bluuebunny/crossref_metadata_2025_split huggingface dataset:
Single molecule studies of spliceosomal RNAs U2 and U6
Authors: Guo Zhuojun , Karunathilaka Krishanthi , Rueda David 
Published: June 2021

Splicing is an essential step in the maturation reaction of eukaryotic pre‐mRNA, in which intervening sequences (introns) are removed from the coding sequences (exons). The spliceosome is a dynamic assembly of five snRNAs and a large number of proteins that catalyzes splicing. U2 and U6 are the only two spliceosomal snRNAs strictly required for both steps of splicing, and they form RNA complex using base pairing. Major conformational changes are expected to take place during the assembly and catalysis of the spliceosome.Here, we have used Single Molecule Fluorescence to study the structural dynamics of a protein free U2‐U6 complex from S. cerevisiae. Our data clearly shows a Mg2+ induced large amplitude conformation change of the U2‐U6 complex. In the absence of Mg2+, helix III and the U6‐ISL are in close proximity, while in the presence of Mg2+, these two helices are far from each other. The conformational change consists of a two‐step process, with an obligatory folding intermediate. We hypothesize that the observed conformational change is essential for spliceosomal activation between the first and second steps of splicing. We have tested this hypothesis by measuring the structural dynamics of a point mutant known to block the second step of splicing and the compensatory mutant. Our data strongly supports this hypothesis. We have also studied the role of the highly conserved base U80 in the folding dynamics of the U2‐U6 complex. The data shows that U80 plays an important role in stabilizing the interaction between helix III and the U6‐ISL and without it, helix III and the U6‐ISL cannot come close to each other. We hypothesize that in the absence of Mg2+, U80 flips out of the U6‐ISL and interacts with helix III to bring them in close proximity.Financial support from Wayne State University (Start‐up funds), the National Science Foundation (CAREER 0747285 to DR), and the NIH (GM085116)
The Regulation of Malate Dehydrogenase by Citrate
Authors: Marion Jimmy , Bell Ellis 
Published: June 2021

Malate Dehydrogenase (MDH) catalyzes the oxidation of malate to oxaloacetate through the reduction of NAD+ to NADH and is vital in a variety of cell processes important for metabolic and energy pathways. Glyoxasomal MDH (gMDH) is a homodimer with each subunit contains a dinucleotide binding domain and a malate binding domain. Citrate has long been suggested as an allosteric regulator of the enzyme but crystal structures show only citrate binding to the active site. To define the effects of citrate both kinetic and biophysical approaches have been used. The ability of citrate to inhibit gMDH is shown to be pH dependent in the range from pH 6‐8. At pH 7 & 8 Citrate only inhibits about 60% of the activity at apparently saturating concentrations suggesting subunit interactions are involved in the citrate effect. The extent of inhibition is also dependent upon substrate inhibition by oxaloacetate. Isothermal titration calorimetry (ITC) studies are also done to investigate the affinity of citrate for the active site of the dimeric gMDH. The effects of citrate on local flexibility and stability of subunit interactions [studied by limited proteolysis and dynamic light scattering] are correlated with the kinetic effects.This research is supported by a Grant from the National Science Foundation: MCB 0448905 to EB
The mechanism of Zinc Inhibition of Glutamate Dehydrogenase Involves Disruption of Subunit Interactions
Authors: Sinanan Leander , Bell Ellis 
Published: June 2021

Zinc is a potent inhibitor of higher eukaryotic glutamate dehydrogenase but the mechanism of inhibition has not been established. The goal of the current study is to correlate zinc binding under various conditions with changes in activity and conformational flexibility. Using Isothermal Titration Calorimetry it is shown that zinc binds to glutamate dehydrogenase with a 1:1 stoichiometry in the presence or absence of amino acid substrates. Using tryptophan fluorescence spectroscopy it appears that zinc affects the most hydrophobic of the three tryptophans in the protein. Examination of the three dimensional structure of the enzyme suggests that zinc may bind at the subunit interface in the "twisty tie" region of the molecule. This is consistent with both the location of the most hydrophobic tryptophan and the fact that zinc appears to inhibit by disrupting subunit interactions necessary for full activity with glutamate as substrate. Differential Scanning Calorimetry and Limited Proteolysis approaches demonstrate that zinc binding induces changes in the conformational flexibility of glutamate dehydrogenase which could be responsible for this effect.This research is supported by a Grant from the National Science Foundation: MCB 0448905 to EB
Effects of Resistant Maltodextrin on Fecal Microbiota Composition
Authors: Mai Volker , Ukhanova Maria , Baer David , Okuma Kazuhiro , Tagami Hiroyuki , Kanahori Sumiko , Henderson Theresa , Gordon Dennis T 
Published: June 2021

Resistant maltodextrin (RM) is a soluble dietary fiber that although resistant to digestion by human enzymes is a substrate for fermentation by the commensal microbiota of the large intestine. We determined the effects of RM on gut microbiota composition in a randomized, controlled dose‐response feeding study in 15 healthy males (14 completed). Subjects were assigned to a treatment sequence that consisted of three 28 day treatments periods with controlled diets: a placebo diet (0 g/d RM + 50 g/d maltodextrin) and two levels of dietary RM (25 g/d RM + 25 g/d maltodextrin and 50 g/d RM + 0 g/d maltodextrin). Fecal samples for the microbiota studies were collected on Days 1, 13 and 24 of each period. Microbiota composition was determined using 16S rRNA based methods that included denaturing gradient gel electrophoresis (DGGE), fluorescent in situ hybridization (FISH), qPCR and 454 based pyrosequencing. Total numbers of bacteria/g of feces increased by 22% in subjects consuming 50 g/d RM (P =0.02). While microbiota diversity appeared unaffected, Bifidobacteria (FISH and qPCR) as well as Rumninococci and Prevotella (454 sequencing) were increased during RM periods. Differences in microbiota composition likely contributed to the variations in energy gain from RM intake observed for study subjects.Supported by USDA, ARS and Matsutani Chemical Industry Co., Ltd.
Phytosterol ester substrate specificity of pancreatic cholesterol esterase
Authors: Brown Andrew William , Hang Jiliang , Dussault Patrick H , Carr Timothy P 
Published: June 2021

Consuming phytosterols and their esters results in decreased LDL cholesterol, an atherosclerotic risk factor. To more completely characterize how phytosterols impart their effects, the present study aimed to determine if pancreatic cholesterol esterase (PCE; EC 3.1.1.13), the enzyme primarily responsible for cholesterol ester hydrolysis in the duodenum, is capable of hydrolyzing various phytosterol esters, and to compare their rates of hydrolysis in vitro. We found that 1) PCE hydrolyzes palmitate, oleate, and stearate esters of cholesterol, stigmasterol, stigmastanol, and sitosterol; 2) specificity was dependent on both the sterol and the fatty acid moieties in the following order of rates of hydrolysis: cholesterol > (sitosterol = stigmastanol) > stigmasterol, oleate > (palmitate = stearate); 3) addition of free phytosterols did not change hydrolytic activity of PCE, while addition of palmitate, oleate, or stearate increased hydrolytic activity. Thus, PCE may play an important but discriminatory role in vivo in the liberation of free phytosterols to compete with cholesterol for micellar solubilization and absorption.Grant Funding SourceUSDA‐NRI competitive grant no. 2007‐35200‐18298
Oxidative stress in cardiac mitochondria caused by copper deficiency may be insufficient to damage mitochondrial proteins
Authors: Johnson William T 
Published: June 2021

Copper (Cu) deficiency may promote the generation of reactive oxygen species (ROS) by the mitochondrial electron transport chain through inhibition of cytochrome c oxidase (CCO) and increased reduction of respiratory complexes upstream from CCO. In the present study, respiration, H2O2 production and aconitase activity were measured in isolated heart mitochondria from weanling rats fed diets that were either deficient (CuD, <1ppm Cu), marginally deficient (CuM, 1.5 ppm Cu) or adequate (CuA, 6 ppm Cu) in Cu for 8 weeks. State 4 respiration (μmole O2 ·min−1 ·mg−1) in CuD rats (2.81±0.71) was lower (P<0.05) than in CuM (3.74±0.88) or CuA rats (3.72±0.78). H2O2 production (nmol · min−1 ·mg−1) in the presence of rotenone was marginally higher (p=0.07) in CuD rats (11.2±4.1) than in CuM rats (8.5±2.4) or CuA rats (8.2±2.6). However, aconitase activity (mU ·mg−1) was higher (P<0.05) in CuD rats (58.5±12.5) than in CuM (25.8±7.0) or CuA rats (28.4±9.3). Aconitase is readily inactivated by ROS and these findings suggest that even though severe Cu deficiency impairs mitochondrial function, mitochondrial oxidative stress is not elevated to a level that leads to oxidative inactivation of susceptible mitochondrial enzymes. Enhancement of aconitase activity may reflect enhanced TCA cycle activity for maintenance of cardiac energy production during Cu deficiency.Supported by USDA‐ARS
Direct and indirect signaling through the epidermal growth factor receptor (EGFr) exerts distinct effects on surface expression of Na+, K+, 2 Cl− cotransporter‐1 (NKCC1) in colonic epithelial cells
Authors: Chappell Alfred E , Barrett Kim E 
Published: June 2021

NKCC1 participates in ion transport in many tissues, including chloride secretion by the intestinal epithelium. EGF regulates intestinal chloride secretion and EGFr can be differentially activated to elicit divergent, stimulus‐specific signaling. Using surface biotinylation, immunoprecipitaion, and western blot analysis in human T84 colonic epithelial cells, we now report that while NKCC1 is internalized in response to carbachol (CCh) through an EGFr‐dependent mechanism, it is not internalized in response to EGF. Thus, after 60 min. basolateral stimulation with 100µM CCh or 100ng/mL EGF, CCh induced significant internalization of NKCC1 (202.3 ± 15.8% vs. control, p<0.001, mean ± SEM, n=11) while EGF did not (n=11). Further, the effect of CCh was blocked by the EGFr inhibitor, AG1478 (1µM, n=6). NKCC1 was ubiquitinated in response to CCh (200.7 ± 41.6% vs. control, p<0.05, n=5) but not EGF (n=3). While it was seemingly not degraded, internalization was proteasome‐dependent and inhibited by MG132 (10µM, n=7). CCh‐stimulated internalization of NKCC1 was also inhibited by PD98059 (50µM) or SB203580 (10µM), indicating involvement of ERK and p38 mitogen‐activated protein kinases. We conclude that the differential effect of direct and indirect activation of EGFr on NKCC1 localization and perhaps activity may have implications for chloride secretion in the colon and in other tissues.Supported by NIH and NSF.
SUMO (small‐ubiquitin‐like modifier) in subcellular fractions of Chlamydomonas reinhardtii reveal potential function
Authors: Miller Amy , Horken Kempton , Matejka Stephanie , Weeks Donald , Bailey Cheryl 
Published: June 2021

SUMO (Small ubiquitin‐like modifier), is a member of the family of ubiquitin‐like polypeptides that become covalently attached to intracellular target proteins. SUMOylation can alter the function, location, and/or half‐life of target proteins. Here we describe three SUMO proteins and three SUMO‐like proteins in Chlamydomonas reinhardtii. The three SUMO genes show differential RNA expression depending on environmental conditions. Western analysis also shows a shift in proteins recognized by the Chlamydomonas reinhardtii SUMO antibodies in response to changes in environmental conditions. The three SUMO‐like proteins are novel fusion proteins with C‐terminal SUMO. Chlamydomonas reinhardtii subcellular fractionation reveals SUMO proteins are conjugated to proteins involved in various cellular processes. Proposed SUMOylated target proteins can be resolved by one‐dimensional and two‐dimensional western analysis.Supported by NSF and the Nebraska Research Council.
The role of methionine metabolism in inflammatory bowel disease
Authors: Benight Nancy Marie , Stoll Barbara , Puiman Patrycja , Burrin Doulgas 
Published: June 2021

Methionine (Met) cycle activity is critical for normal cell functions. Met metabolites S‐adenosylmethionine (SAM) and methylthioadenosine (MTA) are anti‐inflammatory, yet their role in inflammatory bowel disease (IBD) is poorly understood. We hypothesize that active IBD leads to changes in Met metabolites that impact disease and administration of SAM and MTA will reduce inflammation.Study 1 mice received either a control diet (C) or a B6/B12‐deficient diet (D) to disrupt Met metabolism. Mice in both groups were challenged with dextran sulfate sodium (DSS) at 5% acutely or 2.5% chronically. Study 2 mice fed the C diet were then pretreated with control, SAM or MTA supplemented water for 7 days. During days 3‐7, all mice were received 3% DSS. We measured weight change, clinical disease activity index (DAI) and collected colon for analysis in both studies.Study 1 mice fed the C diet had a higher mortality rate of 87.5% after 5% DSS when compared to D diet fed mice 37.5%. Similar results were seen with the 2.5% DSS (25% in C vs. 0% in D) and D diet fed mice had reduction in DAI compared to C fed mice. In Study 2, the MTA group had lower DAI and weight loss. No difference was detected in molecular markers or histology in either study.Met cycle disruption via B‐vitamin deficiency and supplementing MTA were protective during colitis. We postulate that Met metabolites are modulating colonic inflammation. Funded by USDA.
Chemical variance of dry beans determined from UV spectral fingerprints
Authors: Harnly James M , Luthria Dave , Pastor‐Corrales Marcial S 
Published: June 2021

The chemical variance of dry beans arising from three locations (Maryland, Michigan, and Nebraska), 9 varieties, 8 row samples, and triplicate determinations was determined from UV spectral fingerprints analyzed by analysis of variance‐principal components analysis (ANOVA‐PCA). Samples were extracted with 60% aqueous methanol, diluted as appropriate, and spectra (200‐400 nm) acquired by UV spectrophotometry. The data were analyzed using nested one‐way ANOVA where classic single variables were replaced by the sum of results for all wavelengths (200 variables). ANOVA‐PCA allowed pretreatment of the data matrix (648 samples x 200 wavelengths) to yield separate sub‐matrices isolating each experimental factor (location, variety, and row). Visual and statistical interpretation was easily accomplished. The results showed that, for 25 plants per row, 0.3% of the total variance came from location, 10.2% from variety, 70.8% from plant‐to‐plant variation, and 0.9% from analytical uncertainty.This research was funded by USDA and the Office of Dietary Supplements at the National Institutes of Health.
Experimental gas embolism and tissue oxygenation using intravital microscopy
Authors: Torres Luciana N , Kanth Shreya , Torres Filho Ivo , Spiess Bruce 
Published: June 2021

Little is known about microhemodynamic responses to arterial gas embolism (AGE) and associated local O2 tensions (PO2) have never been evaluated in vivo. Therefore, we assessed the adequacy of intravital microscopy to study the microcirculation during AGE, and investigated how bubble‐induced vaso‐occlusion and blood flow redistribution were associated with changes in arteriolar and tissue PO2. Blood pressure and heart rate were not significantly different before and after local AGE in the rat cremaster muscle. The first attempt of embolization did not result in arteriolar occlusion and multiple attempts were needed. Control diameter and blood flow averaged 53 ± 25 and 13 ± 10 nl/s, respectively. After AGE, these parameters changed to 53 ± 24 µm and 1 ± 3 nl/s. Arteriolar PO2 averaged 59 ± 19 mmHg during control and dropped to 34 ± 27 mmHg (upstream) and 24 ± 22 mmHg (downstream) after AGE. Interstitial PO2 changed from 44 ± 22 to 23 ± 20 mmHg and did not follow the drop recorded in the embolized arteriole when the PO2 in nearby flowing vessels remained high. We also measured in vivo bubble reabsorption (volume ≤ 20pL). Our findings suggest that blood redistribution in the muscle prevented severe hypoxia in some tissue areas after AGE and that arteriolar entrapment of air bubbles is associated with prior endothelial cell damage caused by the first bubble passage.Support: NIH, ONR.
Impact of Rat Outer Medullary Architecture on Oxygen Distribution
Authors: Edwards Aurelie , Chen Jing , Layton Anita T 
Published: June 2021

In this study, we examined the impact of the complex structural organization of the rat outer medulla (OM) on oxygen distribution. We extended a previous region‐based mathematical model of the urine concentrating mechanism in the rat OM to incorporate the transport of red blood cells, hemoglobin, and O2. We considered O2 consumption for basal cellular metabolism and for active transport of NaCl across medullary thick ascending limbs (mTALs). Our model predicts that the OM structural organization results in significant oxygen tension (PO2) gradients both in the axial and the radial directions. The segregation of descending vasa recta, the main supply of O2, at the center and immediate periphery of the vascular bundles gives rise to large radial differences in PO2 between regions and helps preserve O2 delivery to the inner medulla. Assuming that the mTAL active Na+ transport rate decreases when mTAL PO2 falls below a critical level, oxygen availability to mTALs has a significant impact on the concentrating capability of the model OM. The model also predicts that when the OM undergoes hypertrophy, its concentrating capability increases significantly only when anaerobic metabolism supports a substantial fraction of the mTAL active Na+ transport, which is otherwise critically reduced by the low interstitial and mTAL luminal PO2 in a hypertrophied OM.Support: NIH Grant DK‐53775 (AE and AL) and NSF Grant DMS‐0701412 (AL).
Adenosine Indexes RyR1‐Agonist‐Induced Calcium Overload In Lymphocytes from Malignant Hyperthermia (MH) Susceptible (MHS) Patients
Authors: Bina Saiid , Capacchione John , Muldoon Sheila , Bunger Rolf 
Published: June 2021

The RyR1 receptor exists in human blood lymphocytes (B‐cells); RyR1 agonists can induce calcium overload in B‐cells from MHS individuals. We hypothesized that RyR1‐agonism is associated with increased adenosine (ADO) formation due to increased ATP degradation, which is enhanced in B‐cells from MHS subjects.MethodVenous lymphocyte cells were isolated in HBSS + 1% BSA. Erythro‐9‐(2‐hydroxy‐3‐nonyl) adenine (EHNA, to block ADO deamination) followed by selective RyR1 agonist 4‐chloro‐m‐cresol (4CmC) were added (37 °C for 30 min). HCLO4 terminated reactions. ADO + inosine (INO) were measured by HPLC.Results(for 106 cells): ADO + INO levels in control (n=7) and MHS (n=11) cells were 2.7 ± 0.5 and 5.9 ± 0.6 μM respectively. After 5mM 4CmC treatment, ADO + INO increased to 5.6 ± 1.0 and 35.6 ± 3.1 µM in normal vs. MHS cells (P<0.002). There were no overlaps in the ADO+INO levels between stimulated MHS cells and normal cells.Conclusionsi) RyR1‐agonism‐induced calcium release in B‐cells produces energetic stress that is indexed by increased ADO formation; ii) in MHS cells, 4CmC greatly increases the ADO formation, consistent with increased ATP catabolism due to loss of calcium control; iii) the lymphocyte cells‐adenosine system has clinical potential as a minimally invasive MH diagnostic test.Supported by USUHS
Functional Studies on Escherichia coli Protein NsrR, a Novel Nitric Oxide Sensitive Transcriptional Repressor
Authors: Bitoun Jacob Philip , Duan Xeuwu , Ding Huangen 
Published: June 2021

NsrR, a novel transcriptional repressor from Escherichia coli, coordinates a solvent exposed [2Fe‐2S] cluster through three highly conserved cysteine residues. In the presence of nitric oxide (NO), the iron‐sulfur cluster in NsrR is modified to form a dinitroysl iron complex, and transcriptional repression of NsrR is relieved, promoting the expression of NO detoxifying and defense genes. Here it is shown that NO modification of purified recombinant NsrR occurs at a higher rate than other iron‐sulfur proteins. This result suggests that the NsrR[2Fe‐2S] cluster could be a primary target of NO cytotoxicity. The redox midpoint potential of the NsrR [2Fe‐2S] cluster was determined to be ‐346 ± 7 mV under anaerobic conditions, indicating that in vivo the [2Fe‐2S] in NsrR could be in its reduced state. The mobility shift analysis indicated that the [2Fe‐2S]‐NsrR has a slightly higher binding affinity for its target DNA under reduced conditions than under oxidized conditions, indicating that NsrR may also regulate its transcriptional activity via the redox state of its iron‐sulfur cluster.This work was supported, in whole or in part, by the National Institute of Health Grant RO1 CA107494 from the United States Public Health Service. This work was also supported by the National Science Foundation Grant MCB‐0416 (to H.D.).
The Effects of Nucleotides on Hexamer Stability of Glutamate Dehydrogenase
Authors: Lekova Virzhiniya , Bell Ellis 
Published: June 2021

Bovine glutamate dehydrogenase which catalyzes the oxidative deamination of L‐Glutamate using either NAD or NADP as cofactor, is extensively regulated by a variety of ligands including the purine nucleotides, ADP and GTP as well as having a regulatory site for NADH. The aims of the current study are to establish whether 1] cofactor substrates, and 2] purine nucleotide regulatory ligands affect the stability of the hexamer. Guanidine Hydrochloride denaturation‐renaturation experiments were performed using the fluorescence of the three tryptophans in each subunit. Fluorescence emission spectra and lifetime measurements were used to follow the effects of denaturation and the impact that added ligands have. Dilution experiments were used to indicate whether the presence of ligands affected refolding of the protein. The results demonstrate that both cofactors and purine nucleotides affect both unfolding and refolding processes. Interestingly in the denaturation steps NADPH which binds only to the active site has similar effects to NADH which binds to both active and regulatory sites.Supported by NSF Grant MCB 0448905 to EB
Consumption of fiber is associated with lower body weight measures in US adults: National Health and Nutrition Examination Survey (NHANES), 1999‐2004.
Authors: Cho Susan S , Nicklas Theresa , O'Neil Carol 
Published: June 2021

The objective of this study was to evaluate the effects of consumption of fiber and whole grain (WG) on body weight measures in US adults. A nationally representative sample of U.S. adults participating the NHANES, 1999‐2004, was analyzed for body mass index (BMI) according to quartiles of dietary fiber and WG intake. Participants were divided into four fiber consumption groups: <8.9g/d, 8.9 to <13.63, 13.63 to 19.96, =19.96g/day. Participants were also divided into four WG intake groups: <0.6 (Quartile (Q)1; control), 0.6 to <1.5, 1.5 to <3.0 and =3.0 servings (Q4). Increased consumption of dietary fiber was associated with lower BMI and WC in women and lower WC in men. Increased intake of WG was associated with lower BMI (Q1‐28.52 vs. Q4‐27.78, p<0.05) and WC Q1‐94.0 vs.Q4‐92.23 cm, p<0.05) in women only, but this association disappeared after adjusting for cereal fiber. The data suggest that dietary fiber is an active component in WG effects.Supported by USDA & Kellogg's Corporate Citizenship Fund.