bluuebunny/crossref_metadata_2025 huggingface dataset:
Antimycin induces apoptosis in rapamycin resistant SW620 colorectal cancer cells
Authors: Teran Blanca Lucila , Thomas Janice
Published: June 2021

Mammalian target of rapamycin, (mTOR), a serine/threonine protein kinase is a central regulator of cell growth and proliferation and is observed to be hyperactive in many common cancers. Rapamycin, a natural macrolide immunosuppressant, forms a complex with FKBP12 and mTOR inhibiting mTOR activity. Classified as Duke's Type C level cancer, SW620s are colorectal, rapamycin resistant, adenocarcinoma cells with widespread metastasis. During a high through put screening of the National Cancer Institute's Mechanistic Diversity Set, we demonstrated that antimycin not only inhibits growth and proliferation but induces apoptosis in SW620 cells. Antimycin, a group of secondary metabolites produced by Streptomyces bacteria, disrupts the proton gradient across the inner mitochondrial membrane which results in the build‐up of the toxic, free radical superoxide. As a result, apoptosis was induced in several other colorectal cancer cell lines suggesting antimycin is a compound that could potentially be used for the treatment of colorectal cancer.
Activation of the hypoxia pathway and glycogen metabolism
Authors: Eppich Erin , Whipple Melissa , Parker Glendon
Published: June 2021

Renal cell carcinoma cells (RCCC) are the cause of around 90% of kidney cancer. This cancer is distinctive in that the hypoxia pathway is dysregulated, often through the absence of von Hippl‐ Lindau protein, and it has excessive levels of glycogen, which leads to the distinctive “clear‐cell” carcinoma histological phenotype. We hypothesized that the two distinctive features of RCCC are causally linked. To test this we treated Caki‐2 cells, an RCCC cell line, for 30 hours with conditions that activated the hypoxia pathway: including: deferoxamine or DFO (100 μM, 300 μM), a hypoxia chamber and cobalt chloride (CoCl2, 100 μM). Deferoxamine (100 μM, 300 μM), which activates PHD by depleting intracellular Fe2+, and the hypoxia chamber treatment, resulted in significantly higher levels of glycogen (control = 1.5 ± 0.5 μg glucose, hypoxia = 7.4 ± 1.1 μg glucose (p < 0.001), 100μM DFO = 5.9 ± 1.6 μg glucose (p < 0.01), 300μM DFO = 6.1 ± 1.7 μg glucose (p < 0.01); n = 4). Treatment with RNAi oligonucleotides targeted to HIF‐1α, or with exogenous iron, eliminated the ability of deferoxamine to induce glycogenolysis. This illustrates a possible casual link between hypoxia and glycogen accumulation.
The Role of SEL1L in Neurodegeneration
Authors: Chang Wei‐Hua , Krumm Christopher , Francisco Adam B. , Long Qiaoming
Published: June 2021

Neurodegenerative diseases such as Alzheimer¡¦s disease (AD) and Parkinson¡¦s diseases (PD) are characterized by the selective loss of subsets of neurons in the central nervous system (CNS). While increasing evidence reveals a combinatory causes involving both genetics and environmental factors, the molecular etiology underlying the pathogenesis of both AD and PD remains incompletely understood. Suppressor enhancer lin‐12 1 Like (SEL1L), which encodes an endoplasmic reticulum (ER) membrane protein, is highly expressed in the CNS neurons. To investigate the potential association between neurodegeneration and dysfunction of SEL1L, we generated and characterized mice with neuron‐specific knockout of SEL1L (Sel1l‐NKO). Sel1l‐NKO mice have an average lifespan of 8–10 weeks and show significant growth retardation. These mice progressively develop motor dysfunction, manifested by abnormal limb clasping, impaired motor coordination, and loss of body strength. In addition, Sel1l‐ NKO mice exhibit spatial memory deficit. Immunohistological analysis reveals a perturbed dendritic network in the cerebral cortex. Cultured SEL1L‐deficient cerebral cortical primary neurons display an impaired dendritic growth. Together, these results indicate that dysregulation or dysfunction of SEL1L due to genetic mutations or aging may be implicated in the pathogenesis of neuodegenerative disorders in humans.
Protein Thiol‐Based Probing of the Cellular and Metabolic Origins of Brain Oxidative Stress
Authors: Stredny Edward S , Gillespie Paul F , Templeton Cory C , Foley Timothy D
Published: June 2021

Improvement in this laboratory of a previously‐described method, exploiting the high affinity of vicinal thiols for phenylarsine oxide (PAO), has led to the development of a procedure for the efficient capture of proteins containing vicinal thiols that have been oxidized, in tissues, to disulfide bonds. Using this enhanced PAO‐affinity method, we have found that about 50% of the glycolytic enzyme triosephosphate isomerase (TPI) contains oxidized vicinal thiols, amidst a very low oxidation of total protein vicinal thiols, in brains from adult rats. Results of preliminary experiments employing a glutathione redox buffer argue that the vicinal thiols of TPI do not have an unusually high propensity for oxidation and support the view presented here that these thiols are highly oxidized in the brains under study because TPI is enriched in a population of cells that is experiencing substantial oxidative stress. We hypothesize that the observed TPI‐associated oxidative stress may result from increased rates of glucose metabolism by glycolysis at the expense of the antioxidant‐generating pentose phosphate pathway in TPI‐enriched cells. Our findings support the notion that protein thiols may serve as intrinsic sensors of the redox environments in which they reside and that the thiol redox states of cell type‐specific and metabolic state‐associated proteins may shed new light on the origins and, possibly, the pathological triggers of oxidative stress in complex tissues.
Characterization of the intestinal tract neuroendocrine cells in normal and regenerating Holothuria glaberrima
Authors: Huang Sunny Qi , García‐Arrarás José E.
Published: June 2021

The neuroendocrine system is composed of cells that release neurotransmitters and neuropeptide hormones in response to environmental stimuli. In the present study we characterize the neuroendocrine system of the sea cucumber Holothuria glaberrima using immunohistochemistry in normal (non‐eviscerated) and regenerating organisms at different stages of regeneration. The intestinal tract of the sea cucumber was divided into four portions: adjacent to the esophagus, descendant and ascendant small intestine, and convoluted large intestine. Specific antisera against‐calbindin, ‐GABA, ‐Nurr 1, ‐Galanin, ‐PAX 6 and ‐ GFSKLYFamide were employed to visualize neuroendocrine cells. These cells are slender in shape, with basally located nucleus, and their main axis perpendicular to the basement membrane. The number of neuroendocrine cells was higher in the descendant portion of the small intestine and in the large intestine than in the ascendant portion of the small intestine. During regeneration, neuroendocrine cells appeared very early with the formation of the luminal layer and were already observed at 10 days post evisceration. In addition, their numbers increased as regeneration proceeded. These results contribute to a better understanding of the neuroendocrine system in the digestive tract of echinoderms and of the cellular events that characterize the regeneration process.
Phylogenetic Analysis of Nuclear, Ribosomal, Mitochondrial, and Chloroplastic DNA in Chlorella and Related Green Algae Species
Authors: Keller Bryant , Jensen Travis , Larson Sabreena , Patel Niraj , Wichers Katie , Freeman Victoria , Stubbendieck Reed , Barnes Austin , Bailey Cheryl
Published: June 2021

In order to develop a further understanding of the evolutionary relationships between different Chlorella algal strains and related species, methods of algal DNA extraction, PCR, sequencing, and analysis were tailored to our project. With the goal of understanding algal relationships and an intent of affecting algal biofuel production, DNA from multiple regions of algal cells was targeted. Ribosomal DNA including the 18S, ITS1, 5.8S, ITS2, and 28S regions; Chloroplastic DNA (the RuBisCO large subunit coding region); Genomic DNA (the RuBisCO small subunit coding region); and Mitochondrial DNA (Cytochrome C Oxidase subunit I coding region) were picked to be analyzed from each of our algal strains. The comparison of the phylogenetic trees is then being used to determine relationship grouping patterns with increasingly robust trees as more sequencing data is obtained. Further analysis and sequencing of the four regions should give a better understanding of relationships and help to determine the proximity of certain chlorella‐like algal species to one another. Furthermore, extended distance between two strains of the same species provides ground for questioning the nomenclature of certain strains with comparison to their actual genetic composition. Funding provided by NSF‐EPSCoR: 1004–094.
A neutron can make the difference: The 15N stable isotope effect on metabolic pathways
Authors: Filiou Michaela , Webhofer Christian , Nussbaumer Markus , Landgraf Rainer , Maccarrone Giuseppina , Turck Christoph W
Published: June 2021

We have investigated the effects of metabolic labeling with the 15N stable isotope in different organisms.

Mice and bacteria were metabolically labeled using a 15N‐labeled diet or 15N‐labeled media, respectively. The 15N‐labeled organisms were then compared with their unlabeled counterparts at the phenotypic and molecular levels with the help of behavioral tests, quantitative proteomics and targeted enzymatic, metabolic and immunochemical assays.

At the phenotypic level, differences in behavioral traits between 15N‐labeled and unlabeled mice were observed. At the molecular level, changes in protein expression, growth rates, pathway activities, metabolite levels and chromatographic peak patterns were found in the 15N‐labeled specimens.

Our results indicate for the first time a global 15N isotope effect on labeled organisms. This information can be used to correlate phenotypic and molecular alterations and track relevant metabolic pathways.

This work was supported by a QuantPro grant and the Max Planck Society
Enhancer‐mediated regulation of developmental gene expression
Authors: Wysocka Joanna
Published: June 2021

Cell‐fate transitions involve the integration of genomic information encoded by regulatory elements, such as enhancers, with cellular and signaling environments. Identification of genomic sequences that control human embryonic development represents a formidable challenge, as they are embedded within a vast noncoding genomic space. However, enhancers share certain chromatin features, including presence of specific histone modifications and general coactivators. We have recently shown that unique chromatin signatures can be used to annotate two functionally distinct classes of enhancer elements in human embryonic stem cells (hESC): active and poised. We have now extended enhancer profiling to other embryonic cell types of unusual developmental plasticity, such as the neural crest. I will discuss our new findings on the dynamics of enhancer regulation during development and on the interplay between transcription factors and chromatin modifiers involved in that process.
The Fusion Protein of Hendra Virus Contains a Leucine‐Isoleucine Zipper in the Transmembrane Domain that is Important for Association
Authors: Webb Stacy , Popa Andreea , Dutch Rebecca E.
Published: June 2021

Viral fusion proteins promote entry of enveloped viruses by fusion of the viral envelope and target cell membrane. Upon triggering, the fusion protein undergoes a large conformational change, resulting in membrane fusion. Although the pre‐ and post‐fusion conformations for paramyxovirus fusion proteins (F) are known, little is currently known about the transmembrane domain (TMD). To clarify its role, the Hendra virus (HeV) TMD was examined. Directly upstream of the TMD, a heptad repeat domain contains a leucine zipper (LIZ) that continues in frame into the TMD. LIZ mutants with alanine substitutions were expressed in Vero cells to analyze folding and fusion activity. Preliminary data suggested that replacing four residues in the TMD LIZ with alanine significantly decreased surface expression and fusion activity. Using a technique to directly analyze TMD interactions using sedimentation equilibrium centrifugation, we showed that the wild type HeV TMD associates in a monomer‐trimer equilibrium. However, the TMD four alanine mutant of the LIZ ablates trimerization, implicating the LIZ in TMD‐TMD interactions. This work implicates that stability of the pre‐fusion F requires TMD‐TMD interactions, so analysis of the role of LIZ in TMD association could offer new insights into fusion and potentially identify targets for antivirals. This work was supported by the National Institutes of Health grant R01AI051517.
Designing a unique reporter based microarray sensor platform for monitoring cell signalling.
Authors: Gibriel Abdullah
Published: June 2021

Purpose

Biological processes in mammalian cells are driven by gene expression that is in turn regulated by transcription factors (TFs). The latter are controlled by a series of complex network of biochemical pathways that represent the core for cell signalling. There has been little success in monitoring cell signalling using traditional methods such as proteomics. In this report we demonstrate the possibility of deconvoluting such complexity using a multiplexed library of plasmids carrying a TF binding site upstream of a unique reporter sequence (UR). A microarray sensor platform was then used to monitor dynamic changes in cells by measuring UR expression levels.

Methods

HEK293 cells, treated with various drugs and inducers, were transfected with the multiplexed plasmid library. mRNA was then purified and reverse transcribed to produce first strand cDNA. The latter was then amplified by PCR using a universal fluorescently labelled primer. Purified PCR amplicons were then hybridized with antisense oligonucleotide captures at 53°C. Epoxy slides were then washed and quantified using the ScanArray Express Scanner (Perkin Elmer).

Results

The microarray platform was optimized to detect down to 1 attomol with a maximal degree of specificity and a minimal probe volume of 2μl. This platform was able to detect changes in the TF activity following treatment of cells with dexamethazone, cadmium, forskolin, TPA, cAMP analogues, cGMP analogues and PDEIs. The data was consistent with qPCR and western blot analysis.

Conclusion

The microarray platform was shown to be robust, sensitive and reproducible in monitoring dynamic changes in mammalian cells. The system can be easily tailored to include transcription factors of interest to fit with the required purposes.
Antibacterial activity and cytotoxicity of Aster sphathulifolius maxim. against oral‐bacteria and oral‐cancer cell lines
Authors: Lee Je‐Hyuk , Kim Gun‐Hee
Published: June 2021

Whole plant‐extract of A. sphathulifolius showed the anti‐bacterial activities against tested oral pathogens except of S. aureus. The anti‐bacterial activities against S. mutans, S. sobrinus, S. pyogenes, and S. epidermidis were observed in most of all parts of A. sphathulifolius. However the growth of S. aureus was inhibited by only root‐extract of A. sphathulifolius. A. sphathulifolius showed the outstanding cytotoxicity against human oral gingival carcinoma cell with approximately 75.1 and 93.1% cell death in 250 and 500 μg/ml of concentrations, respectively. Cell viability of human oral squamous carcinoma cell and tongue carcinoma cell were suppressed to 13–18% at 1000 μg/ml of A. sphathulifolius. We showed that A. sphathulifolius extracts have the anti‐bacteria activity against oral pathogens and the cytotoxicity of oral carcinoma cell lines together. Taken together, it is anticipated that A. sphathulifolius might be used more actively for oral health care, as mouthwashes and toothpastes, even an alternative therapeutic drug.
Induction of Cell Cycle Arrest and Apoptosis by an Extract Created from California Walnuts (JUGLANS SP.) in MDA‐MB‐231, MCF7, and HeLa Cells
Authors: White J Brandon , Pham Anh , Le Vy , Kim John , Okuda Roy
Published: June 2021

Walnuts contain components that may slow cancer growth including omega 3 fatty acids, phytosterols, polyphenols, carotenoids, and melatonin. Previous reports have shown that feeding mice a diet that contains walnuts was able to inhibit growth of MDA‐MB‐231 human breast cancers implanted in nude mice. However, the compounds which elicit this action have yet to be elucidated. We have conducted a pilot to begin characterization compounds isolated from walnuts and their ability to kill cancer cells. A methanol extract was prepared from the kernel of walnuts, solvent evaporated and the powder was dissolved in DMSO to a concentration of 100mg/ml and was used in subsequent assays. The extract induced cell death as measured by the trypan blue exclusion assay in a dose and time dependent manner. Mitochondrial outer membrane potential decreased rapidly with treatment. Phosphatidyl serine flipping increased in a dose‐dependent manner. Our results suggest that compounds isolated from walnuts are capable of inducing cell cycle arrest and apoptosis in a variety of cancer cell lines. Current studies are underway to identify the specific components of the methanol extract which are responsible for these activities. This work was supported by a grant from the California Walnut Commission to JBW.
Phenotype studies of Orf135 CTPase from E. coli and complementation studies with RV1160 from M. tuberculosis
Authors: Gerien Kenneth S. , Thomson Joshua G. , Denial Sarah J. , O'Handley Suzanne F.
Published: June 2021

We are studying the Nudix hydrolases from Mycobacterium tuberculosis as potential novel antibiotic targets. One such potential target is the CTPase Rv1160. Because it has a functional homolog in E. coli, Orf135 CTPase, we can study the phenotype of the orf135 E. coli knockout and carry out complementation studies with rv1160. CTP is a feedback inhibitor of pyrimidine biosynthesis, and a precursor to lipid biosynthesis, including cell membrane formation, thus Orf135 and Rv1160 may help regulate these pathways through degradation of CTP. The orf135 E. coli knockout is less susceptible to streptomycin, a phenotype consistent with a change in the cell membrane. The orf135 knockout mutant, containing a plasmid carrying rv1160, shows the same susceptibility to streptomycin as the wildtype E. coli, thus proving that Rv1160 complements Orf135. This research has been supported by an NIH AREA grant and an RIT College of Science summer fellowship.
AraL from B. subtilis is a sugar phosphatase and member of the nitrophenyl phosphatase family of the HAD superfamily
Authors: O'Handley Suzanne F. , Hill Jacqueline K.
Published: June 2021

The HAD (Haloacid Dehalogenase) superfamily is a diverse superfamily with a majority of the enzymes being phosphatases. One family of the HAD superfamily is the nitrophenyl phosphatase family, so named because the first family members were yeast enzymes found only to hydrolyze p‐nitrophenyl phosphate. Since that time, a number of enzymes with a variety of activities have been identified and characterized. There are pyridoxal phosphatases, a UMPase, phosphoglycolate phosphatases, and most recently, a sugar phosphatase from B. subtilis. We have cloned the araL gene, expressed and purified the AraL protein, and partially determined its activity. Like the other family members, AraL is a phosphatase, but unlike any other member, it is the first sugar phosphatase to be reported for this family. Currently, we have found activity on ribose 5‐phosphate, but this may not be the best substrate, as araL is within the arabinose operon. If AraL degrades sugar phosphates found in the arabinose pathway, its cellular role may be to help regulate this important sugar metabolism pathway. This research was supported by an NIH AREA grant.
SELDI‐TOF MS Combined With Magnetic Beads for Detecting Serum Protein Biomarkers for Diagnosis of Active Tuberculosis
Authors: Liu Jiyan , Wei Liliang , Jiang Tingting , Zhang Xing , Xu Dandan , Wang Chong , Li Zhongjie , Zhong Jianping , Li Ji‐Cheng
Published: June 2021

New technologies for the early detection of tuberculosis (TB) are urgently needed. The aim of the present study was to screen for the potential protein biomarkers in serum for the diagnosis of TB using proteomic fingerprint technology. The surface‐enhanced laser desorption ionization time of flight mass spectrometry (SELDI‐TOF‐ MS) combined with WCX magnetic beads was used to profile the serum proteins from 180 patients with TB, 91 cases of healthy volunteers, 40 cases of lung cancer patients, 40 cases of pneumonia patients, and 40 cases of chronic obstructive pulmonary disease. The protein fingerprint expression of all the serum samples and the resulting profiles between TB and control groups were analyzed with the Biomarker Wizard system. A total of 35 discriminating m/z peaks were detected that were related to TB (p < 0.01). The model of biomarkers based on the four biomarkers (2554.6, 4824.4, 5325.7, and 8606.8 Da) generated excellent separation between the TB and control groups. The sensitivity was 83.3% and the specificity was 84.2%. Blind test data indicated a sensitivity of 75.0% and a specificity of 83.5%. The data suggested a potential application of SELDI‐TOF MS as an effective technology to profile serum proteome, and with pattern analysis, a diagnostic model comprising three potential biomarkers was indicated to differentiate people with TB and healthy controls rapidly and precisely.

This work was supported by grants from National Special Sci‐Tech Projects (No. 2012ZX10005001–006), National Natural Science Foundation of China (No. 81072724, No. 81273882), and Zhejiang Province Special Sci‐Tech Projects (No. 2009C03011–3).
Moonlighting Proteins
Authors: Jeffery Constance , Mani Matt , Amblee Vaishak , Chen Chang , Zhang Zhou
Published: June 2021

The ability of a protein to have multiple functions and be involved in different multi‐protein complexes or pathways can complicate the prediction of the protein function from sequence or structure and the annotation of sequence and structure databases. We are studying moonlighting proteins, a class of multifunctional proteins in which a single polypeptide chain has multiple biochemical functions that are not due to gene fusions, multiple splice variants or multiple proteolytic fragments. Many of the known moonlighting proteins are cytosolic enzymes that are also transcription factors, crystallins, chaperones, extracellular growth factors, or cell surface adhesins. The variety of known moonlighting proteins, the multiple ways in which one protein can have multiple functions, the potential benefits to the organism of combining two functions in one protein, and the methods proposed for a protein to evolve a second function suggest that moonlighting proteins might be common.

To date, most moonlighting functions have been found by serendipity. There is currently no straightforward method to identify which proteins have moonlighting functions, or for determining if a protein of interest is a moonlighting protein. In addition, sequence homologues of moonlighting proteins often do not perform both functions. We are currently organizing information about the sequences, structures, functions and functional sites of the over 200 known moonlighting proteins with the goal of preparing an online, searchable database of moonlighting proteins
Resolving the bacterial selenomethionyl proteome for differential expression proteomic analysis
Authors: Taylor Savannah Joy , Broderick Kathleen Marie , Boles Jeffrey
Published: June 2021

The utilization of selenomethionine (SeMet) as a phasing tool coupled with multi‐wavelength anomalous diffraction has allowed the solution of a multitude of three‐dimensional structures in the last two decades since its development. However, cell growth is often hampered when SeMet is provided as the sole source of authentic methionine in growth cultures, and some purified recombinant selenoproteins have proven to have reduced activity and/or altered three‐dimensional structures. As a first step toward identifying which specific selenoproteins hamper cell growth, the bacterial selenomethionyl proteome has been resolved by a combination of cation and anion chromatographic separation followed with SDS‐polyacrylamide gel electrophoresis.
A Simplistic Bioinformatics Approach to Clade Identification in Chlorella‐like Green Algae
Authors: Lee Jensen Travis , Stubbendieck Reed M , Barnes Austin L , Wichers Katie M , Keller Bryant J , Larson Sabreena A , Patel Niraj S , Freeman Victoria H
Published: June 2021

Using the 18S (partial), ITS1 (Internal Transcribed Spacer 1), 5.8S, ITS2 (Internal Transcribed Spacer 2) regions of the genomic SSU (small subunit) DNA, we are able to use basic bioinformatics tools to create an effective phylogenetic tree. Sanger sequencing produces a 1.2–1.5 kb region for each individual species that is annotated into four elements, (partial 18S, ITS1, 5.8S, and ITS2) and is formatted for stand‐alone MAFFT v6.903b (Multiple Alignment using Fast Fourier Transform). The multiple alignment is then carried out on each element and concatenated to remove the potential overlap between the elements. Stand‐alone PhyML v3.0 (Phylogenetic estimation using Maximum Likelihood) is then used on the multiple alignment data, and a tree can be viewed using FigTree v1.3.1 (a phylogenetic tree editor and viewer program). The results of this method gives well‐defined clades that are now being used in genera and even species level determination. Using our simplistic method is an effective way to engaging the problem of green algae identification.
Quantum analysis/improvement of antipsychotic's docking results
Authors: Nunes Gustavo , Zanatta Geancarlo , Bezerra Eveline M. , da Costa Roner F. , Freire Valder N. , Gottfried Carmem C.
Published: June 2021

Research support: CNPq‐Brazil

Human dopamine receptors are important targets in treatment of Parkinson's disease and Schizophrenia. Thus, the understanding of the binding mechanisms involving selective ligand agents and dopamine receptors D2 and D3 may contribute to pharmacologic strategies. Recently, we have applied quantum mechanics methods to calculate the contribution of each amino acid residue in the binding pocket of D3 receptor in complex with eticlopride (Zanatta, G et al, 2012). In this study, to understand the mechanism of haloperidol interaction, we employed the Molecular Fractionation with Conjugated Caps (MFCC) followed by quantum calculations to analyze the structure of haloperidol docked in D3 receptor using Autodock 4.0 (Fig 1).

It was not possible to obtain the stabilization in total binding energy for a radius of 16 Å (Fig. 2), suggesting that this pose is not yet in the best binding conformation. In order to improve the results, we are performing a series of energy minimization steps using quantum mechanics/molecular mechanics (QM/MM). The establishment of a protocol to improve docking results is important to obtain representative structures of selective ligands to D2 and D3 receptors.
Computational prediction and validation of putative ketosteroid isomerase (KSI) structural genomics proteins
Authors: DeLateur Nicholas A. , Somarowthu Srinivas , Shea Colleen , Naniong Mark , Ondrechen Mary Jo , Beuning Penny J.
Published: June 2021

Ketosteroid Isomerase (KSI) performs an important biological function in the metabolism of many bacteria by degrading steroids. Pseudomonas putida KSI (PpKSI) is strikingly efficient and selective. Three putative KSIs identified from Structural Genomics were analyzed by THEMATICS (THEoretical Microscopic Anomalous TItration CurveS) and POOL (Partial Order Optimum Likelihood) and then characterized in vitro to determine their KSI activity. A putative KSI from Mycobacterium tuberculosis (MtKSI) was predicted to have ketosteroid isomerase‐like activity but putative KSIs from Mesorhizobium loti and Pectobacterium atrosepticum were predicted by these methods not to have such activity. Biochemical experiments reveal that the putative KSI MtKSI does indeed possess KSI activity, although with reduced efficiency compared to PpKSI. The two proteins predicted to be misannotated, MlKSI and PaKSI, exhibited no detectable KSI activity. To investigate this lower efficiency in the correctly annotated KSI, we engineered the MtKSI active site to resemble more closely that of PpKSI, and found that most of these mutations alone or in tandem significantly lowered rather than increased activity.
Cholesterol Binding is a Determinant of Canonical Wnt Signaling Activity of Dishevelled
Authors: Sheng Ren
Published: June 2021

Dishevelled (Dvl) serves as a key scaffold protein in both canonical and non‐canonical Wnt signaling pathways. However, the molecular mechanism that determines how differently Dvl participates in these pathways remains elusive. Here, we show that Dvl is a novel cholesterol binding protein and its cholesterol binding is specifically required for the canonical Wnt signaling branch. Dvl interacts with cholesterol with high affinity and specificity via its PDZ domain. In Xenopus embryo, cholesterol binding of Dvl2 is required for the canonical Wnt‐dependent cellular and developmental processes but not for those mediated by non‐canonical Wnt/planar cell polarity (PCP) signaling. Confocal and single molecule imaging shows that cholesterol binding is essential for sustained association of Dvl2 with the Frizzled (Fz) receptor in response to canonical Wnt ligands, enabling its co‐localization with the activated Wnt receptor complex containing low‐density lipoprotein receptor‐related protein6 (LRP6). Taken together, our results suggest that cholesterol serves as a lipid signal that directs canonical Wnt pathway‐specific functions of Dvl.
PMA stimulates PKCalpha‐dependent phosphorylation of ASAH1 in breast cancer cell lines.
Authors: Escobar Tania , Sewer Marion
Published: June 2021

Acid ceramidase (ASAH1) catalyzes the hydrolysis of ceramide to form sphingosine. Studies have implicated deregulation of ASAH1 in cancer, where overexpression is linked to tumorigenesis. The aim of this study was to define the role of phosphorylation in regulating ASAH1 function in breast cancer cells. We show that phorbol 12‐myristate 13‐acetate (PMA) activated a time‐dependent increase in the phosphorylation of ASAH1 at threonine‐287 (T287) in both MCF‐7 and MDA‐MB‐231 breast cancer cells. Moreover, inhibiting calcium‐dependent protein kinase C (PKC) activity attenuated the ability of PMA to increase phosphorylation of ASAH1. Silencing PKCα protein attenuated ASAH1 PMA‐stimulated phosphorylation at T287. Interestingly, the level of expression of PKCα, which has been reported as a marker for breast cancer aggressiveness, is 6‐fold greater when compared to benign MCF10A mammary epithelial cells. In addition, PMA was unable to promote the phosphorylation of ASAH1 in the MCF10A cell line. Furthermore, alanine substitution at T287 reduced the stability of the protein and led to a 57% decrease in the half‐life of ASAH1. Collectively, these data link post‐translational modification of ASAH1 at T287 to the progression of a tumorigenic phenotype in breast cancer cells. This work is supported by NIH DK084178
Overexpression of lipogenic genes in the heart distinguishes physiologic from pathologic left ventricle hypertrophy
Authors: Dobrzyn Pawel , Pyrkowska Aleksandra , Duda Monika , Maczewski Michal , Bednarski Tomasz , Langfort Jozef , Agnieszka Dobrzyn
Published: June 2021

Molecular remodeling in the heart caused by hypertrophy differs between physiological and pathological stimuli. The main goal of this study was to determine how cardiac hypertrophy that is induced by pressure overload or endurance training influences lipid metabolism in the myocardium. The expression of lipogenic genes was upregulated in exercise‐induced hypertrophy and was accompanied by the activation of fatty acid (FA) oxidation pathways regulated by AMPK and RRARα. Intracellular diacylglyceride (DAG), triglyceride (TG) and free FA were not changed in trained compared with untrained hearts. In contrast, in the heart with abdominal aortic banding (AAB)‐induced hypertrophy, we found an increased accumulation of TG and DAG, although the expression of lipogenic genes and the level of FA transport proteins were not augmented. Cardiac steatosis in AAB‐induced hypertrophy was associated with decreased lipolysis, as evidenced by increased hormone‐sensitive lipase phosphorylation at Ser565 and decreased DAG lipase‐α and ‐β protein levels. Obtained results show that cardiac lipid metabolism is differentially regulated in response to physiological and pathological hypertrophic stimuli and suggest that activation of lipogenesis might be involved in the regulatory mechanisms of heart adaptation to stress. Support: NCN UMO‐2011/01/D/NZ3/04777, NCBR LIDER/19/2/L‐2/10/NCBiR/2011.
Aspirin delays skin wound healing by reducing the production of 12‐hydroxyheptadecatrienoic acid, a ligand for BLT2 receptor
Authors: Yokomizo Takehiko , Liu Min , Saeki Kazuko
Published: June 2021

Aspirin, the most famous non steroidal anti‐inflammatory drug (NSAID), is widely used to kill pain and fever by inhibiting cyclooxygenases to produce prostaglandins (PGs). Aspirin is also famous for its serious side effects including mucosal damage of gastrointestinal tracts and delay in skin wound healing. Although reduced production of prostaglandins by aspirin is generally accepted as a common mechanism for these side effects, several reports using genetically engineered mice that lack PG production and PG receptors failed to prove the mechanism. We found that aspirin‐dependent reduction of 12‐hydroxyheptadecatrienoic acid (12‐HHT), an endogenous ligand for leukotriene B4 receptor type 2 (BLT2), is responsible for the delay in skin wound healing. Aspirin delayed the wound closure after skin punching in mice, which was completely abolished in BLT2‐deficient mice. Furthermore, mice deficient in BLT2 or thromboxane A2 synthase, a responsible enzyme for 12‐HHT production, exhibited delayed wound healing. 12‐HHT is produced during blood coagulation and activates BLT2 expressed in keratinocytes to release tumor necrosis factor α and metalloproteinases to induce keratinocyte migration. A synthetic BLT2 agonist accelerated wound closure in vitro and in C57BL/6J and db/db mice. Our results clarify the novel mechanism of aspirin actions, and BLT2 agonists are possible therapeutic drugs for wounds especially for diabetic ulcer.
Omega 3 polyunsaturated fatty acids modulate cyclooxygenase‐2 induction via interactions with GPR120
Authors: Li Xinzhi , Yu Ying , Funk Colin D
Published: June 2021

Cyclooxygenase‐2 (COX‐2)‐derived prostaglandins are implicated in numerous inflammatory disorders. Omega 3 polyunsaturated fatty acids (ω3 PUFAs) such as docosahexaenoic acid (DHA) are recognized as anti‐inflammatory agents. Here, we explore the potential interaction between COX‐2 induction and ω3 PUFAs via the G‐protein‐coupled receptor 120 (GPR120). Pre‐exposure to DHA for 24 h reduced prostaglandin E2 (PGE2) production primarily through COX‐2 inhibition and partially through down‐regulating microsomal prostaglandin E synthase‐1 (mPGES‐1) expression, but not through affecting COX‐1 expression. DHA could also inhibit activation of a human COX‐2 promoter‐luciferase reporter construct containing NF‐κB response elements. We also demonstrated that COX‐2 induction is attenuated in macrophages obtained from Fat‐1 mice where ω3 PUFAs are synthesized endogenously from ω6 PUFAs. In a FeCl3 femoral artery injury mouse model, post‐injury blood flow in Fat‐1 mice was higher relative to their wild type littermates. Knockdown of GPR120 abrogates DHA effects on COX‐2 induction, PGE2 production and interleukin 6 (IL‐6) gene expression, through ERK, Akt, and JNK signaling pathways. These studies provide a new mechanistic basis for the anti‐inflammatory effects of PUFAs via interactions with GPR120. Supported by a joint grant from the CIHR and NSFC (CCI 117951).
Arachidonic acid increases permeability of HBMEC monolayers via increased production of prostaglandin E2
Authors: Hatch Grant Michael , Dalvi Siddhartha , Mitchell Ryan , Aukema Harold , Miller Donald
Published: June 2021

The blood‐brain barrier, formed by microvessel endothelial cells, is the restrictive barrier between the brain parenchyma and the circulating blood. Arachidonic acid (AA) is a conditionally essential polyunsaturated fatty acid [20:4(n‐6)] and a major constituent of brain lipids. In this study transport of AA in confluent monolayers of human brain microvessel endothelial cells (HBMEC) was examined. Addition of [3H]AA to the apical compartment of HBMEC confluent monolayers cultured on Transwell® inserts resulted in rapid incorporation of [3H]AA into the basolateral medium. Knock down of FATP‐1 or CD36 did not alter [3H]AA movement into the basolateral medium and this was due to a rapid generation of prostaglandin E2 (PGE2), an eicosanoid known to facilitate opening of the blood‐brain barrier. Disruption of HBMEC monolayers following AA exposure was confirmed by an increased movement of fluorescein‐labeled dextran from the apical to basolateral medium. HBMECs expressed PGE2 synthase, cyclooxygenase‐1 and ‐2, PGE2 receptors, tight junction proteins and prostaglandin transporters. Specific cyclooxygenase‐2 inhibitors attenuated the AA‐mediated increase in membrane permeability. The results indicate that AA increases the permeability of HBMEC monolayers via increased production of PGE2. [Funded by NSERC Canada; GMH is a Canada Research Chair in Molecular Cardiolipin Metabolism]
Transport and trafficking of fluorescent sphingosine, sphingolipids, and their metabolites
Authors: Kim Raehyun , Kraft Mary L
Published: June 2021

Bioactive sphingolipids are located and transported in cells according to their metabolic pathway and cellular signaling events. However, absence of appropriate fluorescent tool has impeded understanding dynamic nature of sphingolipids during cellular function. To study sphingolipids trafficking and transport in live cells, it is the most desirable to use a fluorescent analog that follows native sphingolipid metabolism and transport, has high photostability, can be visualized in parallel with other common fluorescent labels, such as green fluorescent protein, and does not effect on cell viability. For this purpose, we recently reported a new fluorescent sphingosine, BODIPY 540 sphingosine, which satisfies these requirements. Here we use BODIPY 540 sphingosine to visualize the intracellular transport of fluorescent sphingosine and its fluorescent metabolites over time. We report the subcellular distribution of sphingolipids and their metabolites in living cells as a function of time. We also demonstrate that this probe allows visualizing the rapid movement of transport vesicles that contain fluorescent sphingolipids and their metabolites in real time.
REGULATION OF PLASMA MEMBRANE‐LOCALIZED STRESS RESPONSE SIGNALING BY INOSITOL‐CONTAINING SPHINGOLIPIDS
Authors: Jesch Stephen A. , Gaspar Maria L. , Henry Susan A.
Published: June 2021

Inositol‐containing sphingolipids at the plasma membrane play important roles in regulating stress response pathways, which are critical for lipid homeostasis and cell viability. In the present study, we show that that inositol starvation of budding yeast results in a dramatic reorganization of composition of inositol‐containing sphingolipids, most notably a 50% reduction in the level of mannosyl‐inositolphosphorylceramide (MIPC). To identify lipid‐mediated signaling networks that respond the reprogramming of inositol‐sphingolipid levels, we carried out gene expression profiling following inositol starvation. These results revealed that genes controlled by three stress response pathways, including protein kinase C, HOG signaling and calcineurin pathways, are highly up‐regulated following inositol starvation. Consistent with these findings, slt2Δ, hog1Δ, and cnb1Δ mutants exhibit inositol auxotrophy. Moreover, cells lacking Csg2p, a Ca2+‐binding regulator of MIPC synthesis, constitutively activate protein kinase C, HOG pathway, and calcineurin signaling, suggesting a critical role for MIPC in stress response signaling. However, regulation of the ER‐localized unfolded protein response was unaffected. Together, these results suggest a feedback mechanism for regulating sphingolipid‐mediated signaling by coordinated regulation of stress response pathways.
Dissecting the regulation of the OLE1 gene in Saccharomyces cerevisiae: examining the role of fatty acid species and concentration
Authors: Willey Marshall , Bedard Lauren , Ochs Matthew , McDonough Virginia
Published: June 2021

The OLE1 gene product, the delta‐9 desaturase, inserts a double bond in saturated fatty acids to create unsaturated fatty acids (UFAs). OLE1 expression is controlled in part through the transcription factors Mga2p and Spt23p in response to the supply of UFAs. These proteins reside in the endoplasmic reticulum, and when insufficient supply of UFAs is detected, they are proteolytically cleaved and translocated into the nucleus, where they activate OLE1 expression. Recently our lab has isolated a mutant that is deficient in regulation of OLE1, called nro1 (no regulation of OLE1). The mechanism for the NRO1 protein's action is unknown. In this study, growth tests and enzyme assays using reporter genes controlled by the OLE1 promoter region suggest that Nro1p responds more strongly to the fatty acids 16:1 Δ9 and 18:2 Δ9, 12, than 18:1 Δ9 and 17:1 Δ9. When wild type cells are fed 16:1 Δ9 and 18:2 Δ9, 12, fatty acid profiles revealed them present at a higher percentage than when fed other UFAs, probably because endogenous UFA production is more severely curtailed. The concentration of the fed fatty acid also impacted the regulation of OLE1, with higher concentrations bringing about more robust regulation. However, in nro1 cells, there was diminished regulation of expression in response to feeding certain UFAs as compared to wild type cells. Western blot analysis showed no evidence that Mga2p and Spt23p regulate OLE1 through the NRO1 signaling system. We conclude that the expression of OLE1 is dependent on properties of the fed fatty acid, the amount in the cell, and that the NRO1 gene product may be working through other regulatory networks besides Mga2p/Spt23p. Supported by an award from the National Science Foundation‐Research Experiences for Undergraduates.
Effect of gamma‐radiation and storage period on the erythrocyte membrane of the blood components
Authors: Sousa Leilismara , Maia Grazielle AS , Silveira Alan B , Cortes Vanessa F , Santos Hérica L , Barbosa Leandro A
Published: June 2021

The use of γ‐radiation in blood products causes T lymphocytes inactivation, which mediated Graft‐versus‐host disease, preventing their development. Thus, it is interesting to understand the structural changes induced by irradiation and storage period, to provide the high quality of blood components. The CPDA‐1 bags of red blood cells (RBC) were separated into two units, being one irradiated with 25 Gy. An aliquot was removed for the experiments under sterile conditions on days 3, 5, 7, 9, 11, 14, 21 and 28. The major phospholipids detected in both samples were phosphatidylcholine and phosphatidylethanolamine, and the radiation caused modification of amount of all phospholipids compared to control samples. We also observed alterations in relation to the storage time and irradiation of cholesterol and methemoglobin levels, but oxyhemoglobin level and hemoglobin structure did not show difference. The Na,K‐ATPase activity decreased in 13% on day 7 and 40% on day 28. The thiobarbituric acid reactive substances level increased with the storage time‐dependent. The results indicated that γ‐radiation on RBC and their storage induced a possible reorganization of the lipid membrane content, but it does not appear to be the cause of structural changes in hemoglobin. Together, these alterations probably modulate the activity of important proteins and ionic homeostasis of irradiated RBC. Supported: FAPEMIG, CNPq, CAPES.
Disruption of phospholipase B‐mediated turnover upon loss of Ypk1 in S. cerevisiae
Authors: Surlow Beth , Patton‐Vogt Jana
Published: June 2021

Ypk1, the yeast homolog of the human serum‐ and glucocorticoid‐induced kinase (Sgk1), is a serine/threonine protein kinase known to affect various downstream processes, including plasma membrane flippase activity and sphingolipid homeostasis. The results described here indicate that Ypk1 also impinges upon the turnover of phospholipids. Pulse‐chase experiments reveal that a ypk1 mutant exhibits alterations in phospholipid deacylation and resultant glycerophosphodiester production as compared to a wild type strain. Furthermore, loss of Plb1, a plasma membrane (PM) associated phospholipase, in a ypk1 background results in further alterations in phospholipid deacylation and a growth defect. We are in the process of examining potential explanations for the altered Plb1 activity in the ypk1 mutant. Strains in which other phospholipase B‐encoding genes are deleted in combination with YPK1 do not display a growth defect, suggesting a specific functional interaction between Plb1 and Ypk1. Preliminary studies have identified model PM proteins, including the Git1 permease, whose activity is affected by loss of Ypk1 and/or Plb1. Our data is consistent with the idea that Ypk1 and Plb1 alter plasma membrane phospholipid composition, which in turn impinges upon the activity of integral plasma membrane proteins.
Transport and Metabolism of glycerophosphocholine by Candida albicans: Role of CaGIT3, CaGIT4, and CaGDE1
Authors: Bishop Andrew C , Ganguly Shantanu , Cooley Ben , Mitchell Aaron , Patton‐Vogt Jana
Published: June 2021

Phospholipase B‐mediated turnover of phospholipids results in the production of glycerophosphodiesters. Candida albicans contains 4 ORFs (CaGIT1–4) predicted to be glycerophosphodiester transporters. Here we have identified CaGit3 and CaGit4 as glyerophosphocholione (GroPCho) transporters, with CaGit3 having the greatest transport activity. Deletion of CaGIT2–4 abolishes the cells ability to utilize GroPCho as a sole phosphate source during early growth (<12 hours after inoculation) and abolishes short term (2 min) transport of [3H]GroPCho. Upon reintegration of CaGIT3 (orf19.1979) and CaGIT4 (orf19.1980), both growth and transport of GroPCho is rescued, albeit minimally for CaGIT4. In addition, we have identified CaGDE1 (orf19.3936), as affecting the catabolism of internalized GroPCho, presumably by acting as a glycerophosphodiesterase. As is the case for CaGIT1, the transcriptional regulator, CaPHO4, regulates CaGIT3–4 expression. To our knowledge, this is the first evidence of a eukaryotic permease specific for GroPCho.
Quantitative Determination of the Alternative Isoprenoid Generation Pathway as a Tool to Exploit the Mevalonate pathway in Cancer Cells
Authors: Onono Fredrick , Subramanian Thangaiah , Sunkara Manjula , Subramanian Leela , Spielmann Peter , Morris Andrew J
Published: June 2021

Cancer stem cells and mutant p53 expressing tumors are highly dependent on the mevalonate pathway. Statins inhibit this pathway resulting in growth inhibition of these cells independent of inhibition of cholesterol biosynthesis and of protein farnesylation. Protein prenylation substrates farnesyl‐ and geranylgeranyl diphosphates are synthesized by the mevalonate pathway without producing their corresponding isoprenols. However, cells incorporate exogenously supplied isoprenols into mevalonate pathway metabolites and proteins using a poorly characterized process. In order to quantify this alternative to the mevalonate pathway, we synthesized natural and unnatural analogs of farnesol and geranylgeraniol, including stable isotope‐labeled farnesol and aniline substituted isoprenols. Using tandem mass spectrometry we quantitatively monitored the formation of diphosphate derivatives of these isoprenols and their utilization as prenylation substrates when incubated with cultured cells. Our results reveal that in some cells the capacity to use exogenous isoprenols is comparable to that of the mevalonate pathway raising the possibility that this alternative pathway could have important therapeutic implications