bluuebunny/crossref_metadata_2025 huggingface dataset:
Silencing the respiratory syncytial virus fusion protein gene using RNA interference
Authors: Vig Komal , Lewis Nuruddeen , Moore Eddie G. , Pillai Sheekumar , Dennis Vida A. , Singh Shree Ram
Published: June 2021

Respiratory Syncytial Virus (RSV) is an important respiratory pathogen of medical significance that causes high mortality in infants. At present, there is no vaccine or antiviral drug against RSV. The fusion (F) protein of RSV is a good target for therapeutic purposes as it is primarily responsible for penetration of the virus into host cells and for subsequent syncytium formation during infection. In the present study, four small interfering RNAs (siRNAs), targeting different regions of the RSV F protein were used to silence the RSV F gene in order to disable membrane fusion, viral penetration and syncytium formation. Silencing of F gene was validated by using indirect immunofluorescence, western blot and RT‐PCR. We also analyzed the relationship between siRNA design and target structure. Our results show by cytoplasmic fluorescence and at 20 nM siRNA concentrations the effective silencing of the RSV F gene by the smartpool siRNA and the individual siRNA. RT‐PCR analysis also revealed a decrease in the F mRNA expression. Among the four siRNAs used, siRNA‐1 was most efficient resulting in complete silencing of the F gene expression at 5 nM siRNA concentration. The RSV mRNA target region had substantial effect on siRNA efficiency. The present study shows the silencing of F gene using siRNA and its possible therapeutic use. Financial support for this work by National Institutes of Health Grants 2S06 GM008219‐200012 and NSF‐CREST (HRD# 0734232 ) is acknowledged.
Selective down‐regulation of anti‐angiogenic splice variants of VEGF by IGF‐1 is mediated by PKC, PI‐3K and SRPK1/2
Authors: Nowak Dawid G , Woolard Jeanette , Ladomery Michael , Harper Steve J , Bates Dave O
Published: June 2021

VEGF, the predominant regulatory molecule in angiogenesis, is differentially spliced in its terminal exon (exon 8) to produce either pro‐angiogenic VEGFxxx or anti‐angiogenic VEGFxxxb isoforms. We have shown that IGF‐1 specifically down‐regulates VEGFxxxb expression in proliferating podocytes. Here, we investigated the role of PKC and PI‐3K in IGF‐1‐dependent down‐regulation of VEGFxxxb expression after 48 hours of treatment. VEGFxxxb proteins were measured in cell lysates by isoform‐specific ELISA. VEGFxxxb expression was decreased by IGF‐1 (0.28±0.05 fold to control). BIMI, an inhibitor of PKC kinases, prevented IGF‐1 down‐regulation of VEGFxxxb expression (1.14±0.04 fold to control). SRPIN340, an inhibitor of SRPK1/2 (serine‐arginine (SR) Protein Kinase 1 and 2) kinases implicated in splicing control by phosphorylating splicing factors, inhibited IGF‐1 down‐regulation of VEGFxxxb expression (1.17±0.14 fold). These results indicate that IGF‐1 decreases the synthesis of VEGFxxxb isoforms in proliferating podocytes through PKC, PI‐3K and SRPK1/2 kinases. Funded by the BHF (FS04/09, and BS06/005).
Structural Features of Lon Proteases Deduced from Investigations of Isolated Domains of the Enzyme
Authors: Wlodawer Alexander , Li Mi , Melnikov Edward E , Rotanova Tatyana V , Maurizi Michael , Gustchina Alla
Published: June 2021

ATP‐dependent Lon proteases can be divided into two subfamilies, LonA and LonB. Crystal structures for several individual domains and sub‐fragments of Lon proteases have been solved. Crystal structure of the proteolytic (P) domain of A. fulgidus B‐type Lon (AfLonB) revealed significant differences in the conformation of the active site compared to the P domains from EcLonA and M. jannaschii (MjLonB), despite the similarity of the overall fold. The differences in the interactions of the catalytic residues in the active sites of AfLonB and the other two Lon proteases are primarily connected to the variable conformational state of the segment that precedes the catalytic Ser509, indicating that in isolated P domains this segment does not have a stable conformation that could maintain proper structure of the active site. Structures of the P domains have also indicated a possible hexameric arrangement of subunits in the native state of bacterial Lon proteases. New insights regarding substrate binding and ATP‐dependent unfolding have been obtained from the structures of the N domain, and from the structure of the α‐domain of the ATPase module. The N domain appears to consist of two subdomains terminating in a very long helix (∼80 Å), the latter most likely providing a scaffold for interactions with the ATPase module. These glimpses of the structure of Lon should lead to fuller description of this ancient family of proteases.
The deubiquitinating enzyme ataxin‐3 edits K63‐linkages in mixed linkage ubiquitin chains
Authors: Winborn Brett J , Travis Sue M , Todi Sokol V , Xu Ping , Peng Junmin , Cohen Robert E , Paulson Henry L
Published: June 2021

The importance of differentially linked ubiquitin chains in physiological processes is emerging. The canonical lysine 48‐linked chain (K48‐Ub) primarily signals for proteasomal degradation while other chains, such as those linked at K63 (K63‐Ub), function in myriad pathways. This complexity is regulated by a diverse set of ubiquitin ligases and deubiquitinating enzymes (DUB). One such DUB is the neurodegenerative disease protein, ataxin‐3. Ataxin‐3 contains a N‐terminal catalytic Josephin domain and a C‐terminal ubiquitin binding region with 3 ubiquitin interacting motifs (UIMs) flanking a polyglutamine tract. The tract is expanded in persons with Spinocerebellar Ataxia Type 3.

We report that ataxin‐3 has novel ubiquitin protease activity, editing K63‐linkages in topologically complex ubiquitin chains. Ataxin‐3 binds both K48‐ and K63‐Ub, yet preferentially cleaves K63‐linkages. Proteolysis of K63‐Ub is restricted to longer chains containing at least 5 ubiquitin moieties. Intriguingly, however, ataxin‐3 readily cleaves K63‐linkages in shorter chains containing a mixture of K48‐ and K63‐linkages. The UIMs appear to gate or orient specific linkages for cleavage by the Josephin domain, since UIM mutations eliminate ataxin‐3's K63‐linkage specificity. Our results establish ataxin‐3 as the first reported, mixed linkage ubiquitin‐editing enzyme.

Funding: R01 NS038712 (HLP)
IDENTIFICATION AND CHARACTERIZATION OF BRANCHED‐CHAIN AMINO ACID (BCAA) METABOLON PROTEINS: ROLE OF GLUTAMATE DEHYDROGENASE
Authors: Islam Mohammad Mainul , Nautiyal Manisha , Mobley James , Hutson Susan
Published: June 2021

The catabolic pathway for branched‐chain amino acids (BCAAs) include deamination followed by oxidative decarboxylation of the deaminated product branched‐chain α‐keto acids (BCKAs), catalyzed by mitochondrial branched‐chain aminotransferase (BCATm) and branched‐chain α‐keto acid dehydrogenase enzyme complex (BCKDC), respectively. We have found that BCATm binds to the E1 decarboxylase of BCKDC, forming a metabolon that allows channeling of BCKAs from BCATm to E1. The metabolon also contains glutamate dehydrogenase (GDH1). We show that GDH1 only binds to the pyridoxamine 5’‐phosphate (PMP)‐form of BCATm (PMP‐BCATm), and does not bind to pyridoxal 5’‐phosphate‐BCATm (PLP‐BCATm) or BCKDC components. Leucine allosterically activates GDH1 and oxidative deamination of glutamate is increased further by addition of PLP‐BCATm. Isoleucine and valine are not allosteric activators; but in the presence of PLP‐hBCATm, convert it to PMP‐BCATm, stimulating GDH1 activity. Kinetic results suggest that α‐ketoglutarate (α‐KG) produced by GDH1‐catalyzed oxidative deamination of glutamate is channeled directly to BCATm. Then BCATm is converted to PLP‐BCATm transferring BCAA nitrogen to α‐KG. Thus GDH1 facilitates regeneration of the form of BCATm that binds to the E1 decarboxylase of the BCKDC, and promotes metabolon formation, BCAA oxidation and nitrogen transfer to glutamate. (NIH DK34738).
Mutagenesis of the S‐adenosyl‐L‐methionine binding site in Escherichia coli biotin synthase
Authors: Farrar Christine , Jarrett Joe
Published: June 2021

Biotin synthase catalyzes the final step of the biotin biosynthetic pathway involving the insertion of a sulfur atom into dethiobiotin. The active site of the enzyme contains the cofactor S‐adenosyl‐L‐methionine (AdoMet), the substrate dethiobiotin and two iron–sulfur clusters. The reaction sequence of biotin synthase is initiated by electron transfer through an iron–sulfur cluster into the AdoMet sulfonium, resulting in the cleavage of AdoMet into methionine and a 5′‐deoxyadenosyl radical. The radical is then used to abstract hydrogen from the dethiobiotin, allowing for the insertion of a sulfur atom. A second equivalent of AdoMet leads to another round of catalysis and the formation of a bond that completes the biotin thiophane ring. In this study, single‐point mutations of two conserved amino acids involved in the binding of AdoMet and dethiobiotin are described. The mutants have similar iron‐sulfur content to that of the wild‐type enzyme and variable binding properties pertaining to AdoMet and dethiobiotin. The mutants are unable to produce biotin, however, some are able to cleave AdoMet, producing 5′‐deoxyadenosine and a non‐biotin product. Mass spectrometry is used to analyze the alternate product providing possible evidence for a reaction intermediate where the mutant enzymes are incapable of completing the correct reaction sequence.
Calcium binding effects on GIVA Phospholipase A2 mapped by Hydrogen/Deuterium exchange mass spectrometry
Authors: Hsu Yuan‐Hao , Burke John E , Woods Virgil E. , Dennis Edward A.
Published: June 2021

The GIVA phospholipase A2 (PLA2) contains two domains: a calcium binding domain (C2) and a catalytic domain. GIVA PLA2 activity is Ca2+ dependent in that calcium binding promotes protein docking to the phospholipid membrane. The C2 domain and possibly the catalytic domain penetrate the lipid surface. We now present studies that explore the dynamics and conformational changes of this enzyme in solution utilizing peptide amide hydrogen/deuterium exchange coupled with liquid chromatography‐mass spectrometry (DXMS) to probe the solvent accessibility and backbone flexibility of the GIVA PLA2. We also analyzed the changes in H/D exchange of the GIVA PLA2 holoenzyme upon Ca2+ binding. The DXMS results showed a fast H/D exchanging lid and a slow exchanging central core. The C2 domain showed two distinct regions: a fast exchanging region facing away from the catalytic domain and a slow exchanging region present in the cleft between the C2 and catalytic domains. The effects of Ca2+ binding on GIVA PLA2 are localized in the C2 domain and suggest that binding of two distinct Ca2+ ions causes tightening up of the regions that surround the anion hole at the tip of the C2 domain. This conformational change may be the initial step for membrane penetration.
Interactions of cytosolic phospholipase A2 with pyrrophenone, ca2+ and phospholipids studied using deuterium exchange mass spectrometry
Authors: Burke John E , Hsu Howard , Deems Raymond , Woods Virgil , Dennis Edward A
Published: June 2021

Phospholipase A2 (PLA2) catalyzes hydrolysis of the sn‐2 fatty acyl bond of phospholipids (PL), liberating free fatty acid and lysophospholipid. We are studying the)mechanism of activation, and membrane binding of the cytosolic GIVA PLA2. This enzyme liberates arachidonic acid from phospholipids, which is a precursor formany inflammatory mediators.

We have used hydrogen‐deuterium exchange mass spectrometry (DXMS) to study this enzyme. The rate of exchange of each amide proton is directly proportional to the accessibility to bulk solvent. Measurements of exchange rates have been used to characterize changes in solvent accessibility that result from protein/lipid binding interactions, and conformational changes.

Group IVA cPLA2's interactions with inhibitors and phospholipid were studied using DXMS. We used pyrrophenone, a potent inhibitor of GIVA PLA2, to probe the changes in conformation upon binding in the active site. Large amide exchange rate changes were seen upon inhibitor binding. We propose that these changes can be explained by the opening of the lid region with inhibitor bound. Large changes were also seen upon calcium‐mediated lipid binding, mainly in the C2 domain. We propose the protein inserting into the lipid membrane causes these changes. These studies show that DXMS is an excellent technique for probing enzyme interfacial activation. This work was supported by NIHGM20,501
Orotidine 5’‐Monophosphate Decarboxylase: Effect of an S154A Mutation on Enzyme‐Catalyzed Decarboxylation of OMP and on Decarboxylation of Complexes that Contain the Substrate Pieces
Authors: Barnett Shonoi A , Wood Bryant M , Gerlt John A , Richard John P
Published: June 2021

Orotidine 5’‐monophosphate decarboxylase (OMPDC) catalyzes the chemically difficult decarboxylation of orotidine 5’‐monophosphate (OMP) to give uridine 5’‐monophosphate (UMP) (Scheme 1). The ratio of the second order rate constants for decarboxylation of OMP and of a truncated substrate 1‐(β‐D‐erythrofuranosyl)orotic acid (EO), (4 × 108)‐fold, gives an intrinsic phosphate binding energy of 11.8 kcal/mol. More than half of the dianion binding energy (7.8 kcal/mol) can be recovered in the binding of phosphite dianion to the (OMPDC•EO)‡ complex. The effect of the S154A mutation of the yeast enzyme on the stability of the transition state generated from free OMPDC and OMP (5.7 kcal/mol) is similar to the effect of the mutation on the transition state generated from free OMPDC, EO and phosphite (6.4 kcal/mol), so that the whole substrate and substrate in pieces react through similar transition states. The effect S154A mutation on the reaction of the pieces has been partitioned into a 3.2 kcal/mol effect on the reaction of EO alone, and a 3.1 kcal/mol effect on phosphite activation. The mutation reduces phosphite activation, even though there are apparently no direct interactions between the excised ‐CH2OH and the phosphate of OMP. We propose that dianion binding drives loop closure around the transition state, which serves to strengthen the network of hydrogen bonding interactions with the ‐CH2OH side chain.
Synthesis and Characterization of a Deacylation‐Defective Mutant of the Class D β‐Lactamase OXA‐1
Authors: Schneider Kyle D. , Davis Chris J. , Smart Robert P. , Leonard David A.
Published: June 2021

β‐lactamases hydrolyze penicillin, cephalosporin and carbapenem β‐lactam antibiotics. The class D β‐lactamase OXA‐1 has a catalytic serine (position 67) thought to be deprotonated and thereby activated by a carboxylated lysine (position 70). We have made several mutations of OXA‐1 at both Lys70 and Ser67 to help elucidate the role of these two critical residues in the catalytic mechanism. We have used the fluorescent substrate bocillin and a novel fluorescent β‐lactam to demonstrate that the Lys70Glu mutant can acylate but not deacylate various substrates. We have been able to isolate and visualize a fluorescent acyl complex of bocillin and Lys70Glu using SDS‐PAGE. The formation of the fluorescent band is prevented by preincubation of the mutant enzyme with excess ampicillin, demonstrating that the complex is specific to the active site.
Enhanced phosphorylation of FOXO and GSK‐3 by organo‐vanadium complexes: potential role in insulino‐mimesis
Authors: Vardatsikos George , Mehdi Mohamad Z , Srivastava Ashok K
Published: June 2021

Several organo‐vanadium complexes (OVCs) have been shown to exert multiple insulino‐mimetic effects in rodent models of diabetes. We have shown that Bis(maltolato) OxoVanadium(IV) (BMOV) activates several components of the insulin signaling pathway such as phosphatidyl inositol‐3 kinase (PI3‐K) and protein kinase B (PKB). Forkhead Box Proteins (FOXO) and Glycogen Synthase Kinase‐3 (GSK‐3), downstream substrates of PKB, are thought to contribute to insulin‐induced gene expression of key gluconeogenic enzymes such as phosphoenolpyruvate carboxykinase (PEPCK) and glucose‐6‐phosphatase (G6Pase). OVCs are known to modulate the expression of PEPCK and G6Pase, hence in the present studies we investigated BMOV‐induced FOXO and GSK‐3 regulation. Treatment of Chinese hamster ovary cells overexpressing human insulin receptor (CHO‐HIR) enhanced FOXO and GSK‐3 phosphorylation. Wortmannin, a PI3‐K inhibitor, decreased FOXO and GSK‐3 phosphorylation induced by both insulin and OVCs, as did AG1024, an inhibitor of IR/IGF‐1R‐PTK activity. In summary, these data demonstrate that OVCs potently enhance the phosphorylation of FOXO and GSK‐3 via IGF‐1R‐PTK/PI3K‐dependent pathways, suggesting that OVCs may contribute towards the insulino‐mimetic effects of BMOV by transcription regulation of gluconeogenic enzymes.

(Supported by grants from Canadian Institutes of Health Research).
Comprehensive plasma acylcarnitine profiles of type 2 diabetic and non‐diabetic subjects with or without an uncoupling protein 3 (UCP3) missense polymorphism
Authors: Adams Sean H. , Hoppel Charles L. , Lok Kerry H. , Newman John W. , Garvey W. Timothy
Published: June 2021

Type 2 diabetes mellitus (T2DM) is often correlated with reduced muscle fatty acid combustion in the fasted state. This may be due to dysfunctional mitochondrial activity and accumulation of specific lipid metabolites implicated in insulin resistance. Biomarkers of muscle fat combustion would be valuable to identify persons at‐risk for developing T2DM and to better understand muscle intermediary metabolism. To address this, we compared >40 individual plasma acylcarnitines in overnight‐fasted adult women harboring a UCP3 premature‐stop allele (50% lower whole‐body fat combustion, apparently due to limited muscle UCP3 activity) to a control cohort. Contrary to expectations, acylcarnitine profiles did not differ by genotype. However, across genotypes, T2DM subjects (n=44) displayed (relative to non‐diabetics, n=12): ~50% increased total acylcarnitines (6.52 ± 0.32 vs. 4.40 ± 0.55 μM; p<0.01), ~60% higher acetylcarnitine (5.35 ± 0.29 vs. 3.40 ± 0.46 μ p<0.01), and ~37% decreased propionylcarnitine (0.24 ± 0.02 vs. 0.38 ± 0.04 μ p<0.001). Such patterns are consistent with a hypothetical model in which fasted T2DM tissues (a) have inordinately low availability of propionyl‐CoA (a component of TCA cycle‐replenishing [anaplerotic] reactions), and (b) increased pools of pyruvate‐derived acetyl‐CoA, presumably due to relatively elevated glycolysis and/or reduced TCA cycle acetyl‐CoA consumption.
Bioinformatic Analyses of Altered Regulation of myo‐Inositol Oxygenase in Diabetes Mellitus
Authors: Bajracharya Jayendra , Klig Lisa S
Published: June 2021

In humans, myo‐inositol (MI) is catabolized by myo‐inositol oxygenase (MIOX). Depletion of MI is seen in tissues affected by diabetes mellitus such as kidney, nerve and retina. Up‐regulation of MIOX is a likely mechanism of MI depletion in these tissues. This study examines three possible mechanisms for up‐regulation of MIOX: 1) DNA regulatory elements, 2) additional copies of the gene, 3) alternate splicing resulting in other active isoforms. In silico experiments revealed known and novel regulatory elements in the MIOX gene, which are also present in other genes up‐regulated in diabetes, such as aldose reductase. A genomic sequence 86 % identical to the MIOX gene (chromosome 22) was identified in chromosome 8. This appears to be an intronless version of the MIOX gene. Further examination of the sequence revealed a TATA box and regulatory elements, but translation of the sequence resulted in short peptides with minimal identity with MIOX protein. So, this sequence may be considered a pseudogene. This study has also identified four physiologically relevant alternate splice events in MIOX transcripts evident in the EST database. The amino acid sequences of the translated products of these transcripts retain high percent identity with the known MIOX protein. Structural analyses of MIOX protein and its isoforms will indicate whether the isoforms are more active than the known MIOX protein.
Effect of Hyperketonemia (acetoacetate) on Apoptosis in Human U937 Monocytes
Authors: Rains Justin L. , Jain Sushil K.
Published: June 2021

Hyperketonemia (ketosis) is a pathological condition observed in patients with type I diabetes. In uncontrolled diabetes, blood concentrations of acetoacetate (AA) and other ketone bodies can be as high as 25 mM compared to 0.5 mM in normal individuals. Frequent episodes of ketosis are associated with higher incidences of long term complications in diabetics. This study examines the hypothesis that ketosis leads to apoptosis in monocytes, which contributes to cellular dysfunction and the development of vascular disease in diabetics. U937 monocytes, purchased from ATCC, were cultured with AA (0–10 mM) for 24 hours. Mitochondrial membrane potential (MMP) was determined by DiOC6 uptake; reactive oxygen species (ROS) by using 10 μM dihydroethidium and apoptosis was detected using Annexin V binding, using flow cytometry. Results (mean±SE) show that there was an AA concentration dependant increase in apoptosis, which was 48±9% (p=0.001) at 4 mM AA in comparison to control cells. There was a concentration dependant decrease in the MMP and 4 mM AA treated cells showed 37±8 % (p=.002) decrease in MMP compared to control cells. Similarly, ROS, as expressed by increasing mean fluorescence, showed a concentration dependant increase in cells treated with AA compared with control cells. β‐hydroxybutyrate, another ketone body, did not cause apoptosis, decrease in MMP, and ROS generation. This suggests that elevated blood level of AA can induce ROS and altered MMP, which can lead to the apoptosis in monocytes and cellular dysfunction in diabetes. (Supported by grant from NIDDK and Office of Dietary Supplements)
Importance of signal duration and the time scale dependence of signal integration in biochemical networks
Authors: Locasale Jason W , Chakraborty Arup K
Published: June 2021

Signal duration (i.e. the time over which an active signaling intermediate persists) is a key regulator of biological decisions in myriad contexts such as cell growth, proliferation, and developmental lineage commitments. Accompanying differences in signal duration are numerous downstream biological processes that require multiple steps of biochemical regulation. Here, we present a theoretical analysis that investigates how simple biochemical motifs that involve multiple stages of regulation can be constructed to differentially process signals that persist at different time scales. We compute power spectra of the dynamic gain within these networks to better understand how biochemical networks can integrate signals at different time scales. We identify the topological features of these networks that allow for differential, frequency dependent signal processing. Our studies suggest design principles for why signal duration in connection with multiple steps of downstream regulation is a ubiquitous control motif in biochemical systems.
Structure and Function characterization of ubiquitin like protein of Singapore grouper iridovirus
Authors: TRAN NGOC BICH , LIU YANG , HEW LEONG CHOY
Published: June 2021

Singapore grouper iridovirus (SGIV) containing 162 open reading frames causes the sleepy disease in grouper fish with unknown mechanism. This virus encodes putative ubiquitin like protein with 75% of similarity with animal ubiquitin. The question is why this virus needs its own ubiquitin? To address this question, we firstly confirmed the biological function of SGIV ubiquitin by in vitro ubiquitination assay. The NMR experiment was conducted to determine the structure of the viral ubiquitin. The structure of SGIV ubiquitin presented the common protein foldings of ubiquitin. Although ubiquitin is one the most conserved protein, our comparative analysis of different viral ubiquitin DNA and protein sequences reveales some viral unique regions. On the other hand, the Itraq experiment interestingly showed that ubiquitin is up regulated after virus infection. To further elucidate the function of viral ubiquitin, we are focussing on distinguishing viral and host ubiquitin. The viral unique ubiquitin peptide sequence is used for raising antibody specific for viral ubiquitin. Western blot experiment has showed bands of ubiquitinated proteins up regulated after virus infection. We are futher investingating those changes using 2D gel and Mass spectrometry approach.
Complex Structures in Thin Films Detected By Infrared Spectroscopy
Authors: Kosower Edward M , Markovich Gil , Borz Galina
Published: June 2021

Thin films are ubiquitous and important in chemistry, materials science, biology and engineering, but are difficult to study on the molecular level. Infrared spectroscopy is useful because of the rich detail in molecular information and the speed of acquisition of spectra. We have developed an attenuated total internal reflectance (ATR) technique for samples in thin films on a planar AgX (X=Cl, Br) fiber that reveals unsuspected interactions. We initially discovered surface enhanced infrared absorption (SEIRA) for trypsin and p‐nitrobenzoic acid and more recently, complex structures in thin films of alkanols (some known) and acetonitrile (previously unknown). The structures are H‐bonded oligomers for alkanols and dipole‐dipole oligomers for acetonitrile. Almost all molecules associate in liquid and solid form (gas phase clusters) as a result of dipole‐dipole, hydrogen‐bonding, polarization, and dispersion interactions. We have generalized previous indications: there is an infrared signature of increased absorption intensity or changes in spectrum for many associations. We now report that the peptide model N‐methylformamide associates rapidly (2 sec) to an α‐helical oligomer (λmax 1652 cm−1) which then is transformed (10 sec) into the weakly absorbing cyclic hexamer. Peaks for monomer, dimer and trimers are observed in the first 0.5 sec. The implications for peptide IR will be discussed.
The Antioxidant Activity of Cerium Oxide Nanoparticles is Size Dependant and Blocks Aβ1‐42‐Induced Free Radical Production and Neurotoxicity
Authors: Singh Neeraj , Amateis Eric , Mahaney James E , Meehan Kathleen , Rzigalinski Beverly A
Published: June 2021

We have demonstrated that a single 10 nM dose of cerium oxide nanoparticles (CeONP) with an average size of 10 nm, extends the lifespan & preserves neuronal function in brain cell cultures. We hypothesize that CeONP act as regenerative free radical scavengers. Here, we investigated the antioxidant activity of CeONP using EPR. Hydroxyl radicals (.OH) were generated via Fenton's reaction. Superoxide radicals (O2.–) were generated by irradiating a solution of riboflavin and DMPO with UV rays. CeONP (10 nm) completely quenched .OH & O2.– formation. CeONP with a particle size of 7 nm were less effective than 10 nm particles. We also examined the effect of Fe‐doped ceria (6% Fe), which was less effective than CeONP. The toxic fragment of Αβ, Αβ1‐42, produces free radicals & is hypothesized to be a contributory factor in Alzheimer's pathology. When incubated with spin trap Αβ1‐42 (1mg/ml) generated a 6 line EPR spectra. Αβ1‐42–induced free radical production was completely blocked by CeONP (10 nm). We also examined the neuroprotective capacity of the 3 groups of CeONP using UV, H2O2, & Αβ1‐42 challenge. In comparison to 7 nm CeONP, cell death induced by UV & H2O2 was reduced more effectively by 10 nm CeONP, whereas Fe‐doping of CeONP abrogated antioxidant protection. Neuronal death associated with Αβ1‐42 was reduced 65% by CEONP (10 nm). Our results suggest that 1) CeONP exhibit potent antioxidant activity, which is dependent on size, composition, & particle surface area and 2) CeONP protect neurons from free radical damage & Αβ1‐42 toxicity.
Cross‐linking of M. tuberculosis Catalase‐Peroxidase (KatG)
Authors: Suarez Javier , Ranguelova Kalina , Magliozzo Richard S
Published: June 2021

The formation of inter and intra‐molecular cross‐links has been reported for many heme and metallo‐proteins that form endogenous radicals. M. tuberculosis catalase‐peroxidase (KatG) is a bifunctional enzyme in which tyrosyl and tryptophanyl radicals are known to be formed during turnover with peroxides. The main goal of the present study was to gain insights into the loci of radical formation through the analysis of cross‐linking during turnover of KatG in the presence and absence of reducing substrate. SDS and Native‐PAGE of KatG treated with peracetic acid or hydrogen peroxide under a variety of conditions demonstrate oligomers of molecular weight greater than that of the native dimmer. Isoniazid, the antibiotic activated by Mtb KatG, completely inhibit crosslink formation. The KatG concentration dependence of the distribution and yield of oligomers strongly suggest that the sites of crosslink formation are solvent exposed though tyrosyl radicals are known to be first formed at buried tyrosines during peroxide turnover. KatG mutants at positions Y197, Y353, Y304, W204 demonstrate that these residues are not involved in crosslinking. The results are consistent with the hypothesis that cross‐linking of KatG can occur in the absence of peroxidase substrates and that under physiological conditions, the activation of INH by KatG may be altered by this process. Experiments for the identification of the residues involved in the crosslinks and to study the properties of the higher molecular weight products are ongoing.
RNA interference of PFUS results in inhibition of secretory vesicle biogenesis
Authors: Satir Birgit H. , Liu Li
Published: June 2021

In protistan evolution, gene duplication gives rise to phosphoglucomutase (PGM) superfamily members which had virtually no PGM activity, including Paramecium parafusin (PFUS) and its Toxoplasma and mammalian orthologs, phosphoglycoproteins whose role in Ca2+ ‐regulated exocytosis has been controversial. We show that RNAi knockdown of PFUS leads to an exo− phenotype. Three different regions of the PFUS sequence were targeted. As a control we used the empty RNAi vector. Exocytosis was scored after stimulation. Overall, exocytosis becomes inhibited with all three RNAi constructs, compared to the control. In order to examine if inhibition correlated with a reduction of PFUS, we performed western blot analysis using a polyclonal antibody which recognizes all Paramecium proteins in the PGM superfamily (pan‐PGM antibody) and a synthetic peptide antibody, PFUS antibody, which recognizes PFUS specifically. Cells exposed to RNAi for 72 hr show that PFUS decreases, whereas no decrease is seen in other PGM isoforms. Double labeling in confocal microscopy for PFUS and secretory content (T‐8) shows a corresponding decrease in dense core secretory vesicles (DCSVs) in the cells. These changes are reversed by re‐feeding in the absence of PFUS RNAi. These data demonstrate that knockdown of PFUS inhibits exocytosis, probably by blocking DCSV packaging.
Nitric Oxide Production from Nitrite Occurs Primarily in Tissues not in the Blood: Critical Role of Xanthine Oxidase and Aldehyde Oxidase
Authors: Li Haitao , Cui Hongmei , Kundu Tapan Kumar , Alzawahra Wael , Zweier Jay L.
Published: June 2021

Recent studies have shown that nitrite is an important storage form and source of nitric oxide (NO) in biological systems. Controversy remains, however, regarding whether NO formation from nitrite occurs primarily in tissues or in blood. Questions also remain regarding the mechanism, magnitude, and contributions of several alternative pathways of nitrite dependent NO generation in biological systems. To characterize the mechanism and magnitude of NO generation from nitrite, Electron Paramagnetic Resonance (EPR) spectroscopy, chemiluminescence NO analyzer, and immunoassays of cGMP formation were performed. Addition of nitrite triggered a large amount of NO generation in tissues such as heart and liver, but only trace NO production in blood. Carbon monoxide increased NO release from blood suggesting that hemoglobin acts to scavenge NO. Administration of the xanthine oxidase (XO) inhibitor oxypurinol or aldehyde oxidase (AO) inhibitor raloxifene, significantly decreased NO generation from nitrite in heart or liver. Isolated enzymatic studies revealed that AO had the similar capability as XO to reduce nitrite with the formation of NO. Overall, NO generation from nitrite mainly occurs in tissues not in the blood, with XO and AO playing critical roles in nitrite reduction; and this process is regulated by pH, oxygen tension, nitrite, and reducing substrate concentrations.
Overexpression of catalase increases p27 expression in mouse aortic endothelial cells
Authors: Onumah Ogbeyalu , Jules George , Zhou LiChun , Yang Hong , Guo ZhongMao
Published: June 2021

Endothelial cell (EC) proliferation contributes to normal vascular homeostasis and to various vascular diseases. Accumulating evidence demonstrates that low concentrations of hydrogen peroxide (H2O2) promote EC proliferation. The objective of this report is to assess the regulatory role of endogenous H2O2 in cell cycle progression during EC proliferation. ECs were obtained from mice overexpressing human catalase transgene (hCatTg+/0) and their wild‐type littermates. Cell growth was determined using an ATP luminescent cell proliferation kit. The distribution of cells in G0/G1, S and G2/M phases of the cell cycle were determined using flow cytometry analysis. The expression of cell cycle proteins was determined using western blot analysis. The hCatTg+/0 ECs displayed a reduced growth rate and a prolonged doubling time compared to wild‐type controls, (44.0 ± 4.7 hrs versus 28.6 ± 0.8 hrs, P<0.05). Phase duration calculations indicated that the retarded growth rate and the prolonged doubling time resulted mainly from an extended G0/G1 phase in hCatTg+/0 ECs as compared to the wild‐type ECs (25.0 ± 0.9 hrs versus 15.9 ± 1.4 hrs, P<0.05). In addition, the delayed cell proliferation of hCatTg+/0EC is associated with a significant increase in p27 expression, which is a critical component requiredfor transition of the quiescent cells from G0 to G1. In contrast, cell cycle proteins, such as cyclin D1, cyclin E, cyckin‐dependent kinase‐4 and cyckin‐dependent kinase‐2, that were responsible for the transition from G1 to S phase showed comparable levels between hCatTg+/0 and wild‐type cells. Since catalase is an H2O2 scavenger, our results suggest that endogenous H2O2 mediates EC proliferation by recruiting quiescent cells to the cell cycle.
Myc upregulates anti‐apoptotic protein, Survivin via a transcriptional activation
Authors: Li Hui , Wei Yun , Tan Yun , Wu Xinxing
Published: June 2021

The Myc has been characterized to be a key regulator of cell growth, proliferation, differentiation, and apoptosis for years. Myc is essential for embryonic development and dysregulation of Myc is observed in most of human cancers. Survivin, a member of the IAP family, inhibits activation of downstream effectors of apoptosis, caspase‐3 and ‐7, in cells exposed to apoptotic stimuli. An elevated survivin is also observed in human cancers. In this study, we found that knockdown of Myc expression with siRNA duplexes dramatically decreased the level of survivin in HeLa (a cervical cancer cell line) and MCF‐7 (a breast cancer cell line). RT‐PCR analysis demonstrated that the decease was due to reduction of survivin transcription in siRNA treated cells. In the other hand, overexpression of Myc led an increase of both survivin mRNA and protein. Finally, Our luciferase reporter assays confirmed that Myc was able to induce the survivin promoter in both cancer and normal cells. Further mutagenesis data suggested that Myc upregulated survivin transcription through a non‐canonical E‐box site (CACGCG) and Sp1 sites at the promoter. Thus, survivin is a Myc target and may contribute some functions of Myc in cancer cells. They both are potential targets for cancer therapy.

This work was supported by grants from the National Natural Science Foundation of China (No. 30500465) and China Hubei Natural Science Foundation.
Differential regulation of adipocyte PDE3B in distinct membrane compartments in response to insulin and CL316243
Authors: Khan Faiyaz Ahmad , Lindh Rebecka , Tang Yan , Ruishalme Lida , Ost Anita , Stalfors Peter , Degerman Eva , Manganiello Vincent
Published: June 2021

PDE3B is activated by insulin and CL (CL316243, a b3‐receptor agonist) in adipocytes via PKB‐ and PKA‐ signaling, respectively. Activation of PDE3B by insulin seems important in its ability to reduce cAMP, and inhibit lipolysis. Using sucrose gradient centrifugation and confocal microscopy techniques, PDE3B was found to be localized in internal membranes and in caveolin‐enriched PM in 3T3‐L1 adipocytes. Stimulation of adipocytes with insulin and CL indicated that insulin and CL preferentially phosphorylated/activated PDE3B associated with internal membranes and caveolin‐enriched PM, respectively. Superose 6 chromatography of solubilized membrane proteins from adipocytes stimulated with insulin or CL demonstrated reversible assembly of distinct macromolecular complexes (MMC) that contain 32P‐phosphorylated PDE3B and signaling proteins involved in its activation. siRNA‐mediated knock‐down of caveolin‐1 resulted in down‐regulation of membrane associated PDE3B; insulin‐ and CL‐induced activation of PDE3B was reduced. Insulin‐ and CL‐mediated MMC formation was significantly inhibited by caveolin‐1 knock‐down. These data suggest that caveolin‐1 acts as a molecular chaperone or scaffolding molecule in cholesterol‐rich lipid rafts that may be necessary for the proper stabilization and activation of PDE3B in response to CL and insulin.
Effects of Dietary Fatty Acids on Hepatic Function and Gene Expression in a Model of Hepatic Steatosis
Authors: Gonzalez Maria Monica , Sealls Whitney , Brosnan Julia , Black Paul N , DiRusso Concetta C
Published: June 2021

NADPH‐cytochrome P450 reductase (POR) is required for the function of many microsomal enzymes, including cytochrome P450 monooxygenases In liver, the P450 enzymes play major roles in the metabolism and detoxification of nutrients, pharmaceuticals and toxic compounds. Deletion of the POR gene in mice (liver‐POR‐null), results in reduced serum cholesterol and triglyceride levels and sizable accumulation of triglycerides resulting in steatosis, which may result in inflammation and fibrosis. In previous work, we reported liver‐POR‐null animals have reduced expression of genes involved in elongation and desaturation of PUFA (polyunsaturated fatty acid), which leads to a reduced capacity to synthesize HUFA (highly unsaturated fatty acid). In the present studies, we have examined the fates and regulatory effects of feeding mice diets that are enriched in saturated fat from lard (16:0 and 18:0), PUFA from canola oil (18:2 and 18:3), and HUFA from a fish and fungal oil mixture (20:4, 20:5 and 22:5). The results point to very profound and unique differences in the storage capacity for these fats in adipose tissue, metabolic fates in liver, and effects on gene expression in liver. The deletion of POR exacerbates the problems associated with dietary fat and leads to additional gene expression changes that favor steatosis and inflammation. This work was supported by a grant from the Charitable Leadership Foundation.
mTOR Activation Protects Against UVB‐Induced Cell Death in Cultured Keratinocytes and Dendritic Cells
Authors: Cao Cong , Dragoi Ana‐Maria , Song Xiuzu , Amaral Ashley , Kivlin Rebecca , Lu Shan , Chu Wen‐ming , Kouttab Nicola , Guan Kunliang , Xu Aier , Wan Yinsheng
Published: June 2021

mTOR (mammalian target of rapamycin) serves as a signal integrator of several upstream signals, including growth factors, nutrients, energy levels, and stresses. We demonstrate in this study that UVB radiation induces mTOR activation in human skin keratinocytes (HaCaT cells) and both cultured mouse dendritic cells (XS 106 cell line) and mouse monocyte derived dendritic cells (MoDC). Using specific inhibitors and gene manipulation methods, we observed that UVB induced mTOR activation is dependent on EGFR‐mediated AKT activation. We also found that TSC2/mTOR, downstream of AKT, is involved in UVB‐induced S6K activation. UVB radiation also induces activation of AMPK (AMP‐activated protein kinase) and p38 as well as its upstream signal LKB1. Our data suggests that LKB1/AMPK/p38 serve as a negative feedback pathway against UVB‐induced mTOR activation. We conclude that mTOR activation, together with its upstream signals, serves as a novel survival signal against UVB‐induced cell death.
Rapamycin Induced Transactivation of EGFR: Implications in the Regulation of Cellular Apoptosis
Authors: Chaturvedi Deepti , Cohen Michael S. , Taunton Jack , Patel Tarun B.
Published: June 2021

The mammalian target of rapamycin (mTOR) signaling network regulates cell growth, proliferation and cell survival. Deregulated activation of this pathway is a common event in diverse human diseases such as cardiac hypertrophy, vascular restenosis, nephrotic hypertrophy and cancers. Because of its anti‐proliferative properties, the mTOR inhibitor, rapamycin (a macrolide antibiotic), has been widely used in conjunction with coronary stents to prevent restenosis in coronary arteries following balloon angioplasty. However, in some cases mTOR inhibition by rapamycin does not attenuate the anti‐proliferative and apoptotic effects. Thus, we undertook a detailed study to elucidate the mechanism by which cells confer resistance to rapamycin. Our studies using primary rat aortic vascular smooth muscle cells (VSMC), mouse lung fibroblasts (B82L), COS7 cells, and primary mouse embryonic fibroblasts (PMEFs) show that rapamycin activates ERK1/2 signaling. Our data demonstrate that rapamycin‐mediated activation of the Erk1/2 signaling requires (a) the expression of the EGFR, (b) its tyrosine kinase activity, and (c) intact tyrosine auto‐phosphorylation sites on the receptor. Indeed, rapamycin causes tyrosine phosphorylation of the EGFR. This transactivation of EGFR requires activation of c‐Src, as Src kinase inhibitor (PP2), but not PP3, obliterates the ability of rapamycin to increase Erk1 or RSK1 phosphorylation. Rapamycin treatment also triggers activation of cell survival signaling pathway by activating the pro‐survival kinases Erk1/2 and p90RSK1. Given these findings we hypothesize that rapamycin‐ mediates transactivation of EGFR and activates the pro‐survival pathway via Src kinase.

Supported by NIH grant GM 079226
Analysis of Signaling Pathways using Antibody Microarrays and RNA Interference
Authors: Narahari Janaki , Morgan Aric , Vattem Krishna , Sullivan Yvonne , Hughes Douglas , Hommema Eric , Kilmer Greg , Webb Brian
Published: June 2021

The study of complex cellular signaling pathways requires powerful and specific tools for the manipulation and quantification of protein levels and activity. We have developed a new set of tools to streamline the experimental approach for pathway analysis. These include a series of validated Thermo Scientific SuperSignal® siRNA/Antibody Kits for monitoring gene‐silencing at the protein level, slide‐based antibody microarrays, and several kits for cell‐ based assays. We have demonstrated target‐specific applications to confirm the functionality of the various kits. For example, the expression profile of various cytokines and MMPs were analyzed by protein microarrays following inhibition of several signaling pathways using RNA interference. Knockdown of NFB protein in A549 cells using siRNA resulted in significant inhibition of cytokine production. The activation of signaling proteins in these pathways was studied using our Thermo Scientific Cellomics® HCS Reagent Kits. Our results demonstrate the high quality and utility of these reagents for the efficient profiling of different pathways.
Mitochondrial ChChD3 acts as a Scaffold for Mitofilin, Sam50 and PKA
Authors: Darshi Manjula , Taylor Susan S
Published: June 2021

Cyclic AMP dependent protein kinase A (PKA) is ubiquitous in mammalian cells and plays a critical role in the regulation of various cellular processes. Thus, it has many substrates which usually are highly tissue specific. ChChd3 is one such substrate which we have recently identified by using analog sensitive catalytic subunit of PKA. ChChd3 contains a myristylation motif followed by DUF domain and a CHCH domain. In mouse fibroblasts, the endogenous ChChd3 is primarily localized to mitochondria, sometimes also seen in nucleus. However, over‐expressed ChChd3 is localized only to mitochondria. Deletion of CHCH domain disrupts mitochondrial localization suggesting that CHCH domain is an essential targeting motif in ChChd3. In vitro protein import studies showed that in addition to cysteines in CHCH domain, myristoylation is also necessary for targeting the protein to the IM of mitochondria. Over‐expressed ChChd3 in HEK 293 cells binds to PKA and shows a clear increase in phosphorylation at Thr10 upon stimulation with 8‐CPT cAMP. Further, immunofluorescence and sub‐cellular fractionation studies showed that the protein is phosphorylated in mitochondria and translocated to nucleus. Co‐immunoprecipitation of mitochondrial proteins, Mitofilin and Sam50 with ChChd3 suggests that ChChd3 may be involved in protein import along with Sam50. Studies are currently underway to see the role of ChChd3 as part of mitochondrial protein import machinery.
Cholesterol Suppresses Cellular TGF‐beta Responsiveness by Increasing TGF‐beta Receptor Accumulation in Lipid raft/Caveolae Microdomains and Alterring TGF‐beta Binding to TGF‐beta Receptors
Authors: Chen Chun‐Lin , Huang Shuan Shian , Huang Jung San
Published: June 2021

TGF‐β responsiveness in cultured cells can be modulated by TGF‐β partitioning between lipid raft/caveolae‐ and clathrin‐mediated endocytosis pathways. The ratio of TGF‐β binding to TβR‐II and TβR‐I on the cell surface has recently been found to be a signal that controls TGF‐β partitioning between these pathways. Since cholesterol is a structural component in lipid rafts/caveolae, we have studied the effects of cholesterol on TGF‐β binding to TGF‐β receptors and TGF‐β responsiveness in cultured cells and in animals. Here we demonstrate that treatment with cholesterol decreases the TβR‐II/TβR‐I binding ratio of TGF‐β while treatment with cholesterol‐lowering or cholesterol‐depleting agents increases the TβR‐II/TβR‐I binding ratio of TGF‐β in all cell types studied. Cholesterol treatment increases accumulation of the TGF‐β receptors in lipid rafts/caveolae microdomains of the plasma membrane. Cholesterol/LDL suppresses TGF‐β responsiveness and statins/β‐CD enhances it, as measured by the levels of P‐Smad2 and PAI‐1 expression. Furthermore, the cholesterol effects observed in cultured cells are also found in the aortic endothelium of atherosclerotic ApoE‐null mice fed a high cholesterol diet. These results indicate that high plasma cholesterol levels contribute to the pathogenesis of atherosclerosis, at least in part, by suppressing TGF‐β responsiveness.