bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sirtuin, an oxidant sensitive deacetylase, is post‐translationally modified and degraded by the proteasome in response to cigarette smoke in lung epithelial cells
Authors: Caito Samuel William , Cook Suzanne , Yang Se‐Ran , Rajendrasozhan Saravanan , Rahman Irfan 
Published: June 2021

Sirtuin1 (Sirt1) is a class III histone/protein deacetylase that has anti‐aging and anti‐inflammatory properties. We have previously shown that cigarette smoke (CS) decreased Sirt1 protein level in rat lung and human monocyte/macrophages leading to increased NF‐κB dependent pro‐inflammatory cytokine release. As CS contains greater than 1015–17 free radicals, we hypothesized that the cell's redox status influences Sirt1 protein level in response to CS, leading to post‐translational modifications and degradation. Human bronchial epithelial cells (BEAS.2B) were exposed for 24 hrs to different concentrations of cigarette smoke extract (CSE 0.5–1%) in the presence or absence of buthionine sulfoximine (BSO) or N‐acetyl‐L‐cysteine (NAC), an inhibitor and precursor for glutathione biosynthesis respectively. CSE decreased nuclear Sirt1 levels; co‐treatment with BSO further decreased Sirt1, whereas treatment with NAC rescued CSE depletion. Furthermore, CSE caused 4‐hydroxy‐2‐nonenol adduct formation on Sirt1, which was increased by BSO treatment and decreased by NAC. Decreased Sirt1 level was also found to be due to proteasomal degradation as proteasomal inhibitors (ALLN and MG132) attenuated CSE‐mediated degradation of Sirt1. These data suggest that oxidants in CSE modify Sirt1 post‐translationally, marking the protein for proteasomal degradation in lung epithelial cells, which may contribute to the pro‐inflammatory effect of cigarette smoke.Supported by the NIEHS Center Grant ES01247 and Toxicology Training Program Grant ES07026.
Effect of Sodium Nitroprusside on pHi Recovery in Porcine Non‐pigmented Ciliary Epithelium
Authors: Shahidullah Mohammad , Delamere Nicholas A. 
Published: June 2021

ObjectiveAtriopeptin, acting through cGMP, inhibits NHE1 in the bovine ocular non‐pigmented ciliary epithelium (NPE)1. Here we examined NHE isoforms and tested whether SNP, a NO donor and a stimulator of cGMP, alters intracellular pH (pHi) responses.MethodsNHE isoforms in native and cultured NPE were detected by immunohistochemistry and immunoblot. BCECF fluorescence imaging was used to study the effect of drugs on pHi recovery after an acid load.ResultsNative and cultured NPE expressed NHE1 and NHE4 isoforms. pHi recovery in NPE was Na‐dependent and had DIDS and amiloride sensitive components. SNP inhibited pHi recovery concentration‐dependently. L‐arginine, the NOS substrate, also slowed pHi recovery. Dimethyl amiloride (DMA), a potent inhibitor of NHE, caused substantial inhibition of pHi recovery at 100μM. In cells exposed to both SNP and DMA, the degree of inhibition of pHi recovery was similar to that elicited by SNP alone. The cyclic GMP analog 8‐pCPT‐cGMP (10μM) mimicked the SNP effect. The inhibitory effect of SNP on pHi recovery was suppressed by 1μM H‐8, an inhibitor of cGMP‐dependent protein kinase.ConclusionPrimary cultures of porcine NPE express NHE isoforms found in native cells. NO donors slow pHi recovery through inhibition of NHE. The effect of NO donors on NHE involves a cGMP‐PKG signal transduction pathway.Funding: NIH‐EY06915
IL‐1 mediates persistent but not acute ozone‐induced airway hyperreactivity
Authors: Blensly Kirsten Colleen , Jacoby David B , Fryer Allison D 
Published: June 2021

Ozone (O3) exposure causes airway hyperreactivity, lasting at least 3 days, and increases interleukin‐1 (IL‐1) in human and animal lungs. Eosinophil depletion prevents O3 induced airway hyperreactivity 1 day post O3 but worsens airway hyperreactivity 3 days post O3. Thus, the mechanism of hyperreactivity changes between 1 and 3 days following O3 exposure in guinea pigs. O3 increases IL‐1a in bone marrow. To test whether IL‐1β mediates airway hyperreactivity 1 and 3 days post O3 guinea pigs were treated with an IL‐1 receptor antagonist (Kineret, 30mg/kg ip) 30 minutes before exposure to filtered air or O3 (2ppm, 4 hours). One or 3 days later electrical stimulation of both vagus nerves, in anesthetized animals, caused bronchoconstriction that was significantly potentiated in O3 exposed animals compared to air exposed controls. The IL‐1 antagonist prevented airway hyperreactivity 3 days but not 1 day post O3. Acetylcholine (1‐10ug/kg, iv) induced bronchoconstriction was not changed 1 or 3 days post O3 but was increased in O3 exposed animals treated with the IL‐1 antagonist. In bronchoalveolar lavage the IL‐1 antagonist did not change inflammatory cell numbers in either O3 or air exposed animals at either time point. These data confirm the mechanism of airway hyperreactivity changes over three days following ozone to become IL‐1 dependent.Funding: Steinberg Fellowship; NIH: HL‐55543, HL54659, HL071795, ES‐014601
Ca2+/Calmodulin‐dependent protein kinase regulation of sarcoplasmic reticulum Ca2+ uptake in airway smooth muscle
Authors: Sathish Venkatachalem , Leblebici Figen , Kip Sertac N , Pabelick Christina M , Prakash Y S , Sieck Gary C 
Published: June 2021

In airway smooth muscle (ASM), the state of sarcoplasmic reticulum (SR) Ca2+ stores is an important determinant of [Ca2+]i response to agonist. We have previously shown that acetylcholine (ACh) induces sustained [Ca2+]i oscillations representing repetitive SR Ca2+ release and reuptake. Thus, SR refilling is a key component in refilling of SR stores. We hypothesized that, like cardiac and vascular smooth muscle, ASM SR refilling is regulated by calmodulin (CaM)‐CaM kinase (CaMKII) dependent phosphorylation of phospholamban (PLB). In ASM cells from fresh porcine trachea, effects of W7 (CaM antagonist) and KN‐93/KN‐62 (CaMKII antagonists) on [Ca2+]i responses to ACh were measured using fluorescence microscopy. Separately, SR‐enriched membranes were isolated to monitor ATP‐energized Ca2+ uptake and in vitro phosphorylation of PLB in the presence of CaMKII inhibitors. KN‐93 significantly slowed decay of [Ca2+]i responses, and slowed or inhibited ongoing [Ca2+]i oscillations, with visible slowing of fall‐times of individual oscillations. Pre‐exposure to W7 or KN‐62 did not prevent initiation of oscillations. In SR‐enriched membranes, addition of KN‐93/KN‐62 resulted in significantly slower Ca2+ uptake, accompanied by a matching decrement in the in vitro phosphorylation of Thr 17‐PLB, indicating the influence of CaM‐CaMKII on this protein. These data suggest that agonist‐induced [Ca2+]i regulation in ASM involves the CaM‐CaMKII pathway which indirectly influences Ca2+ release and reuptake, likely through PLB.Supported by NIH grant HL74309 (GCS)
Interactive clicker technology in teaching nutrition and metabolism in the medical curriculum
Authors: Feinman Richard D. , Accurso Anthony , Makowske Mary 
Published: June 2021

Audience response technology via wireless handheld devices (clickers) allows polling of students and provides immediate feedback. We have used this methodology in a first year medical school course to assess students’ preconceptions about nutrition and to address a unique problem: the inconsistency between biochemical facts and commonly accepted recommendations of health agencies. Representative questions from the first class meeting that provided information on student background, led to critical discussion and were used for focus of the course:
During the epidemic of obesity and diabetes, the macronutrient that increased most: (CHO, fat, protein):
In general, the effect of a low‐fat diet on plasma triglycerides? (increase, decrease, no effect)
The dietary component most likely to raise plasma triglycerides: (CHO, fat, protein).
The major stimulus for insulin secretion: (glucose, fatty acids, amino acids)
The dietary requirement for carbohydrate: ( ~130 g/d, ~ 50 % of calories, none)
CHO intake recommended by USDA, American Diabetes Association, others: ( ~130 g/d, ~ 50 % of calories, none).
A major effect of insulin: (glucose uptake, inhibition of lipolysis, fatty acid uptake, all)
Repression of EGR target genes by NAB2 and the NuRD complex
Authors: Srinivasan Rajini , Ward Rebecca M. , Svaren John 
Published: June 2021

The Early Growth Response (EGR) family of transactivators are critical regulators of immune system regulation, peripheral nerve myelination and prostate carcinogenesis. The NAB1 and NAB2 (NGFI‐A/EGR1‐binding) transcriptional corepressors directly interact with three EGR family members (Egr1, Egr2/Krox20, and Egr3) and repress activation of their target promoters. In particular, NAB proteins appear to play an important role in regulating growth factor and cell cycle control genes, although the underlying molecular mechanisms have been elusive. We previously uncovered two independent repression domains in NAB2, one of which uniquely associates with the CHD4 (Chromodomain Helicase DNA‐binding protein 4) subunit of the repressive NuRD (Nucleosome Remodeling and Deacetylase) chromatin remodeling complex. To probe the role of the NuRD complex in physiological regulation of endogenous EGR target genes, we identified several genes that are regulated by NAB2 and the NuRD complex using a combination of gene expression and chromatin immunoprecipitation assays. Moreover, repression by NAB2 of at least some target genes requires the histone deacetylase subunits of the NuRD complex. Overall, the results indicate that NAB‐mediated recruitment of the NuRD complex is required for repression of EGR target genes that promote proliferation.This work is supported by a grant (HD41590) from NIH.
Assay for monitoring in real time RNA reverse transcription and measurement enzyme inhibition.
Authors: Sun Ye , Golovlev Val 
Published: June 2021

The objective of this study is to demonstrate a novel approach to the detection of RNA transcription in real time. This new technique offers alternative to real time PCR for the quantitative measurement of RNA without transcription to cDNA. The elimination of the transcription to cDNA step became possible by using the Reverse Transcription Quantitative Bioluminescent Assay (RT‐qBLA). We previously reported RT‐qBLA as a new, sensitive method of detection and quantification of RNA in biological samples (1,2). In current research, the use of RT‐qBLA was expanded to the monitoring of reverse transcription reaction (RT) kinetics and the measurement of RT inhibition and reaction constants. The use of RT‐qBLA for measurement of the efficiency of the inhibition of HIV‐1 RT by nucleoside and non‐nucleoside inhibitors (NRTI and NNRTI) was demonstrated for the first time. RT‐qBLA provides a fast, accurate, and cost‐efficient alternative to the established methods in various applications in research and antiviral drug discovery.This project is supported by NIH grant R43 CA126647.
Antrodia camphorate extract preferentially degrades eukaryotic translation initiation factor 5A
Authors: Huang Jenq‐Kuen , Wang Yinglu , Murtaugh Megan L , Soustek Meghan S , Lin Chi‐Tsai , Wen Lisa 
Published: June 2021

Antrodia camphorate (A. camphorate) is a medicinal mushroom found only in Taiwan. The fruiting bodies of A. camphorate have been used for a long time as a remedy for various diseases in folk medicine, including cancer treatment. The objective of this research was to search for inhibitors in A. camphorate extract that can reduce the activity of eukaryotic initiation factor 5A (eIF5A) modification which is essential for its function in cell proliferation and survival.The maturation/hypusination of eIF5A is a two‐step posttranslational modification catalyzed by deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). The eIF5A modification has been implicated to be a suitable target for anticancer therapy. We wish to find ingredient(s) in A. camphorate extract that can inhibit eIF5A modification. We report that the presence of aqueous extract of A. camphorate capsules reduced the bovine DHS and DOHH activities. The inhibition toward DOHH was in a dose‐dependent manner. Further analysis suggested that the reduced activities were due to preferential degradation of eIF5A protein over seven other proteins tested, presumable by a novel protease present in the extract.Supported by University Research Council, Western Illinois University.
Identification of novel β‐secretase inhibitors through the inclusion of protein flexibility in virtual screening calculations
Authors: Cosconati Sandro , Huey Ruth , Marinelli Luciana , Novellino Ettore , Goodsell David S. , Olson Arthur J. 
Published: June 2021

Alzheimer's disease is a neurodegenerative disorder featuring both the aggregation of extraneuronal amyloid plaques and intraneuronal neurofibrillary tangles. The deposition of Aβ peptides in the brain plays a key role in the onset and progression of this disease, and β‐secretase (BACE‐1) is critical in this process. Thus, the inhibition of this enzyme is an attractive approach to anti‐Alzheimer therapy. So far, the rational design of new BACE‐1 inhibitors has been hampered by the intrinsic flexibility displayed by the enzyme which is not included in standard molecular modeling methods.Here, we incorporated protein flexibility into Virtual Screening (VS) in order to discover new BACE‐1 inhibitors in two ways. First, we used an ensemble of static Xray BACE‐1 structures representing the receptor flexibility in a VS of the NCI database. Also, the different conformations of BACE‐1 were combined to generate a unified description of the protein motion. To do this, a set of maps was computed for each protein conformation, combined in a single set using an energy weighting scheme and finally used in a second VS of the NCI database.The enrichment factors from these two VS approaches were used to assess their respective predictive power. The in vitro evaluation of the best ranking ligands finally led to the discovery of new leads which will be subjected to further structural optimization.
Kinetic and Structural Analysis of Tight Complexes of TEM‐1 Beta‐Lactamase and BLIP mutants
Authors: Mai Tu Thanh , Nguyen Toan Duc , Wang Jihong , Pham Ngoc , Vo De , Zhang Zhen , Palzkill Timothy , Chow Dar‐Chone 
Published: June 2021

The S. clavuligerus ß‐Lactamase Inhibitor Protein (BLIP) is an effective inhibitor of class A ß‐lactamases, including the TEM‐1 β‐Lactamase (TEM1). A previous report from our groups shows that Y143A mutant of BLIP binds to TEM1 with similar affinity as the wild type, but with a larger entropy contribution to binding. The Y50A mutant binds to TEM1 50 fold stronger than wild type, which is the result of increased enthalpy driving forces (Wang J., et al. JBC. 2007; 282:17676–84). In addition, binding of BLIP Y50A with TEM1 exhibits a less negative ΔCp than that of the wild type complex. These findings suggest that binding between BLIP Y50A and TEM1 is enhanced by hydrophilic interactions. Interestingly, our stopped‐flow kinetic measurements show that the association rate of BLIP Y50A with TEM1 is similar to that of the wild type, demonstrating the increased affinity is mainly due to the slower dissociation. To understand the changes in binding thermodynamics and kinetics, we initiated structural studies of these complexes. After extensive crystallization screens, some high quality crystals were obtained for the BLIP Y50A/TEM1 complex and the BLIP Y143A/TEM1 complex crystallized in micro‐needle form. We are currently optimizing conditions to obtain high‐quality crystals. Future X‐ray diffraction analysis will determine if the crystals are suitable for structure determination.Support from AHA & UH to D.C. and NIH to T.P.
Relationship of the intravascular and pericellular (interstitial) oxygen pressures in normal and tumor tissue
Authors: Wilson David F , Vinogradov Sergei A , Apreleva Sofia , Lee William M F 
Published: June 2021

Objective: To determine the influence of vessel walls and diffusion distance in delivery of oxygen to cells in normal and K1735 murine melanoma tissue of mice.Methods: Oxygen pressures were measured by oxygen dependent quenching of phosphorescence, using oxygen probes contained in each compartment. Oxyphor G2 was added to the blood plasma and Oxyphor G3, Pd‐tetrabenzoporphyrin encapsulated with a gen. 2 poly‐arylglycine dendrimer and then coated with oligoethylene glycol residues (Av. MW 350), was microinjected into the interstitial (pericellular) space. The oxygen distributions in the two spaces were compared resting skeletal muscle and K1735 tumors in both awake and anesthetized mice.Results and conclusions: For skeletal muscle of awake mice, the lowest 10% of oxygen pressures for the interstitial and intravascular spaces (believed to represent capillary bed) were not different (23.8 ± 4.5 and 25 ± 4.3 mm Hg, respectively) whereas in isoflurane anesthetized mice there was a small but significant (p =0.01) difference (20.4 ± 6.3 and 27.9 ± 3.5 mm Hg, respectively). In K1735 tumors, compared to skeletal muscle, the oxygen pressures are more heterogeneous and averages lower. In addition there are larger differences between the two compartments, consistent with lower vascular density in the tumors. Supported by NS‐31465, HL081273, and U54 CA105008
Proteomic analysis of serum from visceral leishmaniasis (kala azar) patients
Authors: Rukmangadachar Lokesh Ankenahally , Kataria Jitender , Manral Pallavi , Das Utpal , Samantaray Jyotish C , Kaur Punit , Singh Tej Pal , Srinivasan Alagiri 
Published: June 2021

Background: Visceral leishmaniasis is vector borne disease caused by the protozoa, Leishmania donovani. Approximately 500,000 cases are reported annually. The diagnosis is conventionally made by the demonstration of the parasite in the aspirated fluid from the bone marrow or the spleen. We used proteomics technologies to discover protein biomarkers for the disease.Methods: We compared the serum proteome of visceral leishmaniasis patients with normal healthy controls using proteomics techniques. Immunodepleted serum samples were separated using 2D DIGE. Biological variations of the protein expression level were analyzed with DeCyder software and evaluated for reproducibility and statistical significance. Differentially expressed proteins were identified by tandem mass spectrometry.Results: The profile of differentially expressed protein spots between patients and controls revealed α1 anti‐chymotrypsin, Leucine rich glycoprotein, Complement C3, Complement C3α as up regulated and Haptoglobin and Transthyretin as down regulated in the disease.Conclusions: Identification of differentially expressed proteins throws light on the host parasite interaction and these proteins can be potential biomarkers for noninvasive diagnosis and monitoring response to therapy.The work has been supported from a research grant from Department of Biotechnology, Govt. of India to AS
Purification of a Precursor to a Potential Phospholipid Analog of Phosphatidylserine
Authors: Amato Nicholas Jay 
Published: June 2021

The purpose of this project is to develop a method to purify the protected cyclic phosphatidylserine (PcycPS), an intermediate in the multi‐step synthesis of a proposed phospholipid analog (PLA). The PLA is important because it will allow us to study the structure and function of the naturally occurring phospholipid, phosphatidylserine (PS). The PLA will also allow us to study the structural interactions characteristic of the lipid bilayer and proteins in the presence of calcium by mimicking the structure and function of PS.Chromatographic purification methods investigated have used silica gel, reverse‐phase, and cation exchange solid phases. Following the purification, the product was characterized by using 1H and 31P nuclear magnetic resonance (NMR) and thin layer chromatography (TLC) utilizing molybdenum and ninhydrin reagents for visualization. PcycPS was purified to greater than 85% purity. The final step of the synthesis uses hydrogenolysis to yield the final deprotected compound. This research is supported by The College of Wooster.
Evidence for B cell antigen receptor (BCR)‐driven, BAFF‐facilitated positive selection within a unique population of late transitional B cells
Authors: Meyer‐Bahlburg Almut , Andrews Sarah F , Yu Karl OA , Porcelli Steven , Rawlings David J 
Published: June 2021

In addition to negative selection, several lines of evidence suggest that positive selection may also play an important role in shaping the naïve mature B cell repertoire.We have characterized a late transitional (T2) B cell subset with a distinct phenotypic, functional, and gene expression profile. Adoptive transfer experiments identify this subset as an immediate precursor of both follicular mature (FM) and marginal zone (MZ) B cells. Intriguingly, in contrast to the largely quiescent early transitional (T1) and FM B cells, a high percentage of T2 B cells are actively cycling as demonstrated by cell cycle analysis, measurement of kappa‐deleting recombination excision circles (KRECS) and in vivo BrdU labeling. Proliferation of this subset is BAFF‐dependent, consistent with the highest levels of BAFF‐R expression among all splenic B cells. Finally, M167 BCR idiotypic‐specific B cells are first selected by self‐antigen within this subset leading, ultimately, to preferential enrichment within the MZ compartment.In summary, our data indicate that late transitional B cells represent a bipotent precursor for mature splenic B cells; and can function as a target for BCR‐driven, BAFF‐facilitated homeostatic signaling. These combined findings suggest that this small pool of cells may operate at a previously unknown selection point important for establishment of the mature B repertoire. Funded by NIH 5RO1HD37091
A Universal T Cell Vaccine Against Influenza A
Authors: Tai Wendy Man‐Ting , Gilbert Brian Edward , Corry David Brian 
Published: June 2021

A bivalent influenza virus (INFV) A vaccine composed of purified HA and NA has been available for INFV protection. However, this vaccine requires annual re‐administration. We have developed a novel T cell‐based vaccine that obviates this need. Our trivalent vaccine, is comprised of highly conserved peptides derived from H1N1 M2, HA, and NP peptides formulated with the TLR ligands (TLRLs), MPL, TDM and Poly (I:C) in a liposomal matrix.The tripeptide vaccine (50 μl) was given IN to WT mice on d‐21, ‐14, and ‐7 prior to challenge. Additional groups of WT and Rag1−/− mice received only TLRL once ‐2d prior to challenge. All animals were challenged on d0 with LD90 INFV A H3N2. Survival, weight loss, and symptom scores were monitored. On d0, tissues were collected and assessed for secretion of peptide specific IFN‐γ. In addition, lung cells were assessed for NP tetramer+ CD8 cells and also lung histopathology.M2/TLRL and NP/TLRL immunizations provoked marked inflammation of the lung airways. Immunization with the tripeptide vaccine resulted in 100% survival and minimal weight loss and elevation of symptom scores relative to naïve controls (0% survival). M2 and NP specific IFN‐γ responses were observed predominantly in the lungs. Staining of lung cells with NP tetramer demonstrated ≈5% positive T cells. IN vaccination with TLRL ‐2d prior to challenge also protected WT and Rag1−/− mice against lethal challenge.