bluuebunny/crossref_metadata_2025 huggingface dataset:
High glucose inhibits p53 function via Thr55 phosphorylation
Authors: Wu Yong , Lin Joy , Piluso Landon G. , Liu Xuan
Published: June 2021

Emerging evidence has begun to indicate that elevated glucose levels increase the risk for developing cancer. However, molecular mechanisms for this risk are largely undefined. Here we show that exposure of U2OS cells to high glucose (HG) but not its analog 2‐deoxyglucose increased cellular ATP levels and inhibited p21 expression. Further, HG suppressed p21 expression in HCT116p53+/+ but not HCT116p53−/− cells, implying that the inhibitory effects of HG might be dependent of p53. To define the molecular mechanisms, we show that HG stimulated TAF1 kinase activity and resultant p53 Thr55 phosphorylation, leading to p53 degradation and decreased p21 expression. Inhibition of TAF1 activity with apigenin abolished HG‐induced Thr55 phosphorylation, p53 protein degradation and inhibition of p21 expression suggesting that Thr55 plays a role in HG‐induced p21 inhibition. In addition, HG suppressed p21 expression in H1299 cells transfected with wild type p53 (H1299‐p53) but not with T55A (H1299‐T55A). Moreover, HG‐induced inhibition of p21 was blocked by apigenin in H1299‐p53 but not H1299‐T55A indicating that TAF1‐mediated Thr55 phosphorylation was involved in HG‐induced p21 inhibition. We conclude that HG inhibits p53 function, at least in part, via ATP overproduction/TAF1‐mediated Thr55 phosphorylation. This observation may provide a mechanistic explanation for the association between diabetes and cancer.
Modification of the Adiponectin:Leptin Ratio May Underlie Obesity Dependent Prostate Cancer Progression
Authors: Zeppieri Jordan A. , Connor Michael K.
Published: June 2021

Obesity and prostate cancer exhibit a statistical link, which may be due to the paracrine/endocrine cell cycle effects of the adipocyte‐derived peptides leptin (LEP) and adiponectin (ADIPO). ADIPO induced AMPK–dependent phosphorylation of p27 on T198 (T198‐P) and increased total p27 protein and adiponectin receptor (ADIPOR1) protein levels in androgen‐receptor (AR) negative PC3 prostate cancer cells. Conversely, LEP induced the opposite effects on AMPK, T198‐P p27, total p27 and ADIPOR1 protein levels. LEP and ADIPO also had opposing effects in AR‐positive LNCaP cells. As observed in breast cancer cells, LEP treatment overcame the effects of ADIPO on all proteins examined in a concentration‐dependent manner. Furthermore, ADIPO administration was able to counteract the effects of LEP on all proteins measured. Our data suggest that ADIPO and LEP exert antagonistic effects on prostate epithelial cell cycle regulation, and modification of the ADIPO:LEP ratio may be one of the important factors that underlie the molecular link between obesity and prostate cancer progression.
Metabolic Profiling A Drug Subsisting Gram‐Negative Bacteria
Authors: Armstrong Kyle William , Faria Kathryn A , Rodriquez Jaime L , Benner Richard , Allen Mary E. , Piefer Andrew John
Published: June 2021

Drug resistant bacteria are a serious and growing concern facing medicine in the 21st century. Indeed, antibiotic usage provides a real and dangerous example of natural selection with pathogenic bacteria quickly adapting to survive drug treatment. Until recently, hospitals were thought to be the reservoirs of these resistance genes and bacteria were considered resistant if they could avoid death or damage to the cells. However, in 2008 bacteria were isolated from soils that could “eat” antibiotics, presumably using them to support growth. Taking this one step farther, we have isolated from the upper Susquehanna River bacteria capable of growth on penicillin G as a sole carbon source. The objective of our study is to screen the metabolism of one of these isolates by GC‐MS techniques under two different growth conditions: drug as a single carbon source or high nutrient broth. By screening the metabolism, a metabolic fingerprint for each growth condition will be created. This fingerprint can then be used to elicit information about the metabolic mechanism by which the bacterium is subsisting on drug. In addition, since the bacterium found to subsist on antibiotics was isolated from the local watershed, the mechanism could provide information in regard to the metabolic fate of antibiotics in the environment. This project was funded by Hartwick College and the Otsego County Conservation Association.
Partial beta‐oxidation and bilateral alpha‐oxidation of gamma‐hydroxybutyrate (GHB) in perfused rat livers
Authors: ZHANG Guo‐Fang , Sadhukhan Sushabhan , HAN Yong , Tomcik Kristyen , Murphy Anne , Barnoff Stephanie , Tochtrop Gregory , Brunengraber Henri
Published: June 2021

GHB is neurotransmitter (GABA) and a drug of abuse. It is oxidized to succinate an intermediate of the citric acid cycle. We previously showed that GHB forms GHB‐CoA and 4‐phospho‐GHB‐CoA, an apparently dead‐end product (JBC, 2009). To investigate other fates of GHB, we perfused livers with unlabeled, [1,2‐13C2], [3,4‐13C2] or [13C4]GHB. No labeled acetyl‐CoA, acetoacetate or 3‐hydroxybutyrate were formed from the variously labeled GHB. So, there is no complete beta‐oxidation of GHB. We identified 4‐hydroxycrotonate which reflects an acyl‐CoA dehydrogenase reaction. Also, we found 3,4‐dihydroxybutyrate and 2,4‐dihydroxybutyrate which derive from two modes of hydration of the presumed 4‐hydroxycrotonyl‐CoA. The formation of 2,4‐dihydroxybutyrate was unexpected. Also, in livers perfused with [13C4]GHB, M3 4‐hydroxypropionate and M1 formate were formed. This suggests an alpha‐oxidation process. However, both [1,2‐13C2] and [3,4‐13C2]GHB formed small amounts of M1 formate, as well as M1 and M2 3‐hydroxypropionate. Thus, there must be two alpha‐oxidation processes in GHB oxidation (i) from 2,4‐dihydroxybutyryl‐CoA, and (ii) 3,4‐dihydroxybutyryl‐CoA presumably via 3‐hydroxybutanedioyl‐CoA (malyl‐CoA). Supported by NIDDK RoadMap and by NIEHS.
In search for an appropriate model to study the role of MYO5B in apical membrane protein trafficking
Authors: Mackovicova Katarina , van der Goot Annemieke T , Sjollema Klaas A , Nollen Ellen AA , van Ijzendoorn Sven CD
Published: June 2021

Recently, the mutations in MYO5B gene (encoding for actin‐binding motor protein involved in endosomal recycling to apical membrane) were associated with disturbed intracellular protein trafficking to apical membrane. We aimed to establish a model to study the involvement of MYO5B in protein trafficking. Expression of MYO5B, polarization, intracellular trafficking and microvillous ultrastructure were analyzed in 3D cultures of intestinal T84 and Caco‐2 cell lines and in the intestines of hum‐2 (orthologue of human MYO5B)‐deficient C.elegans by fluorescent and electron microscopy. In contrast to Caco‐2, T84 did not express MYO5B, but the ability of both cell lines to polarize, regulate intracellular trafficking and form microvillous brush border was intact. Similarly, hum‐2‐deficient C.elegans showed no intestinal ultrastructural changes or protein mislocalization. MYO5B deficiency alone did not provide the phenotype mimicking disturbed apical membrane protein trafficking in T84 cell line nor in C.elegans. Mechanisms additional to MYO5B malfunction might play role in altered protein trafficking to apical membrane.
ERK and p38 MAPK regulate chaperone‐mediated peroxiredoxin 6 targeting to lysosomal organelles in lung epithelial cells
Authors: Sorokina Elena M. , Feinstein Sheldon I. , Fisher Aron B.
Published: June 2021

Peroxiredoxin 6 (Prdx6), a protein with both GSH peroxidase and phospholipase A2 (aiPLA2) activities, participates in lung surfactant phospholipid turnover and protects lung epithelium against oxidative stress. Prdx6 has been localized to cytosolic and acidic compartments (lamellar bodies and lysosomes) in lung alveolar epithelium. We propose that Prdx6 subcellular localization determines the balance between the 2 activities. We show that ERK and p38 MAPK regulate Prdx6 targeting to lysosomal organelles in MLE12 cells. Treatment of MLE12 cells with inhibitors of ERK or p38 MAPK, or an inhibitor of PKC, an upstream kinase activator of MAPKs, abolished Prdx6 lysosomal targeting. Moreover, inhibition of PKC, ERK or p38 resulted in Prdx6 accumulation in the endoplasmic reticulum (ER), as detected by co‐localization of Prdx6 with the ER marker calnexin. Inhibitors of PKC, ERK or p38 abolished protein‐protein interaction between Prdx6 and 14‐3‐3ε, a member of the 14‐3‐3 chaperone system which is known to facilitate transport of signaling proteins in the early secretory pathway. These findings suggest that ERK and p38 MAPK regulate Prdx6 targeting to lysosomal organelles, possibly by activation of a chaperone protein, i.e. 14‐3‐3ε, that facilitates Prdx6 translocation along the exocytotic pathway, resulting in its sub‐cellular localization to acidic organelles and consequent activation as a PLA2. [HL019737]
The Endoplasmic Reticulum Protein Orm2 is Required for Cold Adaptation in Saccharomyces cerevisiae
Authors: Zhong Jun Wen , Loertscher Jennifer
Published: June 2021

Despite the fact that wild yeast are known to grow under a wide range of environmental conditions, relatively little is known about mechanisms of cold adaptation. We have found that an endoplasmic reticulum‐resident protein Orm2 is required for cold adaptation in yeast as evidenced by poor growth of the deletion mutant orm2Δ at 10 degrees Celsius. In contrast, the homologous protein Orm1 is not required for cold adaptation. To elucidate the role of Orm2 in cold adaptation, we compared the sensitivity of orm2Δ and wild‐type cells to the anti‐fungal drug amphotericin B and found that orm2Δ cells are more sensitive to the drug at 10 degrees, but not at 30 degrees. orm2Δ mutants also displayed differences in relative amounts of the membrane components ergosterol and squalene as compared to wild‐type at 10 degrees, but not at 30 degrees. Taken together, these results suggest a role for Orm2 as a temperature‐sensitive regulator of the sterol biosynthetic pathway.
Lipid peroxidation induced by H2O2 alters the sensitivity of complex III to oxidative stress by increasing iron labile pool
Authors: Cortés‐Rojo Christian , Estrada‐Villagómez M. Mirella , Calderón‐Cortés Elizabeth , Montoya‐Pérez Rocío , Saavedra‐Molina Alfredo
Published: June 2021

The electron transport chain (ETC) is a target for H2O2 due to the presence of thiol groups (−SH) that undergoes reversible oxidation. −SH serves as a redox sensor that modulates complex activity and avoids further ROS production. Our hypothesis is that the increment on unsaturation degree of mitochondrial membranes disrupts −SH‐mediated ETC reversible inhibition by H2O2 due to enhanced membrane damage. To test this hypothesis, we studied the effects of H2O2 on lipoperoxidation levels, ETC activity, ROS production and free Fe concentration on native yeast mitochondria (−C18:3, lipoperoxidation resistant) and yeast mitochondria enriched with linolenic acid (+C18:3, lipoperoxidation sensitive). Sensitivity of complex III to H2O2 and lipoperoxidation was increased in +C18:3 mitochondria, while complex III inhibition by H2O2 was poorly reverted by a −SH reductanct. Furthermore, increases in ROS production and free Fe concentration were detected only in +C18;3 mitochondria. These results suggest that lipoperoxidation promote Fe release from mitochondrial pools enhancing ROS production and sensitivity of complex III to oxidative damage due to ROS attack in other enzyme components different than −SH groups.
ChChd3, an Inner Mitochondrial Membrane Protein is Essential for Maintaining Cristae Integrity and Mitochondrial Function
Authors: Darshi Manjula , Perkins Guy A , Mackey Mason R , Petrosyan Susanna , Murphy Anne N , Ellisman Mark H , Taylor Susan S
Published: June 2021

Coiled‐coil‐Helix‐Coiled‐coil‐Helix Domain containing protein 3 (ChChd3), was identified in our laboratory as a substrate for Protein Kinase A in mitochondria. Although, there is substantial evidence suggesting ChChd3 is a mitochondrial protein, the biological role of this protein is completely unknown. Here, we show that ChChd3 is an essential protein for maintaining cristae integrity and mitochondrial function. ChChd3 is anchored to the IM of mitochondria through its chch domain by associating with the IM protein Mitofilin, which is known to be involved in regulating cristae morphology. Additionally, N‐terminal myristylation motif of ChChd3 recruits ChChd3 to the outer membrane (OM) of mitochondria, where it associates with SAM50, which is known to be involved in the OM beta barrel protein import. Down regulation of ChChd3 in HeLa cells by an RNA interference method resulted in near complete loss of Mitofilin and Sam50 protein levels, accompanied by altered levels in the proteins associated with mitochondrial dynamics. Further, mitochondria in cells lacking ChChd3 showed peri‐nuclear clustering, fragmentation and loss of cristae followed by drastic decrease in both the cellular respiration and glycolysis, and reduced proliferation. Together our results suggest that, ChChd3 is indispensable for maintaining cristae architecture, and thus regulating mitochondrial function and cellular metabolism.
Influence of lipoperoxidation on the generation of mitochondrial membrane potential
Authors: Estrada‐Villagómez M. Mirella , Cortés‐Rojo Christian , Calderón‐Cortés Elizabeth , Montoya‐Pérez Rocío , Clemente‐Guerrero Mónica , Manzo‐Avalos Salvador , Saavedra‐Molina Alfredo
Published: June 2021

Mitochondria are the main site of ATP synthesis, which requires the buildup of a membrane potential (ΔΨ). Reactive oxygen species (ROS) generated during electron transport can damage electron transport chain (ETC) components. Unsaturated lipids from mitochondrial membranes are the main targets of ROS attack, leading to ETC dysfunction due to membrane disruption. The hypothesis of this work is that lipoperoxidation resistance may also confer resistance to other mitochondrial parameters (i.e. ΔΨ generation) under oxidative stress conditions. We used the yeast S. cerevisiae as model due to the intrinsic resistance of yeast to lipoperoxidation. Also, the sensitivity of yeast to lipoperoxidation can be enhanced through incorporation of linolenic acid (C18:3) to membranes. Our results shown that in −C18:3 cells, the cytoplasmic and mitochondrial ΔΨ was observed even at concentrations of Fe2+ up to 50 μM, while in +C18:3 cells, lower Fe2+concentrations inhibited ΔΨ buildup. As comparison, ΔΨ from rat liver mitochondria, which are prone to lipoperoxidation, was also inhibited by Fe2+ treatment. These results suggest that membranes resistant to lipoperoxidation preserve its structural integrity even under strong oxidative conditions and allow a better function of mitochondrial ETC than the observed in lipoperoxidation‐prone mitochondria.
Mechanistic studies on UDP‐Galactopyranose mutases from Aspergillus fumigatus and Trypanosoma cruzi
Authors: Oppenheimer Michelle L. , Blumer Allison , Poulin Myles B. , Helm Richard F. , Lowary Todd L. , Sobrado Pablo
Published: June 2021

UDP‐Galactopyranose mutase (UGM) is a flavoprotein that catalyzes the conversion of UDP‐galactopyranose to UDP‐galactofuranose. UGM serves as the sole biosynthetic source of galactofuranose, which is found on the cell surface of many bacteria, fungi, and parasites. UGM is a unique flavoprotein since it requires the flavin to be reduced for activity, yet no net redox change occurs during catalysis. Currently, the exact mechanism of isomerization is unknown. UGM is also of interest because it is a novel drug target for several diseases due to the importance of galactofuranose for the survival of M. tuberculosis and for virulence of the fungus, A. fumigatus, and the human parasites, such as L. major and T. cruzi. While many studies have been done on the prokaryotic UGM, little is known about eukaryotic UGM. We present, for the first time, the recombinant expression, purification, and characterization of eukaryotic UGMs from A. fumigatus and T. cruzi. We also report limited proteolysis, steady‐state kinetics, rapid‐reaction kinetics, and site‐directed mutagenesis data that demonstrate significant differences between prokaryotic and eukaryotic UGMs.
Liver regeneration after partial hepatectomy is regulated by a change in sulfur‐containing substance metabolomics in rats
Authors: Kim Sun J , Jung Young S , Kim Young C
Published: June 2021

We determined the change in liver regeneration after partial hepatectomy (PH) in rats treated with buthioninesulfoximine (BSO) or phorone (PHO). BSO administration inhibited the progression of liver regeneration as determined by relative liver weight increase, cyclin D1 and PCNA expression. In rats treated with PHO, however, liver regeneration after PH was enhanced significantly. BSO depressed the elevation of SAM and increased the concentrations of cysteine and its catabolic products, hypotaurine and taurine, in hepatectomized rats. In contrast, hepatic SAM was increased whereas cysteine, hypotaurine and taurine were decreased by PHO treatment. In hepatectomized liver, concentrations of putrescine and spermidine were elevated, but spermine was decreased. BSO administration further increased hepatic putrescine, but decreased spermidine and spermine levels, which were all blocked or reversed in the PHO‐treated rats. GSH‐depleting activities of BSO and PHO in hepatectomized liver were comparable under the conditions used. The results indicate that the progression of liver regeneration after PH is not determined by a change in hepatic GSH level. Altered polyamine levels via a change in hepatic SAM availability may account for the contrasting effects of the two GSH‐depleting agents on liver regeneration.
Paneth Cell α‐Defensin Polymorphisms in C57Bl/6 Mice and Identification of Vestigial Myeloid α‐Defensin Genes in the Mouse Genome
Authors: Ouellette Andre J , Tanabe Hiroki , Shanahan Michael T.
Published: June 2021

Enteric α‐defensins are antimicrobial peptides that are secreted into the small bowel lumen by crypt Paneth cells. Mice express many α‐defensin isoforms (cryptdins, Crps), and certain α‐defensins in C57BL/6 mice are unique to the strain. Abundant native Crps from C57BL/6 small intestine include Crp3 and Crp5 found in all reference strains and five Crps that are unique to the C57BL/6 strain that have in vitro bactericidal activities that are similar to known Crps. Comparisons of C57BL/6 (NIH) and mixed‐strain (Celera) genome assemblies revealed the genetic basis for the differences. The C57BL/6J genome includes the Defcr16, Defcr20, Defcr21 and Defcr24 genes that are absent from the Celera assembly, and, conversely, the NIH assembly lacks Defcr4 (Crp4) and Defcr7 (Crp7) genes found in Celera strains. Analyses of α‐defensin gene loci in mouse and rat suggested that expansion of α‐defensin gene isoforms in both species occurred in part by tandem duplication of a multigene cassette. Although mice are unusual in lacking myeloid α‐defensins, both mouse assemblies include rat myeloid α‐defensin orthologues that are apparent vestigial myeloid α‐defensin genes due to interruption by stop codons. The genetic background of mice exhibiting α‐defensin dysregulation should be considered in the interpretation of Paneth cell responses to varied stimuli. Supported by NIH grants AI059346 and DK044632.
Proteomic identification of hemolymph proteins involved in early stages of immune response in the insect Manduca sexta
Authors: Ragan Emily J , Kanost Michael R
Published: June 2021

Manduca sexta is a large insect useful for biochemical studies. We are interested in the early stages of M. sexta immune response, when the insect recognizes an invading pathogen and triggers effector responses such as activation of phenoloxidase and subsequent production of quinones and melanin. We have identified M. sexta plasma proteins that bind to the surface of microorganisms or form complexes on such surfaces. M. sexta plasma was incubated with killed bacteria or curdlan, a preparation of β‐1, 3‐glucans. Proteins bound to the bacteria after washing were eluted and separated by SDS‐PAGE. 52 bands were identified by MALDI‐TOF/TOF mass spectrometry. We identified potential players in early stage of immune response, including leureptin (a leucine rich repeat protein), hemocyte aggregation inhibitor protein, phenoloxidase, and lysozyme, which bound to all the bacterial samples tested. Mass spectrometry results were confirmed by immunoblot if possible. All the required proteins in the pathway for phenoloxidase activation bound to curdlan. We are investigating the specific protein interactions required for this immune complex formation. This work increases our understanding of the proteins involved in the early stages of insect immune response. Research supported by NIH R01‐GM41247
Targeted nanoparticle delivery of tumor suppressing UDP‐glucuronosyltransferase (UGT) genes into cancer cells
Authors: Radominska‐Pandya Anna , Karmakar Alokita , Bratton Stacie M. , Yang Xu , Mahmood Mena W. , Gallus‐Zawada Anna E. , Biris Alexandru S.
Published: June 2021

We propose the use of nanostructural materials as carriers for the delivery of proteins, genes, growth factors, or other biological systems into cancer cells. Normal cells express a set of UGTs that control the steady state concentration of ligands for nuclear receptors. However, UGT mRNA and protein levels are downregulated or completely absent in several cancer tissues such as those in the breast, ovary and pancreas. We hypothesize that specific UGTs, which catalyze the detoxification and deactivation of biologically active compounds, can be specifically targeted to cancer cells using nanoparticles both in vitro and in vivo and can become an effective tool in cancer treatment. For these studies, we have used single wall carbon nanotubes (20 μg/ml) attached to a UGT expression plasmid to form the CNT/UGT active bio‐nano systems. Specific in‐vitro delivery of these nanosystems by using growth factors and monoclonal antibodies to various cancer cell lines (pancreatic Panc1) was also proved. The incubation of CNT/UGT particles with Panc1 cells for 24 hours resulted in phenotype changes in the cells and eventually death as measured by Caspase 3 activity. It can be concluded that the CNT/UGT system was extremely bio‐active and induced all the cancer cells to die. Experiments with ovarian and breast cancer cell lines are underway. (NIH‐GM075893 to AR‐P; ASTA 08‐CAT‐03 to ASB).
Beta‐1,3‐D‐glucan nanoparticle mediated delivery of siRNAs to professional phagocytes for inhibiting Leishmania infection
Authors: Rana Tanu , Chaudhuri Evan , Chaudhuri Gautam
Published: June 2021

Nanoparticle‐based delivery systems represent a new paradigm for drug design. Many pathogenic diseases, such as leishmaniasis, can be efficiently treated by localized, macrophage‐targeted therapies. The parasitic protozoan Leishmania invades mammalian macrophages to establish infection. We reported previously that Leishmania manipulates the expression of several non‐coding RNA genes (e.g., Alu RNA, B1 RNA and SRP RNA) in the macrophages to favor the establishment of their infection in the phagolysosomes of these cells (Ueda Y., and Chaudhuri, G. J. Biol. Chem. 275, 19428, 2000; Misra, S., Tripathi, M. K., and Chaudhuri, G. J. Biol. Chem. 280, 29364, 2005). Our recent data suggests that Leishmania exposure to the macrophages leads to the decrease in several ncRNA levels including various miRNAs. Replenishment of these RNAs in macrophages can make them relatively resistant to Leishmania infection. Here we report the use of hollow yeast cell wall particles (YCWPs) caging the above ncRNAs for delivery to macrophages in order to make them resistant to Leishmania infection. Our findings support the idea that YCWPs mediated delivery to macrophages can beused to replenish RNA or knock down specific genes within these cells to exert therapeutic effects against Leishmaniasis. Supported by NIH NIAID grants R01AI042327 and R21AI076757 to GC.
A Sensitive Fluorimetric Assay for Quantifying Maleimide Groups in Biopolymers
Authors: Liao Jinfang , Han Xing , Diwu Zhenjun
Published: June 2021

Maleimide groups rapidly and selectively react with thiols, forming stable thioether bonds. This characteristic has led to their widespread use in the formation of stable proteins to proteins or proteins to other biomolecules conjugates. Sensitive assays of maleimide and thiol groups are required for the efficient conjugation of proteins that are expensive and available only in small amounts. The existing methods of quantifying maleimide groups are insensitive and tedious, requiring a large amount sample size. We have developed a sensitive and continuous fluorimetric method for quantifying maleimide groups in proteins, DNA and other biopolymers. This fluorimetric quantitation of maleimide groups is based on Maleimide Green dye that has enhanced fluorescence upon reacting with a maleimide. The method can detect as little as 10 picomoles of maleimide in a 0.1 mL assay volume. It can be performed in a convenient 96‐well or 384‐well microtiter‐plate format and easily adapted to automation with no separation steps required. Its signal can be easily read by a fluorescence microplate reader with Ex/Em = 490/520 nm.
Divergence in proteins‐ Study of Cytochrome b5 isoforms using core‐swap mutants
Authors: Parthasarathy Sudharsan , Kuczera Krzysztof , Benson David R
Published: June 2021

Two isoforms of mammalian Cytochrome b5 exhibit remarkably different biophysical properties in spite of similar sequence and structure. The Outer mitochondrial membrane cytochrome b5 (OMb5) is presently the most stable isoform when compared to its evolutionary counterpart, the micrososmal cytochrome b5 (Mcb5). In order to understand the biophysical properties of the individual proteins, we have constructed a core swap mutant (hybb5) incorporating features from these two isoforms. The X‐ ray crystal structure, at 1.7 Å, of hybb5 has been solved and shows structural features similar to its parent proteins. The thermal stability, stability towards chaotropic agents, redox potential and heme transfer properties of hybb5 seems to have shifted in the direction of OMb5. In parallel we have performed a 10 ns Molecular dynamics simulation of OMb5, Mcb5 and hybb5. The results concur with the experimental data in that hybb5 behaves with characteristics of the OMb5 sub family. Circular Dichroism experiments of the heme free proteins (apo‐form) indicated the presence of tertiary structure. We are at present trying to further understand the apo form of hybb5 and linking it to functional divergence of OMb5 and Mcb5. The subtle structural changes that contribute to different properties in the two isoforms are being investigated via MD simulations.
Proteomic profiling of unnatural amino acid toxicity through differential expression of wild type vs. telluroproteins in Eschericia coli DL41(DE3)
Authors: Brinkley Sarah Christine , Boles Jeffrey O
Published: June 2021

Three‐dimensional conformation of proteins can be solved with greater ease by utilizing unnatural amino acids (UAA), along with x‐ray crystallography. Because atoms naturally occurring in proteins do not have sufficient mass to interact with x‐ray radiation UAA, telluromethionine (TeMet), offers a solution to the “phasing problem”. Hijacking the molecular machinery of cells makes incorporation of TeMet easy, unfortunately, cytotoxicity of TeMet substantially reduces in vivo uptake. Differential expression of a methionine auxotroph, Eschericia coli DL41, can show differences between cells forced to use TeMet in the translation of proteins and cells grown in an authentic methionine environment. Protein profiling of these systems can reveal a molecular snapshot of E. coli DL41 at any given time. The condition of the cell, whether sick or healthy, young or old, can be viewed, quite literally, through proteomics. Detection with UPLC/qTOF MS enables the identification of changes in normal verses diseased states, allowing for the identification of proteins responsible for cell death. Toxic effects due to TeMet can then be reduced, thereby, providing a new tool for the elucidation of 3D structure.
Polymorphisms in UEA‐1 Lectin Binding to Human Buccal Cells and Salivary Fluid
Authors: Vanderpuye Oluseyi Adewale
Published: June 2021

Human saliva may bind various microorganisms, but little is known about the surface proteins of buccal cells which are major constituents of this fluid. To further characterize buccal cell and cell‐free salivary fluid proteins and their variations among individuals, their reactivity with Ulex europaeus –I lectin (UEA‐1) was studied.

After biotin‐UEA‐1 staining of nitrocellulose transfers of Laemmli SDS gels of 48 salivary fluid samples from different individuals, 42 had staining of the stacking gel. A 150 kDA band was present in 20 samples, a 75‐100 kDA was found in 22 samples and 6 samples had UEA‐1 staining in the region 40–50 kDa. Six out of 9 buccal cell samples had clear UEA‐I binding in the stacking gel of nitrocellulose transfers and differed in staining of 130kDa to 200 kDa proteins.

Five of eight salivary fluid and 3 of four buccal cell samples coated onto microtiter plates bound biotin‐UEA‐1. By fluorescence microscopy, five out of seven buccal cell preparations bound UEA‐1. This study thus found differences in reactivity with UEA‐1 among different individuals. This polymorphism may be involved in differential binding of microorganisms to oral components.
Decoding proline‐rich sequence recognition by epitope‐based proteomics
Authors: Freund Christian , Kofler Michael , Schlundt Andreas , Kosslick Daniela , Sticht Jana , Schuemann Michael , Krause Eberhard
Published: June 2021

Multifunctional protein surfaces pose a major challenge for the understanding of an individual protein's contribution to cellular signalling. Deconvolution of network connectivities for individual epitopes by combining site‐specific inhibition with stable isotope labelling of amino acids in cell culture (SILAC)/mass spectrometry provides a powerful tool of investigation and has been applied to signalling adaptors of the proline‐rich sequence (PRS) recognition family. We show that formation of the pre‐spliceosome and of mRNA surveillance complexes depend on PRS recognition and that PRS hubs within these complexes drive the assembly of dynamic initiator complexes (Kofler et al., Mol. Cell. Proteom 8, 2461–2473; Schlundt et al., Mol. Cell. Proteom. 8, 2474–2486 (2009)). Our approach thereby provides complementary information to knock‐down and knock‐out models of protein function and it also reveals the “moonlighting” potential of a given protein surface within the cellular context. This work was supported by grants FG806, SFB740 and SFB765 of the Deutsche Forschungsgemeinschaft.
Scorpion venom peptide accelerating the proliferation of irradiated M‐NFS‐60 cells
Authors: Dong Weihua , Qiu Yifang , Kong Tianhan
Published: June 2021

Persistent myelosuppression occurring in some patients after radio‐ and chemotherapy is an indication of injury to HSCs. There is no available means to stimulate the full‐lineage blood cell recovery. M‐NFS‐60 cells (purchased from ATCC) can reflect proliferating characteristics of HSCs and were used in this study to evaluate the hematopoietic stimulating effects of SVP(separated from scorpion venom in our Lab). AlamarBlue assay, FCM, immunofluorescence and Western blotting methods were used to evaluate proliferating state, IL‐3 receptor (IL‐3R) protein expression and phosphorylating STAT5 levels in non‐irradiated or irradiated M‐NFS‐60 cells after it treated by SVP. The cell proliferation was accelerated, and the expression levels of IL‐3R and phosphorylating STAT5 protein were raised by SVP. The effects were more obvious in irradiated cells. SVP possessed co‐effects with IL‐3. The S phase percentage of M‐NFS‐60 cells was increased but cell cycle was retarded at G2/M phase in irradiated‐cells by SVP. It is suggested that SVP possesses hematopoietic growth factor‐resembling effects.

Supported by National Natural Science Foundation of China Grant 30670906
Myricetin inhibits Akt survival signaling and induces Bad‐mediated apoptosis in a low dose ultraviolet (UV)‐B‐irradiated HaCaT human immortalized keratinocytes
Authors: Yang Hee Jung , Kim Wanyeon , Youn HyeSook , Yun Young Ju , Seong Ki Moon , Youn BuHyun
Published: June 2021

Deregulation of cell survival pathways and resistance to apoptosis are generally accepted as crucial aspects of tumorigenesis. As in many tumors, increasing occurrence of human skin cancer and other conflicting effects of solar ultraviolet radiation enhance the demand for novel chemoprevention agents. Myricetin, a naturally occurring phytochemical, is potent in anti‐cancer promoting activity and affords to the chemopreventive potential of several healthy‐foods. We demonstrate that myricetin inhibits Akt activity to induce apoptosis in a low dose UVB‐irradiated keratinocytes. Treatment of UVB‐irradiated HaCaT cells with an apoptosis‐inducing concentration of myricetin resulted in a decrease in phosphorylation of Akt leading to inhibition of its kinase activity, followed by a decrease in phosphorylation of Bad (a pro‐apoptotic protein), a direct target of Akt in signaling pathway. Comparable to these results, myricetin promoted mitochondrial translocation of Bad, loss of the mitochondrial membrane potential, and release of the mitochondrial apoptotic proteins. In addition, myricetin‐induced apoptosis in UVB‐irradiated cells was attenuated in the presence of caspase inhibitors. Together, these results indicate that myricetin might take on potent chemopreventive activity by targeting the Akt‐mediated apoptotic signaling axis in UVB‐induced skin carcinogenesis.
Effects of specific phytocompounds on induction of immunogenic tumor cell death and its application as adjuvant of dendritic cell‐based vaccine
Authors: Chen Hui‐ming , Chen Swey‐shen , Yang Ning‐sun
Published: June 2021

An emerging concept of ¡§a key‐lock paradigm¡ between the immunogenic cell death and dendrite cell (DC) is recently proposed. Immunogenic cell death is characterized by immunogenic factors (HMGB1, calreticulin, heat shock proteins), which are helpful for the maturation, antigen uptake and presentation of DC and serve as powerful immunological adjuvants. Whether some specific phytocompounds claimed to have anti‐tumor effects can deliver the immunogenic signals and keep expending the tumor‐recognition factors is a challenging question. To address this possibility, we first determined the expression profiles of immunogenic factors in tumor cells lysate (TCL) obtained from B16 cells treated with test phytocompounds and chemotherapeutic drugs at varying concentrations. Shikonin can induced tumor cell death and specific immunogenic factors expression in TCL in a dose‐dependent manner. The cytotoxic effects of splenocytes on target cancer cells were much higher in TCL‐shikonin‐DC vaccinated mice than in TCL‐DMSO‐DC vaccinated mice. TCL‐shikonin‐DC vaccination can substantially decrease the tumor growth and maintain over 70% of survival rate of tumor‐bearing mice after 35 days of tumor cell injection. In conclusion, shikonin is shown here to sensitize tumor cells to the lytic effects of DC‐activated immune effector cells, which may warrant future evaluation as adjuvant of tumor lysate‐pulsed DC vaccine.
Effect and mechanism of action of pine bark extract on glucose uptake by Caco‐2 cells
Authors: El‐Zein Ola Mohamad , Kreydiyyeh Sawsan Ibrahim
Published: June 2021

This work investigated the effect of pine bark extract (PBE) on glucose uptake by Caco‐2 cells. Cells were incubated in presence of PBE and [14C] 3‐O‐methyl‐D‐glucose and the change in radioactivity of the incubation medium was measured. PBE reduced significantly glucose uptake but did affect neither the activity nor the expression of the Na+/K+ ATPase suggesting that PBE affects the level of apical glucose transporters. To test this hypothesis, glucose uptake was measured in presence of inhibitors of Sodium‐dependent Glucose Transporter 1(SGLT1) and Facilitated‐Diffusion Glucose Transporter 2 (GLUT2), as well as different signaling mediators known to be involved in glucose transport. The results showed that PBE acts by activating p38 Mitogen‐activated protein (MAP) kinase which in turn activates SGLT1 transporters and two different pathways that target GLUT2: an inhibitory one involving Phosphatidylinositol‐3 kinase and a stimulatory one involving MAP/Extracellular signal‐regulated kinase. The effect of PBE was not attributed to the major ingredient, proanthocyanidins, since glucose uptake was increased in its presence. Supported by URB.
Modulatory effects of Emblica officinalis (Amla) on adipocytokines in mice fed high‐fat‐diet (HFD)
Authors: Kipyakwai Gideon Kipkoech , Buesa Lance Martinez , Nerurkar Pratibha V.
Published: June 2021

Overnutrition is correlated with obesity and associated metabolic disorders including type 2 diabetes (T2D) and cardiovascular diseases. Studies indicate that adipocytokine leptin is a key element in regulating food intake and obesity. Plasma levels of leptin are also known to inversely correlate with body fat mass. The objective of our study was to investigate the effects of Ayurvedic medicine Emblica officinalis (amla, EO) on adipocytokine regulation in mice fed 58% kcal high‐fat‐diet (HFD) for 16 weeks. Male C57BL/6J mice were randomized into four groups: 1) control, 2) EO (10% w/w), 3) HFD and 4) HFD + EO. After 16 weeks, EO had no significant effect on daily food intake but improved body weights and plasma levels of leptin and resistin. We are currently investigating the effects of EO on leptin and resistin signaling in adipocytes. Such studies offer mechanistic evaluations for potential use of alternative medicine. [Grants: NCCAM (R21AT003719), USDA‐CREES (2004‐34135‐15182)].
Efflux pump upregulation in DTAC Resistant Salmonella
Authors: Dvorzhinskiy Aleksey , Kautz Megan , Herson Diane S.
Published: June 2021

Salmonella spp. are gram‐negative pathogens estimated to cause 1.4 million cases of food poisoning annually in the United States and are increasingly becoming resistant to disinfection in a variety of environments. In this study, Salmonella enterica 4931 strains were developed which were able to grow in the presence of 500–600 ppm Dodecyltrimethylammonium chloride (DTAC), a quaternary ammonium compound (QAC) and also displayed a cross resistance to penicillin. Later, these strains with reduced susceptibility (SRS) were passaged in the absence of DTAC to mimic environmental conditions after exposure an antimicrobial. The minimum inhibitory concentration (MIC) of DTAC for these strains (DSRS) did not vary appreciably from the SRS level for forty passages. The results from our microarray studies support the findings that reduced susceptibility to QACs is linked to an upregulation in the transcription of acrB, an efflux pump. This study also showed that SRS and DSRS strains in the presence of the efflux pump inhibitor showed an even greater sensitivity to DTAC than the parental strain. The discovery of similar efflux pump inhibitors could lead to advances in combating SRS strains in clinical and natural environments.
Creativity and Innovation in Teaching and Learning: The Significant Need for Educational Reform
Authors: Kader Shereen Abdel
Published: June 2021

Creativity has been thought to be associated with the arts in numerous contexts for many years. Applying and appreciating creativity in sciences, engineering, math, and other subjects is still limited till present because of many misconceptions about the creative thinking, process, product, evaluation, and value. This researcher believes that creativity is very valuable as a significant life skill that everyone at the global, societal, and individual levels need to acquire in order to effectively meet the challenge in the next century. This researcher created a new theory that introduces a new profile to understand creativity in children and adults. “Education for Creativity rather than Education for achievement” is a new educational reform that this researcher initiated. Implementing creativity in all life aspects including our daily life activities in addition to all areas of study and research are other issues that are needed to be highly considered in order to produce an innovative society. Recognizing and rewarding creative behavioral expressions in students’ creative process rather than teaching for the product is one of the most critical factors that leads to innovative research and impressive learning outcomes. Examining students’ learning style in connection to their creativity profile in the subject area is another critical factor to help the students to reach their maximum potentials. It has been proven according to this new theory that most students who are highly interested in conducting research in science, math, technology, and engineering belong to the right brain visual spatial non verbal learning style which has its own thinking and personality profile in addition to its own challenges.
Real Time PCR Quantification of p21 Upregulation in UV‐Irradiated HaCat Cells
Authors: Middleton Jesse Lee , Meads Jessica , Waikel Rebekah
Published: June 2021

The p21 protein, which is upregulated in response to UV‐light, halts cell cycle and can promote apoptosis. Levels of p21 mRNA can be quantified by Real‐Time PCR. We have designed a protocol using Real‐Time PCR to detect the upregulation of p21 transcript in HaCat cells after UV‐irradiation to be used in an undergraduate laboratory course. Cultured HaCat cells were exposed to 15mJ/cm2 UV‐B light, total RNA was extracted and reverse transcribed, and Real‐Time PCR with SYBR Green was performed. Following this level of exposure, levels of p21 were significantly higher in irradiated cells than non‐irradiated cells. This protocol can easily be adapted to be used in any Molecular Biology or Biochemistry Laboratory Course.
A Summer at the Bench instead of the Beach; Integration of High School Teachers and Students in the Research Laboratory
Authors: Wallert Mark A , Richter Stan , Vicki Welke , Hammes Dan , Provost Joseph J
Published: June 2021

This past summer, two Detroit Lakes High School Seniors and two of their teachers drove 50 miles each morning from their rural lakeside community to devote their days to pursue questions of how cells coordinate movement. The students were teamed with an undergraduate, where they learned the scientific skills to investigate the phosphorylation sites for the sodium hydrogen exchanger. The research team of students, teachers and professors worked to design several site‐directed Ser/Thr – Ala mutants of the carboxyl terminus of the exchanger using mass spectrometry data. In conjunction with this work, the MSUM team hosted two day‐long outreach events at Detroit Lakes where students learned about protein structure, function and molecular biology. The combined research team introduced a portion of the research project into the classroom by expressing the mutants made by the students in two high school courses as part of their capstone project. The goal of this two‐year project is to promote research‐based educational activities; building connections between teachers and students in secondary schools and colleges; to present students with role models; and provide faculty and their students with meaningful service‐learning opportunities. This project was funded by the ASBMB Teacher Research Award, NSF‐MCB‐081778, & NSF‐RUI‐MCB 0930432
STRUCTURING MULTIDISCIPLINARY BIOLOGY COURSES ‐ A case study: Cellular Microbiology
Authors: Candas Mehmet
Published: June 2021

Biology curricula have been traditionally based on disciplinary approach to instructional topics like biochemistry, cell biology, immunology, microbiology, etc. However, biological sciences knowledge is revised with an increasingly rapid rate due to multidisciplinary approaches in research, which involves concepts from many fields of biology. This necessitates implementation of multidisciplinary biology courses for preparing students. To address this need, we incorporated an upper‐level elective course, Cellular Microbiology that integrates topics from a number of biology disciplines. The course complements the scientific knowledge and principles established in eukaryotic cell biology, molecular microbiology, immunology, microbial genomics and proteomics. The course topics establish a framework for reviewing fundamental biological mechanisms and processes with relevance to research along with appropriate relevance to clinical applications involving medical microbiology, parasitology and infectious disease control. The course provides an opportunity for learning outside the disciplinary thinking and recruits students’ interest in areas where they develop capacity for critical thinking and problem‐solving. Multidisciplinary biology courses like Cellular Microbiology are essential to achieving greater synergy and efficiency in the continuum of biomedical education.
Undergraduate Laboratory: Increasing Awareness of the Role of Lipids in Biochemistry
Authors: Stahelin Robert V , Cherney David , Kanovsky Amelie
Published: June 2021

Cellular membranes harbor receptors, ion channels, lipid domains, lipid signals, and scaffolding complexes, which function to maintain cellular growth, metabolism, and homeostasis. Moreover, abnormalities in lipid metabolism attributed to genetic changes among other causes are often associated with diseases such as cancer. Thus, there is a need to comprehensively understand molecular events occurring within and on membranes as a means of grasping disease etiology and identifying viable targets for drug development. The lipid bilayer has a highly polarized structure that consists of a central hydrocarbon core and two flanking interfacial regions that are highly dynamic and may contain >10,000 different lipids. This dynamic variety of glycerolipids, sphingolipids, and sterols in the membrane organelles provide spatial and temporal architecture to direct signaling processes through target proteins. The central lipid bilayer viewed as a hub for biological interactions can serve an integral role in biochemistry education. To increase what has traditionally been an underserved area of biochemical education, we have designed a lipid‐based curriculum for the undergraduate classroom and laboratory. An overview of the curriculum and laboratory experiments will be given.
Genetic Determinants for Yeast's Resistance to Copper, Iron and Zinc Overload
Authors: Jo William , Loguinov Alex , Chang Michelle , Wong Peony , Wintz Henri , Zhang Luoping , Smith Martyn , Vulpe Chris
Published: June 2021

The metals iron, copper and zinc, although essential for life, are toxic when present in excess. We have conducted a parallel analysis of yeast deletion mutants under metal overload conditions to simultaneously evaluate the growth phenotype of more than 4700 strains. We established the genetic requirements of the yeast Saccharomyces cerevisiae when grown in the presence of toxic concentrations of iron sulfate, copper sulfate and zinc chloride. These screens identified distinct but overlapping mutant sensitivity profiles. In general, genes encoding proteins involved in intracellular membrane trafficking and vacuole morphogenesis were essential in all three conditions. Highly essential genes included SUR1 and CSG2 in iron, SXM1 in copper and ZRC1 and DAP1 in zinc. YDR186C and YHR045W, encoding putative proteins of unknown function, were identified as potentially novel genes for yeast resistance to zinc overload. Our data suggest that any of these metals, when present in excess, induce secondary metal deficiency, strengthening the connection between iron, copper and zinc metabolism in yeast. In conclusion, our results indicated the requirement of highly‐conserved biological processes in response to iron, copper and zinc toxicity, and identified potentially novel genes that may play a role in metal homeostasis and/or detoxification in yeast. Funded by NIEHS Superfund Basic Research Program [P42 ES004705‐19] and the UC Berkeley Faculty on Research Grant