bluuebunny/crossref_metadata_2025 huggingface dataset:
Microwell‐based 3D co‐culture model for drug screening by shotgun metabolomic profiling
Authors: Lu Xiyuan , Lodi Alessia , Tiziani Stefano
Published: June 2021

The three‐dimensional (3D) cell culture systems, which mimic the in vivo cell environments, are well recognized as tissue and organ substitutes. High‐throughput metabolomics is increasingly applied to investigate different individuals and time‐dependent responses to environmental stimuli. Rapid data acquisition and improved detection limits of direct‐infusion mass spectrometry (DIMS) are paving the way for high‐content metabolomics in preclinical screening, opening new opportunities in drug discovery and personalized medicine.

Here, we investigated cell viability and induction of reactive oxygen species in stromal cells cultured on various 3D systems as well as standard monolayer culture (2D) to evaluate which system provides the most favorable growing conditions. The selected 3D system was then tested for use in 3D co‐culture of leukemia and stromal cells for DIMS‐based high‐throughput/high‐content metabolic drug screens. The nanobiomatrix‐polycaprolactone (NBM‐PCL) induced the highest cell viability and the lowest induction of ROS and was suitable for use in a 96‐ and 384‐ well plate format. The DIMS results show that very relevant metabolic modulations are associated with co‐cultured cells. Doxorubicin treatment in leukemia co‐cultured with stromal cells induces some unique metabolic responses that drastically different from those observed in leukemia cells alone thereby showing the value of the 3D co‐culture high‐content metabolic drug screen.

In conclusion, we optimized a high sample throughput method for the use of 3D co‐culture with a DIMS‐based high‐content metabolic drug screen and drug development.

Support or Funding Information

This research is supported by the MD Anderson Cancer Center Leukemia SPORE P50 CA100632, R01 CA206210‐01, and CPRIT RP180309.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
NaAtm1: Studying a Heavy‐Metal ABC Transporter System
Authors: Hicks Dennis , Yang Janet
Published: June 2021

Adenosine Triphosphate Binding Cassette (ABC) Transporters are a superfamily of integral membrane proteins that have been found embedded in both the cellular membranes and internal organelles of all species yet analyzed. This extreme conservation throughout the tree of life is due to their vitally critical and broad task of transporting all manner of things into and out of the cell and related organelles. This broadness of responsibility has led to an abundant diversity of this family, which in turn, has allowed life to adapt to the many environments found on Earth. While the importance and variability of this family is abundantly clear, many mechanistic and regulatory questions remain. The diversity which has made this family so useful in the natural world has resulted in a huge challenge in the scientific world: elucidating the many mechanisms this family has developed to perform its many functions. The scientific community has so far been able to identify overarching features of all ABC transporters such as the Walker A and Walker B motifs; however, the mechanisms of individual transporters remain undiscovered. In our research, we focus on a specific ABC transporter called NaAtm1 (Novosphingobium aromaticivorans ABC transporter of mitochondria 1), which has been shown to mediate transition metal export in its host species.

While some transition metals (e.g. iron and copper) are essential for certain cellular processes such as respiration and enzyme catalysis, other metals (e.g. cadmium and mercury) can be very toxic to life. Due to this extreme dichotomy, a cell's ability to distinguish between friend and foe is of the utmost importance, and therefore should be a highly regulated process. Our goal is to better understand the full mechanism of the NaAtm1 transporter, to determine why it exports some metals while leaving others alone, and to understand the regulatory process that controls it. Toward this goal, we are conducting site‐directed mutagenesis, Fluorescent spectroscopy, and quantitative analyses of the NaAtm1 transport system. Here we present our preliminary work on examining NaAtm1 transport activity of substrates.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exploring the functional role of an ancient mitochondrial fatty acid synthesis pathway
Authors: Dove Katja K , Kadirova Malika F , Nowinski Sara M , Ouyang Yeyun , Van Vranken Jon G , Rutter Jared
Published: June 2021

Fatty acid synthesis plays a crucial role in the cellular energetic landscape. Cytoplasmic Fatty Acid Synthesis (FAS) generates fatty acid intermediates that are used to store energy, build cellular structures such as membranes, and act as precursors for the biosynthesis of hormones. In addition to the cytoplasmic fatty acid synthesis pathway, eukaryotic cells have maintained a second, highly conserved mitochondrial fatty acid synthesis pathway (mtFAS), reminiscent of prokaryotic fatty acid synthesis pathways. Here, we explore the functional role of this ancient mitochondrial fatty acid synthesis pathway. Historically, a major function of mtFAS was considered to be the production of lipoic acid which is an essential co‐factor for key mitochondrial, metabolic enzymes such as pyruvate dehydrogenase. However, recently our lab has shown that mtFAS also plays a critical role in mitochondrial biogenesis by coupling Acetyl‐CoA levels with iron‐sulfur biogenesis as well as electron transport chain complex activation by promoting late‐stage assembly (Van Vranken et al., 2016; Van Vranken et al., 2018). These findings expanded the functional roles of the so far underappreciated mtFAS pathway into its broader role in the regulation of mitochondrial biogenesis and metabolic function. The identification of human patients with mutations in mtFAS genes further highlights the importance of the mtFAS pathway and the need to understand all aspects of its functions. However, many facets, especially regarding its own regulation, are still unknown. Here we present putative players in the regulation of the mtFAS pathway and their impact on cellular function.

Support or Funding Information

Cardiovascular Training Grant, University of Utah, 5T32HL007576‐32, Howard Hughes Medical Institute

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identifying the Regions Involved in the Interactions between CYP1A2 and CYP2B4
Authors: Saha Aratrika , Connick J. Patrick , Reed James R , Backes Wayne
Published: June 2021

The cytochromes P450s (P450 or CYP) that are involved in drug metabolism are membrane‐bound heme‐containing proteins that reside primarily in the endoplasmic reticulum (ER) of the liver, small intestine, and lung. P450s interact with limiting levels of cytochrome P450 reductase (CPR) to oxidize endogenous and exogenous substrates, converting them to water‐soluble products. Apart from competing for CPR, P450s also have been shown to form both homomeric and heteromeric complexes. Using Bioluminescence Resonance Energy Transfer (BRET), our lab has shown that CYP1A2 and CYP2B4 form physical complexes that, when formed, alter their metabolic characteristics. Interestingly, CYP1A2 and CYP2B4 form high affinity complexes. CYP1A2 also is capable of forming homomeric complexes; however, CYP2B4 does not appear to form such complexes. The goal of this study is to identify the regions that participate in the formation of CYP1A2•CYP1A2 and CYP1A2•CYP2B4 complexes, focusing on surface residues on both proteins. This will be accomplished by generating chimeric proteins and inserting a fragment of CYP2B4 into CYP1A2. The N terminus of the P450 sits in the ER membrane and we have divided the protein into 5 faces, namely the proximal face (region close to heme that binds to CPR), distal face (opposite to heme) and three lateral faces (DE, GE, and KL1). Using molecular cloning we have generated chimeric proteins. In these chimeric proteins, parts of the proximal, distal and lateral regions have been mutated. These chimeric proteins are predominantly CYP1A2 with fragments of CYP2B4 inserted in them and vice‐versa. These chimeras were expressed in HEK‐293T cell line followed by BRET to determine whether complexes are formed. Preliminary data shows that regions of the proximal and lateral faces that were tested did not appear to be involved in the complex formation. Other fragments in these regions remain to be examined. However, we inserted the region K59‐D75 of CYP2B4 into the corresponding region of CYP1A2 and tested for both CYP1A2•CYP1A2 and CYP1A2•CYP2B4 interactions. Surprisingly, substitution of the K59‐D75 fragment of CYP2B4 into CYP1A2 enhanced the BRET signal for formation of the CYP1A2‐CYP1A2 complex, suggesting that this lateral face participates in CYP1A2•CYP1A2 complex formation. Subsequent experiments will be performed to examine whether protein function is affected.

Support or Funding Information

Supported by NIH GM123253 & ES013648

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanistic insight into oxidized ribonucleotide (8‐oxo‐GTP) insertion by a DNA polymerase
Authors: Smith Mallory
Published: June 2021

During each cell division, more than 1 million ribonucleotides are misinserted into the genome. A close examination of the ribo‐ and deoxyribo‐nucleotide pools reveals the presence of damaged and potentially mutagenic nucleobases. In particular, 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐oxo‐dG) is well‐known as a prevalent and mutagenic oxidized nucleobase that contributes to carcinogenesis and several neurological diseases. However, the mutagenic properties of its ribonucleotide equivalent (r8‐oxo‐GTP) remain poorly understood. Furthermore, the biological relevance of r8‐oxoGTP is underscored by the attenuated ability of nucleotide pool sanitizing enzyme MTH1 to catabolize r8‐oxo‐GTP. Using model DNA polymerases, DNA polymerase beta and Bst DNA Polymerase I, we comprehensively characterized r8‐oxoGTP insertion opposite cytosine (non‐mutagenic) and adenine (mutagenic) using both kinetic and structural techniques. Our results show that the 2′‐OH, unique to the ribo‐ form of 8‐oxo‐GTP, heavily diminishes the rate of insertion for r8‐oxo‐GTP opposite adenine and nearly eliminates its insertion opposite cytosine. These results provide novel insight into the mechanistic strategies employed by DNA polymerases to minimize the insertion of damaged ribonucleotides into the genome.

Support or Funding Information

This work was supported by the National Institutes of Health (grant number R01‐ES027558).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular Crowding Modulates Actin Filament Mechanics and Structure
Authors: Castaneda Nicholas , Lee Myeongsang , Rivera‐Jacquez Hector J , Marracino Ryan R , Girardi Eric , Tang Jay X , Merlino Theresa R , Kang Hyeran
Published: June 2021

The cellular environment is crowded with high concentrations of macromolecules that significantly reduce the accessible volume limiting biomolecule interactions. Reductions in cellular volume can generate depletion forces that affect protein assembly and stability. The mechanical and structural properties of actin filaments play critical roles in various cellular functions including structural support, cell movement, division, and intracellular transport. Although the effects of molecular crowding on actin polymerization have been shown, how crowded environments affect filament conformations, dynamics, and mechanical properties is unknown. In this study, we investigate the effects of solution crowding on the modulations of filament mechanics and structure both in vitro and in silico. Direct visualization of filaments in the presence of crowding agents is achieved by fluorescence microscopy imaging, allowing for the quantification of filament thermal bending dynamics and mechanics. Biophysical analysis indicate that molecular crowding modulates thermal fluctuations, enhances filament's effective bending stiffness, and reduces average filament lengths. Utilizing the explicit molecular dynamics simulations, we demonstrate that molecular crowding alters filament conformations and structural properties indicating overall compaction and stabilization that are directly coupled to filament mechanics. Taken together, our study suggests the interplay between excluded volume effects and non‐specific interactions raised from molecular crowding may modulate actin filament mechanics and structure.

Support or Funding Information

This study was supported by the UCF start‐up fund and In‐House grant for H.K. The authors would like to thank the National Science Foundation for support of this work through REU site EEC 1560007. We acknowledge the computational time from UCF stokes cluster.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Fascia of the Abdomen and Pelvis
Authors: Brueckner Jennifer
Published: June 2021

This presentation will review the key components of the abdominopelvic fascia, with particular emphasis on the clinical relevance of each subdivision. By the end of this session, participants should be able to describe the organization of the superficial and deep fascial layers of the abdominal wall, along with the underlying subserous transversalis fascia and parietal peritoneum. Two types of pelvic fascia will be reviewed, including the membranous pelvic and endopelvic fascia, along with the ligaments formed by each.
Unexpected complexity of the budding yeast transcriptome
Authors: Ito Takashi
Published: June 2021

Budding yeast Saccharomyces cerevisiae is the most extensively studied organism in functional genomics, but transcript structure has remained largely elusive for most of its 6,000 genes. To complement this least explored field, we sequenced cDNA libraries generated by using a vector‐capping method that allows accurate mapping of transcription start sites (TSSs). Consequently, we assigned 11,575 TSSs to 3,638 genes, demonstrating that most yeast genes have two or more TSSs. We also revealed alternative splicing events joining two adjacent genes. Furthermore, we isolated full‐length cDNAs of ∼1,800 novel transcripts including small transcripts derived from “intergenic” regions, antisense transcripts to known genes, and sense transcripts starting from inside the known genes. Extrapolation of these results suggests the presence of ∼10,000 transcription units in total. Thus, the yeast transcriptome is considerably more complex than previously thought and shares many recently uncovered characteristics with those of mammals. Pervasive transcription to generate a plethora of noncoding RNAs is not specific to higher eukaryotes but likely an intrinsic feature of the eukaryotic system. Accordingly, the yeast would provide a good model to study noncoding RNAs, which may represent the largest part missing from our current parts list.
Effects of radiation on pro‐inflammatory pathways in rat brain
Authors: Lee Won Hee , Sonntag William E. , Lee Yong Woo
Published: June 2021

Pro‐inflammatory pathways in the brain have been implicated in the progression of neurological disorders. The present study was designed to investigate our hypothesis that radiation‐induced brain injury is mediated by pro‐inflammatory mechanisms. Four month old F344×BN rats received either whole brain irradiation with a single dose of 10 Gy γ‐rays or sham‐irradiation, and were maintained for 4, 8, and 24 h following irradiation. The expression levels of various pro‐inflammatory mediators such as cytokines, chemokine, adhesion molecules, and matrix metalloproteinases were analyzed by real‐time reverse transcriptase‐polymerase reaction. A significant and marked up‐regulation of TNF‐α, IL‐1β, IL‐6, MCP‐1, ICAM‐1, VCAM‐1, E‐selectin, MMP‐2, and MMP‐9 was observed in hippocampal and cortical regions isolated from irradiated animals. To elucidate the molecular mechanisms of radiation‐induced brain inflammation, effects of radiation on the DNA‐binding activity of pro‐inflammatory transcription factors were also examined. A series of electrophoretic mobility shift assay demonstrated that irradiation significantly activated AP‐1, NF‐κB, and CREB. These data suggest that irradiation induces pro‐inflammatory pathways in the brain through overexpression of pro‐inflammatory mediators and activation of transcription factors AP‐1, NF‐κB, and CREB. (This work was supported by NINDS R01NS056218)
Cloning and expression of Scp1, a yeast metacaspase homologue, from Schizophyllum commune
Authors: Hussnain Syed Amal , Gulack Brian C , Fox Kristin M
Published: June 2021

Past research has supported the existence of caspase‐dependent apoptosis in unicellular organisms, and the first caspase‐like gene in yeast was identified and named YOR197w. Due to criticism underlying the study of mammalian apoptosis in unicellular organisms such as yeast, we took the initiative to study a YOR197w homologue in the simple, multicellular fungus Schizophyllum commune. Previously, we had successfully isolated and cloned a YOR197w homologue from the cDNA library of this organism, and named it Scp1. A careful analysis of the sequence showed that the isolated Scp1 had acquired a set of mutations, requiring us to start the project afresh by isolating a new copy of the Scp1 gene from the cDNA library. To date, we have again successfully cloned Scp1 in the pGEM® ‐T Easy (Promega) cloning vector. In the near future, we expect to clone this putative metacaspase gene in the pET‐15b (Novagen) expression vector, induce expression, and study the structure and function of the resulting protein.
Transcriptomic analysis of the program mediating enterocyte differentiation by HNF4, GATA4, or CDX2 in the rat ileal crypt cell line IEC‐6
Authors: Asselin Claude , Fleet James C. , Delabre Jean‐François , Lévy Émile
Published: June 2021

HNF4α (H), GATA4 (G), and CDX2 (X) are transcription factors (TF) proposed as central regulators of enterocyte differentiation. We studied the impact of these TF on enterocytes using stable IEC‐6 cell lines expressing H, G, or X. H increased microvilli density when grown on Matrigel and increased lipid/apolipoprotein synthesis. X‐cells had columnar morphology while G‐cells were larger and formed multiple layers at confluence. Array analysis on RNA from confluent cell lines (Affymetrix Rat 230 2.0 GeneChips, n=3/line) revealed many changes for each TF vs. control IEC‐6: at 1% false detection rate H, 3767 (52% up); G, 2409 (55%); X, 1977 (34%). Common transcript signatures may be due to cross‐regulation of the TF: H increased G (3.4x) and X (2.7x) mRNA; X increased H mRNA (2.1x) and protein; G increased X mRNA (3x) but suppressed H mRNA (70%). Cluster analysis revealed unique TF responses. Functional analysis showed that H increased lipid metabolism and tight junction transcripts, reduced cell cycle transcripts, and altered matrix protein transcripts. G reduced cell cycle and tight junction transcripts but increased those for apoptosis. X increased glycolysis and clathrin‐coated vesicle transcripts but reduced those for MAPK signaling and Golgi vesicle transport. Our data suggest that individual TF are critical drivers for specific enterocyte phenotypes. Supported by Showalter Trust (JCF), NSERC and CCFC (CA).
The tumor suppressor Ikaros transcription factor is crucial for the anti‐proliferative Vasoactive Intestinal Peptide Receptor‐1 expression in developing thymocytes
Authors: Sandy Ashley Rose , Failing Jarrett , Vomhof‐Dekrey Emilie , Hoselton Scott , Schuh Jane , Dorsam Glenn
Published: June 2021

The Leukemia and Lymphoma Society states more children die of leukemia yearly than any other cancer, therefore effective treatments are crucial. In the absence of the tumor suppressor Ikaros (IK), mice develop T cell leukemia with 100% penetrance. We demonstrated that IK regulates a gene termed vasoactive intestinal peptide receptor‐1 (VPACR‐1), when overexpressed in NIH‐3T3 cells. In T cells, VPACR‐1 suppresses interleukin‐2 and subsequent proliferation. Therefore, we hypothesize that IK tumor suppressor activities are manifested by maintaining high levels of VPACR‐1. Thus, an IK null, double negative stage 3 (DN3) thymocyte cell line, JE131, showed a significant reduction in VPACR‐1 compared to wild type (WT) thymic tissue. To obtain an accurate comparison to JE131 cells, thymic tissue was purified by AutoMACS and FACS for the DN3 subpopulation with a similar phenotype to JE131 cells (CD44Lo/CD25Hi/CD4−/CD8−). By qPCR, WT DN3s expressed VPACR‐1 ≤20% that of total DN cells, however this low level is ≥16X that of JE131 cells, and strongly supports IK as a positive potentiator for thymocyte VPACR‐1 expression. Future studies will reintroduce IK into JE131 cells by lentiviral transduction to rescue VPACR‐1 expression and support VPACR‐1 as an IK gene target. Another major goal is to map VPACR‐1 expression levels in all DN populations. Supported by NIH/NIDDK Career Award (KO1 5K01DK64828‐2) and COBRE at NDSU.
Production and characterization of ZFP36L1 antiserum against recombinant protein from Escherichia coli
Authors: Cao Heping , Lin Rui , Ghosh Sanjukta , Anderson Richard A. , Urban Joseph F.
Published: June 2021

Tristetraprolin (TTP/ZFP36) family proteins are anti‐inflammatory. They bind and destabilize some AU‐rich element‐containing mRNAs such as tumor necrosis factor mRNA. In this study, recombinant ZFP36L1/TIS11B (a TTP homologue) was over‐expressed in E. coli, purified, and used for polyclonal antibody production in rabbits. This antiserum, and an antiserum against recombinant mouse TTP, was used to detect ZFP36L1 and TTP in mouse 3T3‐L1 adipocytes and RAW264.7 macrophages. Immunoblotting showed that ZFP36L1 was stably expressed, but TTP was induced by insulin and cinnamon and not by lipopolysaccharide (LPS) in 3T3‐L1 adipocytes. In contrast, ZFP36L1 was undetectable but TTP was strongly induced in LPS‐stimulated RAW264.7 cells. Quantitative real‐time polymerase chain reaction (PCR) confirmed higher levels of ZFP36L1 mRNA in adipocytes and TTP mRNA in RAW264.7 cells. Low levels of ZFP36L1 expression were also confirmed by northern blotting in mouse embryonic fibroblasts. These results demonstrate that TTP and ZFP36L1 are differentially expressed at the mRNA and protein levels in mouse adipocytes and macrophages (supported in part by the Intramural Research Program of the NIH, NIEHS and USDA‐ARS Human Nutrition Research Program).
Directed Evolution of Arginine Kinase Oligomerization
Authors: Swegal Warren , Fraga Dean , Edmiston Paul L
Published: June 2021

A new technique for directed evolution of protein oligomerization was developed. The system is based on fusing a target protein to the DNA binding domain of a cI lambda phage inhibitor protein lacking an oligomerization domain required for function (Mariño‐Ramírez, et al. J. Bact. 186:1311). Activation of the λ inhibitor can only proceed if the target undergoes mutational events that lead to dimerization, thus providing a selectable means for protein oligomerization. The phosphogen kinase (PK) family was used as a model system to test the technique. Within the PK family, arginine kinase (AK) is a monomer, yet the other prominent member, creatine kinase, is a homodimer. The two share a high degree of tertiary structure similarity making them a target for directed evolution to create a dimer from monomeric AK. Prior to random mutagenesis and screening, a cloned variant of L. polyphemus AK that possessed ten site‐directed mutations was created to incorporate key residues found within the dimer interface of CK, however these mutations alone do not produce a dimeric AK. Ethylenenitrosourea was used to randomly mutate the AK gene in vivo. After mutation steps, cells containing the fusion were exposed to λKH5‐h80 with increasing levels of stringency by adding variable levels of ITPG as a second means of inhibitor activation. The AKs of surviving clones were sequenced to determine the number and identity of the mutational events.
Substrate Recognition by Bacterial Enoyl Reductases
Authors: Zhang Xujie , Rafi Salma , Tonge Peter J
Published: June 2021

Tuberculosis (TB) kills over two million people every year. We are currently studying a series of known or putative enzyme drug targets from MTB that includes InhA, the NADH‐dependent enoyl‐ACP reductase from the MTB fatty acid biosynthesis (FASII) pathway.

InhA is a validated target for drug discovery and we are investigating the interaction with the natural acyl carrier protein substrate (AcpM). Based on studies with the E. coli enoyl reductase homologue, we hypothesized that three basic residues close to the substrate binding loop in InhA, R195, R225, and K233, interact with acidic residues in the AcpM recognition helix. We also have evidence that an additional set of basic residues close to AcpM Helix II, R45, R49, R53, play a critical role in AcpM recognition by InhA. Our studies also have implications for the ability of acyl carrier protein to recognize and interact with a diverse array of intracellular protein targets.
Identification of an allosteric binding site in gamma‐glutamylcysteine ligase
Authors: Rozenboom Kelly , Boettcher Colin , Kelly Brenda
Published: June 2021

Glutathione has been implicated in chemotherapeutic resistance due to its function in detoxication and its high abundance in cancer cells. By inhibiting glutathione synthesis selectively in cancer cells, these cells may become depleted of glutathione and more susceptible to chemotherapeutic agents. The enzyme γ‐glutamylcysteine ligase (γ‐GCL) catalyzes the first and rate‐limiting step in the synthesis of glutathione. Our objective was to identify non‐substrate amino acids that alter enzyme activity as a function of α‐aminobutyric acid (α‐ABA) concentration. Our results show that most of the non‐substrate amino acids examined inhibit the enzyme at high, near‐saturating concentrations of α‐ABA but activate the enzyme at low concentrations of α‐ABA. Fluorescence spectroscopy was used to probe the effect of these non‐substrate amino acids on the binding of substrates. Resultant plots of non‐substrate concentration versus fraction protein bound are sigmoidal in shape and dissociation constants increase for each non‐substrate amino acid in the presence of α‐ABA. We hypothesize there are two distinct binding sites in γ‐GCL that may bind either substrate or a non‐substrate and that the two sites have an allosteric relationship. This work was supported through the Greater Gustavus Fund, Langsjoen Fund, and Sigma Xi Fund at Gustavus Adolphus College.
Human γ crystallins form fibrillar amyloid aggregates through a partially unfolded intermediate
Authors: Wang Yongting , Drahos Kate , King Jonathan
Published: June 2021

The transparency of the human eye lens depends on the stability and solubility of the lens crystallins. Age‐related cataract is a result of aggregation of damaged or misfolded forms of crystallins. The most abundant monomeric crystallins, namely human γC, γD, and γS, have been found in the insoluble fraction of age‐related cataracts. Mutations in the genes encoding these proteins are associated with congenital cataracts. Here we examined the aggregation of wild type and a number of congenital mutants of γ crystallins under acidic conditions (which might be encountered in lysosomes of differentiating lens fiber cells). When incubated without denaturant at pH3 and 37°C, γ crystallins assembled into fibril structures imaged with TEM. These fibrils also bound Thioflavin T, measured by fluorescence at 485nm. Congenital mutants, especially those which destabilize the protein (L5S and V75D of γD), aggregated more readily than their wild type counterpart. The tryptophan fluorescence of these proteins exhibited a red‐shift under aggregation‐prone conditions, suggesting the presence of a partially unfolded intermediate. The aggregation of γ crystallins strongly depended upon the pH of the solution, correlating with the fluorescence red‐shift. Such correlation suggests that this partially unfolded intermediate is on the aggregation pathway. This research was supported by NIH (GM17980) and NEI (EY015834) to J.K.
Identifying Biomarkers of the MHC Gene to Investigate Chemosensory Discrimination
Authors: Bazzoli Tyler Cross , Brown Steven D
Published: June 2021

To better understand the mechanisms underlying the major histocompatibility complex (MHC)‐dependent chemosensory signals, it is critical to chemically identify MHC‐dependent odorants. Urine samples from MHC‐genotyped mice were analyzed using capillary gas chromatography. Employing multivariate analysis and pattern recognition techniques on the chromatographic data, it is possible to classify mice by their MHC genotype and explore which chemical compounds are biomarkers capable of discrimination. Soft Independent Modeling of Class Analogy (SIMCA) of the data was performed and indicated that the observed genotypes were easily distinguishable and well separated, a strong indication that specific compounds differentiated the genotypes. Based on the SIMCA model, a graph of the discriminating power of each compound within the model was generated. The compounds exhibiting high genotype‐discriminating power were dimethyldisulfide, methyl methylsulfenylmethyl disulfide, Z‐5,5‐Dimethyl‐2‐ethylidenetetrahydrofuran, and E‐5,5‐Dimethyl‐2‐ethylidenetetrahydrofuran. The structural similarity between the two disulfide compounds along with the similarity of the two tetrahydrofuran compounds suggest that there may be a biochemical explanation for the role that these four compounds play in differentiating mice with dissimilar MHC genes. Funded by HHMI Undergraduate Science Education Program.
The Role of N‐glycosylation in Drosophila Development: Characterization of alg10
Authors: Dominica Carolyn Reeves , Lebois Evan P. , Selva Erica M.
Published: June 2021

Proteins that function extracellularly must undergo the process of secretion and are subject to posttranslational modifications before cell signaling can occur. In this study, Drosophila harboring mutations in alg10 are characterized. alg10 encodes a glycosyltransferase that catalyzes addition of the terminal glucose to the growing dolichol‐linked oligosaccharide just prior to its en masse transfer to nascent polypeptides. Our study of P‐element excision alleles of alg10 shows that this gene product is of critical importance during Drosophila development. Removal of both zygotic‐ and maternal‐derived alg10 results in severe and pleiotropic embryonic deficits, demonstrating the importance of regulated N‐glycosylation during early Drosophila development. Removal of alg10 from larval eye imaginal discs leads to a disordered eye of reduced size. These effects may be mediated through the Sevenless activated MAP kinase cascade, as we find alg10 mutant eye discs display pathway gain‐of‐function phenotypes. These findings indicate that regulated N‐glycosylation of a component of the Sevenless receptor tyrosine kinase pathway is an important target of Alg10 function. Together, our data suggest that tissue specific addition of terminal glucose to the dolichol‐linked oligosaccharide is an essential regulatory event in Drosophila development. Sponsored by HHMI Biological Sciences Education Program
Identification of Novel Posttranslational Modifications of Caveolin‐3
Authors: Fuhs Stephen Rush , Insel Paul A.
Published: June 2021

Caveolin, a critical protein component of caveolae, serves as a scaffold for a large number of signaling molecules. This study asks the question: What determines the dynamic nature and extent of localization of signaling components in caveolin/caveolae? Posttranslational modifications (PTMs) influence protein binding and thus we asked if PTMs of caveolin‐3, a muscle‐specific caveolin, help regulate its interactions with signaling proteins. Multiple phosphorylation sites have been identified in caveolins‐1 and ‐2, but not caveolin‐3. To determine whether cav‐3 is phosphorylated, we isolated adult rat cardiac myocytes and immunoprecipitated (IP) with anti‐phosphoamino acid antibodies. Immunoblotting of IPs with anti‐caveolin‐3 antibodies revealed that caveolin‐3 was phosphorylated on Tyr and Thr. In addition, other data has shown that caveolae‐localized signaling components can be regulated by sumoylation (e.g., GLUT4, PTP‐1b and Dynamin). We find that caveolin non‐covalently binds the Small Ubiquitin‐related Modifier (SUMO), implying that caveolin binds sumoylated proteins. Caveolin‐3 co‐immunoprecipitates with Ubc9, the E2 conjugating enzyme, and is itself sumoylated in vitro. We speculate that phosphorylation and sumoylation are PTMs that contribute to the ability of caveolin‐3 to regulate signal transduction. (supported by NIH grants).
Specific Binding of GAPDH in the Presence or Absence of Selenium to SPS Monitored by Affinity Chromatography
Authors: Galloway‐Yunusah Michelle , Stadtman Thressa C.
Published: June 2021

The methods of synthesis and insertion of selenium into selenocysteine (Sec)_containing enzymes has been recognized in prokaryotes, eukaryotes and archaebacteria. Yet, the transport and intracellular trafficking of selenium is incompletely characterized. Therefore, considerable attention has reallocated to the identification and characterization of selenium adducts or delivery proteins required in specific metabolic pathways. It has been documented the Escherichia coli glyceraldehyde_3_phosphate dehydrogenase (GAPDH; EC ) actively bound selenium possibly as a perselenide derivative following in vivo labeling with 75Se[selenite] [Lacourciere, G.M., Levine, R.L. and Stadtman, T.C. (2002) PNAS USA 99, 9150_9153]. Subsequently, Yuki and coworkers [Ogasawara, Y., Lacourciere, G.M., Ishii, K. and Stadtman, T.C. (2005) PNAS USA 102, 1012_1016] provided in vitro evidence that each subunit of human erythrocyte GAPDH possessed a reactive cysteine residue, which can act as the binding site for selenium supplied as selenodiglutathione from selenite and glutathione. These findings suggest GAPDH participated as a component of a selenium delivery system for the biosynthesis of selenophosphate, a reactive selenium compound, catalyzed by the E. coli selenophosphate synthetase (SPS). To date, there are no reports relating to the binding capability of SPS to native and 75Se_labeled GAPDH. Consequently, the work presented herein reports the application of affinity chromatography as a screening tool to emphasize the specificity of the Se enzymes. This research was fully supported by the National Heart, Lung, and Blood Institute, National Institutes of Health.
Epac1, an exchange protein activated by cAMP, increases cell migration through syndecan clustering, PI3K activation and heparan sulphate production.
Authors: Baljinnyam Erdene , Iwatsubo Kousaku , Wang Xu , Ulucan Coskun , Lagunoff David , Ishikawa Yoshihiro
Published: June 2021

Background: The role of epac1, an exchange protein activated by cAMP, in melanoma migration is largely unknown. Heparan sulfate (HS), a major ECM, and syndecan2, a HS‐binding protein on the cell surface, play important roles in regulating such migration. Syndecan2, when activated, translocates to lipid‐rich plasma microdomains, known as lipid rafts. We thus examined the role of epac1 in HS production and syndecan2 translocation in melanoma migration.

Methods: SK‐MEL‐2, a human melanoma cell line, and migration assays with Boyden chambers were employed.

Results: Activation of Epac1 by adenoviral Epac1 overexpression or 8pMeOPT, an Epac‐specific cAMP analog, significantly increased cell migration in a time‐ and dose‐dependent manner. Epac1 also increased HS production by 3.5‐fold, and the removal of HS abolished Epac1‐induced migration. Epac1 also increased expression of N‐deacetylase/N‐sulfotransferase‐1 (NDST‐1), a key synthetic enzyme for HS. Sucrose gradient fractionation showed that Epac1 markedly increased syndecan2 translocation to rafts. In addition, disruption of rafts with lipid depletion abolished Epac1‐induced migration.

Summary: Epac1 increased migration in malignant melanoma through HS production and syndecan2 translocation to rafts.
Effects of perinatal and maintained calorie restriction to mitochondrial level of oxidative stress in rat liver
Authors: Albarrán‐Bravo Sacnité , Pérez‐Gallardo Rocío V. , Clemente‐Guerrero Mónica , Manzo‐Ávalos Salvador , Saavedra‐Molina Alfredo
Published: June 2021

Calorie restriction (CR) is known to extend the lifespan of a number of organisms including yeast, worms, flies, rodents and perhaps non‐human primates. The mechanisms through which occurs have been unclear. CR during gestation led to growth retardation at birth. If CR was prolonged throughout lactation, adult body weight was permanently reduced. There is also evidence that the intrauterine environment of the fetus may be of greater importance in the etiology of chronic diseases in adult fetus.

The aim of this project is to determine the effect of a perinatal and maintained calorie restriction regimen in oxidative stress in rat liver mitochondria. Offsprings of one month of age showed a decreased in body weight of 48% in female and 56% in male rats. Levels of NO• in CR rats increased 180% in females and 282% in male rats compared to controls. Lipoperoxidation showed an increase of 211% in female and 116% in male rats under CR regimen against controls. Female and male rats of five months of age weighed 35% and 50%, respectively, less than controls. The determination of NO• shows a diminution of 56% in female and 36% in male rats under calorie restriction versus controls. The same can be seen in determination of lipoperoxidation with a diminution of a 54% in male rats but, in contrast, an increased in lipoperoxidation levels in female rats of 97% were observed versus controls. Our initial conclusion is that oxidative and nitrosative stresses are affected in offsprings of one month and in five month‐old. Supported by Fondos Mixtos CONACYT‐ Gob. Edo. de Michoacán (64277, 64308), COECYT and CIC‐UMSNH (2.16) grants.
Structure and mechanism of a soluble diacylglycerol kinase
Authors: Jerga Agoston , Miller Darcie J , White Stephen W , Rock Charles O
Published: June 2021

DgkB is an essential diacylglycerol kinase (Dgk) that participates in lipoteichoic acid biosynthesis in gram‐positive bacteria. The structure of Staphylococcus aureus DgkB, a prototypical member of the Dgk superfamily (Pfam00781) was solved both as the free enzyme and in complex with ADP. DgkB is an asymmetric dimer, and each monomer has nucleotide binding P‐loop in domain 1 located across from a second domain the presents the catalytic base and substrate to the active site. A distinctive feature of DkgB is the structural Mg2+ site consisting of several conserved aspartate residues and structured water molecules. The enzyme requires both ATP•Mg2+ plus free Mg2+ for activity and site‐directed mutagenesis is consistent with the Asp•water•Mg2+ network functioning to correctly orient the catalytic base, Glu273, in the active site. DgkB is activated and directed to membrane diacylglycerol through its interaction with anionic phospholipids. The nucleotide binding residues, the acidic amino acid ligands binding the structural Mg2+, and the active site base, Glu273, are conserved in the catalytic cores of the mammalian signaling Dgks, indicating that these enzymes use the same mechanism and have similar structures as DgkB. (Supported by NIH GM34496 and ALSAC)
Arachidonate distribution in resting and proliferating primary human T cells
Authors: Robichaud Philippe Pierre , Boulay Katherine , Poirier Samuel , Surette Marc E
Published: June 2021

Arachidonate‐phospholipid remodelling is characterised by the transfer of arachidonate (AA) between cellular phosphatidylcholine (PC) and phosphatidylethanolamine (PE) species. It has been suggested that AA‐phospholipid remodelling may control cellular AA distribution and availability.

In this study, the cellular distribution of AA was measured in resting and in anti‐CD3+IL‐2 ‐stimulated primary human T cells. In pulse‐chase experiments a very rapid redistribution of [3H]AA from PC to PE was measured in stimulated proliferating cells compared to resting cells. This increased remodelling was accompanied by a marked shift in the mass distribution of AA into 1‐alkyl‐ and 1‐alk‐1‐enyl‐linked PE species in stimulated cells. Minor differences in AA distribution were measured in cells arrested in different phases of the cell cycle (G1/S, G2/M and S), with the most marked changes observed when the cells transitioned from G0 into the cell cycle. The re‐distribution of AA in proliferating T cells was reversed when the stimulus was removed and the cells ceased to proliferate. Previous studies linked the expression of group IVC phospholipase A­2 with a redistribution of AA into PE species suggesting a role for this enzyme in remodelling. However, remodelling activity was not modified in HEK293 cells overexpressing this enzyme, suggesting that a different isozyme is likely involved. Microarray experiments are now underway with the aim of identifying which enzymes are possibly involved in the remodelling of arachidonate in cycling human T cells. (Supported by the Canadian Institutes of Health Research and the Canada Research Chairs Program).
The One Room School House Model
Authors: Henderson LaRhee Henderson Lucille , Buising Charisse
Published: June 2021

We’ve developed a one room school house model to teach undergraduate research in the biochemistry, cell and molecular biology program at Drake University. It uses apprenticeship, cooperative‐style learning and peer mentoring in a cross‐disciplinary and cross‐community educational program. Because it is based in the principles of a learning community, we've found that this approach allows a small number of faculty to engage large numbers of students in research and maintain a productive scholarship program. The model is cost effective, aids learning, supports development of science and transferable management skills, is productive, and supports diversity. It provides students with skills in scientific research and transferable skills that they take to apply to a wide set of careers.
Biochemistry/Molecular Biology and undergraduate liberal education
Authors: Wolfson Adele J.
Published: June 2021

In November of 2006, ASBMB received a grant from the Teagle Foundation. The Foundation hopes to engage disciplinary societies in assessing the relationship of the goals of undergraduate concentration in their discipline to those of liberal education. This grant provides an opportunity for ASBMB to examine undergraduate programs in biochemistry and molecular biology and evaluate the success of their graduates. The Teagle Working Group has conducted interviews and surveys. Results so far confirm that graduate faculty and employers value research experience above all other types of preparation. The chairs’ survey indicates that about half of departments are working with the ASBMB guidelines, while the other half were unaware of these recommendations. Since the introduction of the guidelines, about one‐third of departments have added specific coursework and more undergraduate research; under 20% have added skills‐based requirements, and the same number reported a movement away from lecture‐based teaching. About one‐quarter have paid more attention to assessment of student learning. We will highlight examples of courses that emphasize skills rather than content. Following the education award presentation, we will hold an open‐mike session on undergraduate education in BMB fields: the relevance of ASBMB guidelines, how to assess students’ success, and the interaction with other goals of a liberal education.
Profiling of lipid mediators released spinally in response to peripheral painful inflammation
Authors: Buczynski Matthew W. , Svensson Camilla I. , Stephens Daren L. , Yaksh Tony L. , Dennis Edward A.
Published: June 2021

A facilitated processing of noxious input at the spinal level contributes to tissue injury and inflammation‐induced hyperalgesia. Lipid metabolism in the spinal cord is of particular interest because of the high concentration of lipids in the central nervous system, where lipid mediators such as eicosanoids, ethanolamines and free fatty acids may play important roles in the processing of pain signals at the spinal level. Previous work has demonstrated that lipid mediators can have both hyperalgesic and anti‐hyperalgesic properties. To study the production of lipid mediators during hyperalgesia, we employed a model of inflammatory pain in which carrageenan is injected to the hind paw of rats, and cerebrospinal fluid and spinal cords were harvested at different time points over a 24 hour period. Using GC‐MS and LC‐MS/MS methodology capable of analyzing over 150 different lipid mediators, we have detected over 40 different metabolites derived from each of these classes. Application of this method may allow for the detection of lipid mediators with previously unappreciated functions, as well as determining unidentified connections between these classes of signaling molecules. This work was supported by the LIPID MAPS Large Scale Collaborative Grant GM069338 (MWB, DLS, EAD), NIH NS16541 (TLY), DA021654 (CIS), 2T32DK007202‐32 (MWB), and the Arthritis Foundation (CIS).
EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF A DROSOPHILA QC‐LIKE PROTEIN
Authors: Parker Amanda Thigpen , Bateman Robert C
Published: June 2021

Glutaminyl cyclase, the enzyme responsible for N‐terminal pyroglutamyl residue formation, is found in a broad range of living species from yeast to humans. A BLAST analysis using the human glutaminyl cyclase protein sequence has revealed a family of QC‐like proteins in organism from human to Drosophila with high homology to glutaminyl cyclase in several organisms including humans. In Drosophila, the QC‐like protein and QC protein have greater than 53% sequence identity. The predicted tertiary structure among the two proteins is highly conserved. To investigate the function and structure of these QC‐like proteins, bacterial fusion proteins were developed for the Drosophila QC‐like protein containing a purification tag. Purification resulted in a 40 kD protein which was apparently devoid of glutaminyl cyclase activity. Currently Drosophila QC and QC‐like proteins are being expressed in parallel to clarify their structural and functional relationship.