for the quantification of cytoskeletal organization and dynamics in populations of cancerous cells with low variability. Anisotropic Solid Microetching (ASoMiC) was used to constrain cells onto transparent microislands and subsequently imaged. This methodology creates a unique platform for studying cell motility and identifying therapeutic targets.Triangular microislands spatially segregate cytoskeletal components and allow for study of microtubule (MT) growth trajectories in defined geometric confines. We show that growing MTs are targeted towards focal adhesions (FAs). RNAi studies reveal that myosin IIA dependent f‐actin bundles are integral to this process. Since MT targeting of FAs is important in metastasizing cancer cells, this system can be used to identify new cancer therapeutic targets.To study cell motility, we used micropatterned linear tracks to constrain cells to 1‐D. We found that metastatic cells were “superdiffusive” vs. “diffusive” non‐metastatic cells. This quantitative description of a cell's motility leads to the systematic characterization of metastatic potential and response to therapeutic agents.ASoMiC allows the study of cytoskeletal organization and dynamics of cancerous cells with minimal cell‐to‐cell variability. These systems provide a platform for the study and manipulation of the cytoskeletal organization.Funding: NIH Award 1R21CA137707‐01
PDGF‐BB induces the cell surface co‐localization of laminin and dystroglycan and fibronectin and α5‐integrin
Authors: Kastanis George John , Hernandez‐Nazara Zamira , Nieto Natalia , Rincón‐Gallardo Ana Rosa , Popratiloff Anastas , Dominguez‐Rosales Jose Alfredo , Lechuga Carmen , Rojkind Marcos 
Published: June 2021

Hepatic stellate cells (HSC) are embedded in a laminin‐rich matrix in the Space of Disse of the normal liver. During injury HSC migrate to the injured sites and produce scar collagen. One of the first proteins produced during liver injury is fibronectin. Therefore we investigated the role of laminin, fibronectin and its receptors in HSC migration.METHODSWe used mouse HSC treated with PDGF‐BB and used Northern and Western analysis to study the expression of dystroglycan (DG) and α5‐integrin (α5‐Int), the respective receptors for laminin (LN) and fibronectin (FN). We also used confocal microscopy to investigate the cellular localization of DG and α5‐Int.RESULTSPDGF‐BB induced the cell surface colocalization of α5‐Int and FN as well as DG and LN. While DG and LN were localized to the projections of the HSC, FN and α5‐Int were present along the body of the cells. PDGF‐BB also induced significant changes in the expression and regulation of DG and α5‐Int. While the expression of α5‐Int was up‐regulated that of DG and LN was down‐regulated. However, DG RNA levels were sustained due to the increased transcription of the gene with a 50% decrease in its mRNA t1/2. Antibodies to DG or α5‐Int prevented PDGF‐BB–induced HSC migration.CONCLUSIONOverall, our findings suggest that both LN and FN are required for HSC migration. However, while the cells migrate on a FN carpet, they appear to be anchoring on LN.
New Evidence for a Novel Biosynthetic Pathway to Plasmalogens in Anaerobic Bacteria
Authors: Goldfine Howard , Guan Ziqiang , Johnston Norah C. , Aygun‐Sunar Semra , Daldal Fevzi , Raetz Christian R.H. 
Published: June 2021

Study objectiveA study of the polar lipids of Clostridium novyi NT and C. tetani was undertaken as part of an ongoing exploration of plasmalogen biosynthesis in anaerobic bacteria.Approach and resultsOur study of the polar lipids of two species of clostridia by thin‐layer chromatography and mass spectrometry has revealed the presence of phosphatidylethanolamine (PtdEtn), cardiolipin and phosphatidylglycerol (PtdGro), as major phospholipids with small amounts of lysyl‐PtdGro and alanyl‐PtdGro in C. novyi. PtdEtn, PtdGro and aminoacyl‐PtdGro were present in both the diacyl and alk‐1′‐enyl acyl (plasmalogen) forms and cardiolipin plasmalogens were found to contain one or two alk‐1′‐enyl chains. In contrast, the precursor lipids phosphatidic acid, CDP‐diacylglycerol and phosphatidylserine were present almost exclusively as diacyl phospholipids.ConclusionsThese findings are consistent with the hypothesis that plasmalogens are formed from diacylated phospholipids at a late stage of phospholipid formation in clostridia. This novel pathway begins with acylation of glycerol‐3‐P and contrasts with the route in animals in which a saturated ether bond is formed at an early stage of plasmalogen biosynthesis from acyl‐dihydroxyacetone‐P and the alk‐1‐enyl bond is formed by an aerobic mechanism. Research support: GM‐069338, DOE‐ER‐20052, and the University of Pennsylvania.
Examination of NAK‐associated protein‐1 homo and hetero‐interactions
Authors: Call Richard Jason , Bell J. Ellis , Bell Jessica K. 
Published: June 2021

Protein‐protein interactions form the communication link from membrane‐bound receptors to their downstream cellular targets.ObjectiveOur long‐term goal is to examine the protein‐protein interactions responsible for the activation of the Toll‐like receptor 3 (TLR3) signaling cascade that leads to type I interferon production. In the current study, we have targeted the kinase scaffold protein, NAK‐associated protein 1 (NAP1).MethodsCFP/YFP/Alexa Fluor 546 fusion proteins of the NAP1 kinase binding domain (KBD) and scaffold binding motif (SBM) of the kinases, TANK Binding Kinase‐1 (TBK‐1) and IκB kinase epsilon (IKKε), were generated to assess interactions using fluorescence resonance energy transfer (FRET). Biochemical characterization of recombinant targets was completed using gel filtration (GF).ResultsBy gel filtration studies, NAP1 KBD elutes as a dimeric species. NAP1 KBD directly interacts with TBK1 and IKKε, with low micromolar affinity in vitro. Mutagenesis studies to identify the residues 1ecessary for the kinase recognition sequence are ongoing.ConclusionsNAP1 KBD forms stable homo‐oligomers and directly interacts with TBK1 and IKKε. Funding was provided by the American Cancer Society.
Modulation of the Switch Regions in Ras GTPase
Authors: Holzapfel Genevieve A , Buhrman Gregory , Mattos Carla 
Published: June 2021

Ras is a monomeric GTPase which in its GTP‐bound form can bind various effectors in signal transduction pathways. Through its interaction with the effector Raf and associated Ras/Raf/MEK/ERK cascade it is directly involved in the control of cell proliferation and thus implicated in many human cancers. Switch I and switch II undergo conformational transition during hydrolysis and contains active site residues and the effector binding sites. In the Ras/Raf complex only switch I is involved in the interaction. We have a crystal form of Ras in which switch I has the conformation seen in its complex with Raf and switch II is disordered and free of crystal contacts. This facilitated discovery of an allosteric switch, activated in our crystals by calcium acetate, that places the catalytic residue Q61 in the active site. In this crystal form Ras can adopt both the active and inactive states of the allosteric switch, where the activated state is characterized by a shift in helix3/loop7 toward helix 4 and ordering of switch II for catalytic activity. Here we show that DTT stabilizes the inactive form of the switch by occupying a pocket between switch II and helix 3, resulting in an ordered anti‐catalytic conformation of switch II. This anti‐catalytic conformation is also seen in highly transforming mutants, such as RasQ61L. By changing the relative concentrations of calcium acetate and DTT in Ras crystal soaks we show transition between the on and off states of the allosteric switch. Our results provide a proof of concept that the allosteric site and switch II/helix3 pocket can be used to modulate the state of Ras and should therefore serve as novel targets sites in ligand design.NIH R01 CA096867
A novel protein engineering approach for investigating GPCR/ligand interactions
Authors: Witteveen Natalie Anne , Gooley Paul R , Stone Martin J , Bathgate Ross A D 
Published: June 2021

Relaxin family peptide receptors (RXFP) 1 and 2, receptors for relaxin and insulin like peptide‐3 (INSL3) respectively, are G‐protein coupled receptors possessing large N terminal domains consisting of 10 leucine rich repeats (LRRs) and a low density lipoprotein Class A (LDLa) module. Ligand mediated activation requires primary binding to the LRRs as well as a secondary interaction involving the extracellular loops (ELs) of the transmembrane domains. The LDLa is necessary for activation possibly by a specific interaction with the ELs.The project aims to identify residues of RXFP1 and RXFP2 ELs involved in ligand/LDLa binding by mounting them onto a soluble protein scaffold and mapping specific ligand interactions using NMR. We have produced soluble scaffolds containing RXFP1 and RXFP2 EL1 and EL2 and are conducting ligand/LDLa titrations for NMR analysis before residue assignment. Preliminary NMR results show peak changes upon relaxin titration with the RXFP1 scaffold suggesting specific interactions are occurring. Identified interacting residues will be mutated in RXFP1 and functional characterisation will confirm their importance for receptor function.Understanding mechanisms of RXFP1 and RXFP2 activation will aid design of relaxin and INSL3 analogues for therapeutic use. This work is supported by project grant 628427 (to RADB and PRG) from the National Health and Medical Research Council, Australia.
Vitamin B12, Cognition, and Brain Magnetic Resonance Imaging Measures
Authors: Tangney Christy C , Aggarwal Neelum T , Li Hong , Wilson Robert S , Evans Denis A , Morris Martha Clare 
Published: June 2021

BackgroundLow total brain volume (TBV) and high white matter hyperintensity volume (WMHV) have been related to cognitive impairment; vitamin B12 (B12) deficiency has been associated with cognitive decline.ObjectiveTo investigate whether serum markers of B12 nutriture (B12, homocysteine, methylmalonate, cystathionine, 2‐methylcitrate) are related to brain volumes and to cognitive performance in a bi‐racial population sample in four Chicago neighborhoodsDesignCross‐sectional analysis of a prospective bi‐racial study of 121 older adults of the Chicago Health and Aging ProjectOutcome MeasuresMagnetic resonance imaging measures of TBV, WMHV and cerebral infarcts and tests of cognitive performanceResultsBoth serum homocysteine and methylmalonate levels were inversely associated with global cognitive function; only methylmalonate levels were associated with episodic memory and perceptual speed. Homocysteine was related to WMHV and levels of both markers were inversely associated with TBV. The association between homocysteine and TBV was stronger among persons with hypertension.ConclusionsPoor vitamin B12 status as evidenced by elevated homocysteine and methylmalonate levels may be associated with reduced TBV and impairment in several cognitive domains. This common, treatable condition may be important role in cognitive decline with aging.
The Effects of Cholesterol‐Lowering Diets with Lean Beef on Blood Pressure and Vascular Health: Results from the BOLD (Beef in an Optimal Lean Diet) Study
Authors: Roussell Michael Antonio , Gaugler Trent , West Sheila , Heuvel John Vanden , Kris‐Etherton Penny 
Published: June 2021

Observational and clinical studies show benefits of additional plant and dairy protein on systolic blood pressure (SBP). However, the effects of a moderate protein diet rich in animal protein on SBP has not been examined. We hypothesized that a moderate protein DASH‐like diet containing lean beef would significantly reduce SBP.Forty moderately hypercholesterolemic individuals (LDL‐C >110 mg/dL) were enrolled in a randomized crossover controlled feeding study. Participants were fed an Average American control diet (AAD; 33% total fat, 12% SFA, 49% carbohydrate (CHO), 19% protein (PRO), 0.7 oz beef/d], DASH (27% total fat, 6% SFA, 50% CHO, 19% PRO, 1.0 oz beef/d), BOLD (28% total fat, 6% SFA, 54% CHO, 19% PRO, 4.0 oz beef/d), and BOLD+ (28% total fat, 6% SFA, 46% CHO, 28% PRO, 5.4 oz beef/d) diets for 5 weeks with a 1 week washout period between diets. In addition to SBP, endothelial function and vascular elasticity via EndoPat were measured. SBP was significantly decreased on the BOLD+ diet (−4.24 mm Hg) compared to AAD. Augmentation Index (AI) was significantly reduced in participants on the BOLD diet (−4.1%) compared to AAD. There were no significant effects of the dietary treatments on reactive hyperemia index.In conclusion, a moderate protein diet including lean beef improves SBP. The inclusion of lean beef in a DASH‐like diet also improves vascular elasticity. Supported by: Beef Checkoff.
Relationship between body mass index and perceived body size among African American churchgoers in Baltimore City
Authors: Ganz Ollie T , Pamela Surkan , Martins Paula , Summers Amber , Gittelsohn Joel 
Published: June 2021

Perceptions of body image vary between different ethnic groups. We explored the relationship between body mass index (BMI) and body image among African‐American churchgoers. Participants included 139 adult respondents, 32% males and 68% females from 6 African‐American churches in Baltimore. The data was collected as part of the Healthy Bodies Healthy Souls study, a program aimed at preventing diabetes by improving diet and exercise among African‐American church goers in Baltimore City. Body self image, preferred body type, and healthy body type were assessed at baseline using a 9‐item scale of figure drawings intended for urban African‐Americans. We calculated discrepancy between the individual's assessment of his/her own body type to what he/she considered ideal and healthy. For participants with higher BMIs there was a larger discrepancy between his/her perceived body size to preferred body type (R =0.56, p <0.001) as supposed to those with lower BMIs. Higher BMI was also associated with large discrepancies between perceived body size and healthy body size (R =0.64, p <0.001). African‐American congregants with higher BMIs had a larger discrepancy between their perceived body self‐image and the body type they consider desirable and healthy, implying that such individuals may be motivated to adopt health behaviors to lower BMI.Grant Funding Source: American Diabetes Association
Relationships between income, food expenditures and purchasing choices in African American churchgoers in Baltimore City
Authors: Graham Karlene , Martins Paula , Rikoon Amy , Mahendraratnam Nirosha , Frick Kevin , Rowan Megan , Gittelsohn Joel 
Published: June 2021

Healthy Bodies Healthy Souls, a church‐based diabetes prevention program, will be evaluated in a sample of 375 non‐diabetic adult respondents, using psychosocial (knowledge, self‐efficacy, intentions), behavioral (diet, physical activity) and health (BMI, blood pressure, waist circumference) measures. Analysis of the baseline data on the first 138 respondents showed that over 78% have a high school degree and 73% have household incomes more than $30,000. Using the Chi‐Squared test, an association was found between having an annual income greater than $30,000 and monthly grocery expenditures greater than $180 (p=0.004). Expenditures on single meals at carry out, fast food, and sit down restaurants did not vary significantly with household income. Respondents from households that grossed more than $30,000 annually were more likely to purchase 100% whole wheat bread at least once (p=0.03) and fresh fruit greater than three times (p=0.045) in the 30 days prior to responding to the questionnaire. However, no association was observed between the purchasing frequency of high fiber cereals, multigrain bread, brown rice, and cooking spray. These findings can help guide future programs aimed at influencing food purchasing decisions in African American communities.Grant Funding Source: American Diabetes Association
Depressive symptoms in urban African‐American churchgoers in relation to self‐efficacy for healthy behaviors
Authors: Surkan Pamela , Chen Lenis , Martins Paula , Flamm Laura , Gittelsohn Joel 
Published: June 2021

Overweight and obesity are at epidemic levels in the US, with especially high rates in African Americans. Given the associations between depression and many health behaviors, we sought to understand its relationship to antecedents overweight and body mass index (BMI). Using baseline data from a church‐based diabetes prevention program in Baltimore, Healthy Bodies Healthy Souls (N=133), we studied how depressive symptoms are related to self‐efficacy and behavioral intentions. The ten‐item Center for Epidemiologic Studies Depression Scale was used to assess depressive symptoms. As dependent variables, we created self‐efficacy and behavioral intention scales related to healthy eating and engaging in physical activity (Cronbach's α=0.54 and 0.40, respectively). In adjusted regression models, higher depressive symptoms were associated with lower self‐efficacy for healthy eating and physical activity (β= −0.3, p<0.001). No relationship was observed between depressive symptoms and intentions to perform these behaviors (adjusted β= −0.009, p=0.8). Likewise, higher depressive symptoms were not associated with participant BMI. Results imply that depressive symptomatology may be a risk factor for low self‐efficacy related to healthy eating and physical activity. Given the lack of association with BMI, larger prospective studies are needed to assess actual health behaviors and to confirm these results.Grant Funding Source: American Diabetes Association