bluuebunny/crossref_metadata_2025 huggingface dataset:
4.0 Å Resolution Cryo‐EM Structure of the Mammalian Chaperonin TRiC/CCT Reveals its Unique Subunit Arrangement
Authors: Cong Yao , Baker Matthew L. , Jakana Joanita , Woolford David , Miller Erik J. , Reissmann Stefanie , Kumar Ramya N. , Redding‐Johanson Alyssa M. , Batth Tanveer S. , Mukhopadhyay Aindrila , Ludtke Steven J. , Frydman Judith , Chiu Wah
Published: June 2021

TRiC is a eukaryotic chaperonin essential for de novo folding of ~10% newly synthesized cytosolic proteins, many of which cannot be folded by other chaperones. This is likely linked to TRiC's unique subunit organization, whereby each ring consists of eight different subunits in an arrangement that remains uncertain. Using single particle cryo‐EM without imposing symmetry, we determined the mammalian TRiC structure at 4.7 Å resolution, which is the highest resolution asymmetric cryo‐EM structure to date. This revealed the existence of a two‐fold axis between its two rings. A subsequent two‐fold symmetrized map yielded a 4.0 Å resolution structure that evinces the densities of a large fraction of side‐chains, loops and insertions, which permitted unambiguous identification of all eight individual subunits. Independent biochemical near‐neighbor analysis supports our TRiC subunit arrangement. This allowed the optimization of a Cα backbone model of the entire TRiC complex from the homology models against the cryo‐EM density. A refined atomic model for one subunit showed ~95% of the dihedral angles in the allowable regions of the Ramachandran plot. Our model reveals an unevenly distributed positively charged wall lining the closed folding chamber of TRiC is strikingly different from those of prokaryotic and archaeal chaperonins. These interior surface chemical properties likely play an important role in TRiC's cellular substrate specificity.

Supported by Nanomedicine Development Center (PN1EY016525) and NCRR (P41RR02250).
ATS fusion human growth hormone has anti‐ageing effects on old mice
Authors: Lee Saet‐byul , Park Hyunju , Kim Jongsun
Published: June 2021

Protein aggregation is a major stability problem of therapeutic proteins. Previous studies showed that the ATS (acidic tail of synuclein)‐hGH (human growth hormone) fusion protein was more stable and soluble than wild type hGH. However, bioactivity of the protein was not confirmed in in vivo experiment. We executed a comparative study for hGH and ATS‐hGH in old mice. As shown in preliminary data, we verified that hGH and ATS‐hGH had bio‐activity in cell line experiment. Then, we executed in vivo animal studies, 30 μg/100 μl of hGH and ATS‐hGH were subcutaneously injected to 8 month female mice twice per a week for 14 weeks. As a result, mouse had no change in body weight. However, fat content of abdominal region and triglyceride levels in blood were more significantly reduced in hGH and ATS‐hGH group than those of control group. Most of all, fat content of abdominal region of ATS‐hGH was more reduced than hGH treated group. Also, creatinine was induced, but, urea nitrogen was not increased in blood of hGH and ATS‐hGH group. These results suggest that hGH and ATS‐hGH increased muscle capacity. In addition, immunohistochemistry result through PCNA stanning showed that proliferation of liver cell was increased in hGH and ATS‐hGH group. These result suggest that bioefficacy of ATS‐hGH is better than hGH, particulary effect in reducing the abdominal fat and in increasing the muscle content of old female mice.
Elucidation of the mechanism for TAO import in T.brucei mitochondria
Authors: Hamilton VaNae Nierra , Singha Ujjal , Sharma Shvetank , Chaudhuri Minu
Published: June 2021

TAO is the only terminal oxidase in the mitochondria of the bloodstream form of T.brucei. It is a nuclear encoded protein that needs to be imported in mitochondria to be functional. TAO possesses a predicted N‐terminal mitochondrial targeting sequence (MTS). Unlike other MTS containing proteins TAO does not need mitochondrial membrane potential (ψ) for its import in mitochondria of the bloodstream (BS) form but depends on ψ in the procyclic (PS) form of the parasite. To understand the import mechanisms of TAO in the two developmental forms, N‐terminal deletion mutants of this protein were generated. In vitro import of radiolabelled wild type and mutant TAO proteins into mitochondria from BS and PS forms has shown that deletion of the first ten amino acids did not affect the import of TAO in mitochondria from either form, whereas deletion of the first twenty amino acids inhibited import in mitochondria of the PS form only. Unlike the full‐length protein, the first ten amino acid deletion mutant of TAO depends on membrane potential for import in mitochondria of the BS form. These results suggest that length of and basic residues within the MTS of TAO play important roles in requirement of ψ during import.

This project is supported by NIH Grants 1SC1GM081146 and 1F31 AI083011‐01.
Towards understanding how lipid variations and membrane proteins may influence the biophysical chemistry of biomembranes
Authors: Heikal Ahmed A
Published: June 2021

Biomembranes in living cells are complex, heterogeneous and dynamic systems that regulate numerous biological processes such as cell signaling, endocytosis and exocytosis, and protein trafficking. Cholesterol‐rich lipid domains have been hypothesized to exist in a liquid‐ordered phase and play an important role in cellular functions. Yet, these direct observations of these lipid domains in the plasma membrane of living cell remain elusive. In addition, the relevance of model membranes to elucidate the structure‐function relationship of cellular membranes has been debated recently. Here, we test the hypothesis that cholesterol diffuses as a complex with other lipids in membranes, both model and natural ones. We also examine the role of lipid variation and proteins on the biophysical properties on biomembranes using comparative studies on giant unilamellar vesicles (GUVs) and plasma membrane vesicles (GPMVs) isolated from Hs578Bst living cells using newly developed Bodipy‐cholesterol derivatives. The fluorescence dynamics assay used here includes two‐photon fluorescence lifetime imaging, fluorescence resonance energy transfer, and fluorescence polarization as well as correlation spectroscopy. Our comparative studies on GUVs and GPMVs serve as a platform to test our understanding of lipid‐lipid and lipid‐protein interactions in the plasma membrane.
Sphingosine Kinase 2 and S1P in the Nucleus Regulate Histone Acetylation by Inhibition of Histone Deacetylases
Authors: Hait Nitai Chand , Allegood Jeremy , Maceyka Michael , Strub Graham M , Harikumar Kuzhuvelil B , Kordula Tomasz , Milstien Sheldon , Spiegel Sarah
Published: June 2021

Sphingosine‐1‐phosphate (S1P) is a potent sphingolipid mediator formed inside cells by phosphorylation of sphingosine catalyzed by two sphingosine kinase (SphK) isoenzymes, SphK1 and SphK2. We have now shown SphK2 is associated with nucleosomal histone H3 and regulates histone acetylation. S1P specifically binds to the histone deacetylases, HDAC1 and HDAC2, and inhibits their enzymatic activity preventing the removal of acetyl groups from lysine residues within histone tails. When S1P is added to isolated nuclei or SphK2 is overexpressed, increased acetylation occurs at the same histone lysines. SphK2 associates with HDAC1/2 in repressor complexes and is selectively enriched at the promoters of the genes encoding p21 and the transcriptional regulator c‐Fos, where it enhances local histone H3 acetylation and transcription of these genes. Further studies are underway to examine the specificity of the S1P effects. We are proposing that HDACs are direct intracellular targets of S1P, the first identified endogenous small molecule regulator of these enzymes, linking nuclear S1P to remodeling of chromatin and epigenetic regulation of gene expression.
Distinguishing Biochemical Activities of Splice Variants of Human FATP2
Authors: Melton Elaina Marie , Arias‐Barrau Elsa , Srinivasan Padmamalini , DiRusso Concetta C. , Black Paul N.
Published: June 2021

Fatty acid transport proteins (FATPs) represent a family of integral membrane proteins, which function in transport and activation of exogenous fatty acids (FA). Splice variants of human FATP2 have been identified that differ in the ATP binding region, which is required for adenylate formation. FATP2a contains the entire ATP/AMP motif while FATP2b lacks exon 3 resulting in a protein lacking critical residues within this motif. Using T‐REx 293 cells expressing FATP2a or FATP2b, we have [1] defined their individual roles in long chain FA transport and long and very long chain FA activation and [2] initiated studies addressing whether they direct specific classes of exogenous FA into downstream acyl CoA pools using stable isotopically labeled FA and high resolution mass spectroscopy. Our current data is consistent with the postulate that FATP2a activates exogenous very long‐chain FA via vectorial acylation. FATP2a is able to activate the very long chain FA, lignoceric acid (C24:0), while FATP2b is devoid of this activity. Expression of both FATP2a and FATP2b, however increase the import of the fluorescent long‐chain FA C1‐BODIPY‐C12, which indicates probable interaction with an unknown long chain acyl CoA synthetase. These findings are consistent with the hypothesis that different FATP isoforms function in the trafficking of specific classes of very long chain FA. [Supported by NIH grants: GM056840 and DK071076].
Genistein Regulates Ceramide Metabolism by Inducing Acid Ceramidase Expression in MCF‐7 Breast Cancer Cells
Authors: Lucki Natasha Chrystman , Sewer Marion B
Published: June 2021

Aberrant estrogen receptor (ERα) expression plays a role in the pathogenesis of most breast cancers. Genistein (gen) is a major isoflavone that stimulates tumor growth by inducing the transcription of many estradiol (E2)‐sensitive genes. Conversely, micromolar concentrations of gen inhibit cell growth and induce apoptosis. Ceramide (cer) and sphingosine‐1‐phosphate (S1P) have been extensively studied for their opposing roles in cancer pathogenesis. Cer inhibits cell growth and promotes apoptosis while S1P is a potent modulator of cell proliferation. Acid ceramidase (ASAH1) directly regulates the concentrations of these bioactive molecules by hydrolyzing cer to form sphingosine, which is phosphorylated into S1P. Based on the critical role that the cer/S1P rheostat plays in regulating cellular homeostasis, we hypothesized that gen modulates sphingolipid metabolism by controlling the expression of ASAH1. We show that 20 nM gen induces ASAH1 mRNA and protein expression by 4.2‐ and 2.3‐fold, respectively, concomitant with an increase in ceramidase activity. Reporter gene deletion studies localized the gen‐responsive region to within 500 bp of the transcription start site. In addition, EMSA and ChIP studies revealed that ERα binds to the gen‐responsive region of the ASAH1 promoter. Our results reveal a novel mechanism through which gen promotes cell proliferation, by activating cer metabolism.
Hepatic long‐chain acyl‐CoA synthetase 5 (ACSL5) partitions fatty acids between anabolic and catabolic pathways
Authors: Bu So Young , Mashek Douglas G
Published: June 2021

Long‐chain acyl‐CoA synthetases (ACSL) and fatty acid transport proteins (FATP) catalyze the initial step of fatty acid metabolism. Of numerous ACSL and FATP isoforms, ACSL5 is expressed predominantly in tissues with high rates of TAG synthesis suggesting it may have an anabolic role in lipid metabolism. To characterize the role of ACSL5 in hepatic energy metabolism, we knocked down ACSL5 by using siRNA in rat primary hepatocytes. Compared to cells transfected with control siRNA, knockdown of ACSL5 significantly decreased fatty acid or glucose induced lipid droplet formation observed by Oil‐red O staining. These findings were further extended with metabolic labeling studies showing that ACSL5 knockdown resulted in decreased [1‐14C] oleic acid or acetic acid incorporation into intracellular glycerolipids (30–50%) without altering fatty acid uptake. ACSL5 knockdown increased acid soluble metabolites (ASM), a measure of fatty acid β‐oxidation, without changing PPAR‐α activity and target gene expression indicating that the effect was not mediated by transcriptional regulation. These changes in fatty acid metabolism were similar during pulse and chase periods, which indicate that ACSL5 may not affect intracellular lipid turnover. These results suggest that ACSL5 has a unique role in channeling both exogenous and endogenous fatty acid towards glycerolipid synthesis rather than catabolic pathways.

This study was supported by American Diabetes Association.
Characterization of the human LPIN1‐encoded phosphatidate phosphatase isoforms
Authors: Han Gil‐Soo , Carman George M
Published: June 2021

The mammalian LPIN1 gene encodes the Mg2+‐dependent phosphatidate (PA) phosphatase, which catalyzes the dephosphorylation of PA to produce diacylglycerol (DAG), and its expression by alternative splicing produces the α and β isoforms. Here, we found a novel splice form (γ) of human lipin 1, which differs from the α form by containing additional 26 amino acids. In addition, we characterized the enzymological properties of human LPIN1α, β, and γ‐encoded PA phosphatases purified after heterologous expression in Escherichia coli. The LPIN1‐encoded PA phosphatase enzymes followed surface dilution kinetics with respect to PA using Triton X‐100/PA mixed micelles. These enzymes had similar Km values (4.2–4.5 mol %) for PA, but their Vmax values were in the order of α (34.9 μmol/min/mg) > β (24.9 μmol/min/mg) >> γ (3.4 μmol/min/mg). Although the lipin 1 isoforms showed a significant difference in catalytic activity, they were similar with respect to reaction conditions for optimum activity, inhibitors, and effectors. Supported by NIH grant GM‐28140.
THE LINKING OF BETA‐OXIDATION TO CARDIOLIPIN REMODELING
Authors: Hatch Grant M , Taylor William A
Published: June 2021

We previously identified a human linoleoyl‐Coenzyme A monolysocardiolipin (MLCL) acyltransferase activity (MLCL AT‐1) (Taylor and Hatch 2009 J. Biol. Chem. 284: 30360‐30371). This 59 kDa human protein was identical to the 74 kDa human α‐subunit of trifunctional protein (αTFP) minus the first 237 amino acids. Here we characterize the MLCL AT activity of αTFP. The 74 kDa αTFP exhibited MLCL AT activity and western blot analysis under non‐denaturing conditions revealed the presence of a 150 kDa protein indicative of the dimeric form of αTFP. NAD stimulated the MLCL AT activity of αTFP. Recombinant human αTFP exhibited specificity for linoleoyl‐CoA in the acylation of MLCL to cardiolipin (CL). Expression of αTFP increased MLCL AT activity and [1‐14C]linoleate incorporation into CL in Hela cells, whereas, RNAi knock down had the opposite effect. RNAi knock down of the β‐subunit of trifunctional protein increased MLCL AT activity indicating that αTFP has MLCL AT activity and the β‐subunit is a negative regulator of activity. Thyroxine‐treatment of rats, which increases MLCL AT activity, increased αTFP mRNA expression compared to euthyroid controls, whereas, propylthiouracil‐treatment, which reduced MLCL AT activity, had the opposite effect. The results clearly indicate that the 74 kDa human αTFP has MLCL AT activity and thus linking an enzyme of CL remodeling with β‐oxidation.
A novel microdissection model for probing cellular interactions. I. alpha methyl mannose blocks the cellular interaction
Authors: Coyle‐Thompson Catherine , Askari Tina , Mkrtchian Mary , Portillo Georgina , Manukyan Mariam , Abramian Sarineh , Qubrosi Mirey , Makhoul Elias , Frederick Robert , Allen Jenieke , Dreyfuss Justin , Oppenheimer Steven B.
Published: June 2021

Adhesion between the archenteron tip and blastocoel roof in the NIH designated sea urchin embryo model is a cellular interaction that has interested investigators for over a century, but its molecular basis is not understood. Here we microdissect the two components of this cellular interaction and use alpha methyl mannose to map molecular groups that may be involved in mediating the cellular interaction. Whole 48–54 hour fixed Lytechinus pictus embryos were washed three times and then dissected in pH 8.0 artificial sea water (ASW), 15°C. The roofs of the blastocoel and the tips of the archenteron were put together and observed to stick together. In over 50 separate trials using bound sets of roof and archenterons attached together, either 0.2 M or 1.0 M alpha methyl mannose was added to the pieces. The pieces remained together. However in 100% of the cases, manually separating the pieces in alpha methyl mannose at either molarity resulted in blocking the rebinding of the blastocoel roofs and archenteron tips.

Re‐binding occurred in the absence of the sugar. The results suggest that alpha methyl mannose binding receptors are involved in mediating the cellular interaction. These studies offer a novel approach to map glycans and glycan binding partners that may be functionally important. By microdissecting the components of cellular interactions out of the embryo proper, these components can be probed in pristine media away from factors in intact embryos that could confuse results (Supported by NIH NIGMS SCORE S0648680, MARC, RISE, the Joseph Drown Foundation, the Sidney Stern Memorial Trust, and CSU Northridge Biology Full Immersion Research Experience (FIRE) course funding).
Localization of human sperm PMCA4b which is required for normal motility in mouse sperm
Authors: Shelly Katharine , Aravindan Rolands , Martin‐DeLeon Patricia
Published: June 2021

Progressive and hyperactivated motility in sperm are both dependent on Ca2+ and ATP to effect fertilization, with hyperactivated motility utilizing more ATP than progressive motility. The ATP‐driven enzyme Plasma Membrane Calcium/calmodulin‐dependent ATPase isoform 4b (PMCA4b) is the principal calcium efflux pump in mouse sperm, and its absence results in the loss of hyperactivated motility and fertility. Thus, PMCA4b plays an important role in murine sperm function. Protein BLAST analysis illustrates 84% identity and 91% similarity between the mouse and human PMCA4b sequences. Analysis of the functional domains suggests conservation of PMCA4b's activity in human sperm. The objective of this investigation is to characterize the presence of PMCA4b in human sperm by Western blotting, as well as to determine its subcellular localization by immunocytochemistry (ICC). Flow cytometric analysis serves to confirm the presence of PMCA4b on the membrane. ICC studies should elucidate whether or not localization of human PMCA4b is present on the principal piece of the flagellum, which would coincide with findings in the mouse and suggest its involvement in human sperm motility. This work was supported by the Charles Peter White Fellowship and the NIH‐COBRE grant #5P20RR015588‐07.
Fluoresceination of FepA during colicin B killing: effects of temperature, toxin and TonB
Authors: Smallwood Chuck Randall , Marco Amparo Gala , Xiao Qiaobin , Trinh Vy , Newton Salete M. C. , Klebba Phillip E.
Published: June 2021

We studied the reactivity of 35 genetically engineered Cys sulphydryl groups at different locations in Escherichia coli FepA. Modification of surface loop residues by fluorescein maleimide (FM) was strongly temperature‐dependent in vivo, whereas reactivity at other sites was much less affected. Control reactions with bovine serum albumin showed that the temperature dependence of loop residue reactivity was unusually high, indicating that conformational changes in multiple loops (L2, L3, L4, L5, L7, L8, L10) transform the receptor to a more accessible form at 37 degrees C. At 0 degrees C colicin B binding impaired or blocked labelling at 8 of 10 surface loop sites, presumably by steric hindrance. Overall, colicin B adsorption decreased the reactivity of more than half of the 35 sites, in both the N‐ and C‐ domains of FepA. However, colicin B penetration into the cell at 37 degrees C did not augment the chemical modification of any residues in FepA. The FM modification patterns were similarly unaffected by the tonB locus. We did not detect TonB‐dependent structural changes in FepA, either alone or when it interacted with colicin B at 37 degrees C. The only changes in chemical modification were reductions from steric hindrance when the bacteriocin bound to the receptor protein. The absence of increases in the reactivity of N‐domain residues argues against the idea that the colicin B polypeptide traverses the FepA channel.
Design of a Three Dimensional Physical Model of P‐glycoprotein
Authors:   Lincoln SMART Team, Colton Shannon , Herman Tim , Giles Kurt , Koo Ben , Jeske Sabine , Grover Anita , Gin Richard
Published: June 2021

Expressed in a large variety of tissues, the transporter P‐glycoprotein (P‐gp) facilitates small molecule efflux from cells as a detoxification mechanism for a remarkable range of drug substrates. Of interest to clinicians and drug development scientists is the number of drug‐drug interactions mediated by competitive inhibition of the protein. Similarly, as it is highly expressed in enterocytes, P‐gp can be a significant barrier to drug absorption, and, therefore, considerable effort has been spent on the discovery of a non‐absorbed P‐gp inhibitor. Traditional methods for visualizing protein structures and interactions with small molecules are limited in the amount of information that can be conveyed in two dimensions. Using rapid prototyping technology, we have built tangible 3D models of P‐gp using the recently resolved structure (PDB: 3g5u), allowing students and scientists alike to more fully explore the transporter protein and its small molecule substrates and inhibitors.

The SMART (Students Modeling A Research Topic) program was established by theCenter of BioMolecular Modeling at the Milwaukee School of Engineering and involves partnerships between university researchers and local high schools. This work is funded by NIH‐NCRR‐SEPA and HHMI.
Role of PDZ proteins in the differential regulation of the proximal tubule NaPi transporters
Authors: Giral Hector , Caldas Yupanqui , Lanzanó Luca , Gratton Enrico , Sutherland Eileen , Blaine Judith , Okamura Kayo , Verlander Jill , Levi Moshe
Published: June 2021

The Na‐dependent phosphate (NaPi) transporters NaPi‐2a and NaPi‐2c play a major role in the renal reabsorption of Pi and maintenance of Pi homeostasis. The transporters show differential regulation under dietary and hormonal stimuli suggesting different regulatory pathways controlling the endocytosis, stability, or functionality of the transporters. The scaffolding NHERF family of PDZ proteins has been involved in the regulation of NaPi‐2a and NaPi‐2c. We propose that differences in the molecular interaction with these PDZ proteins are related with the differential adaptation of the transporters. We studied the specific interaction of NaPi‐2a and NaPi‐2c with NHERF‐1 and −3 in the OKP cells by FLIM‐FRET lifetime measurements. Results showed a direct interaction of NaPi‐2a with NHERF‐1 (FRET occurrence), while there was no interaction between NaPi‐2c and NHERF‐1. In contrast NaPi‐2c showed significant FRET with NHERF‐3 protein. According with these results, adaptation of the NHERF‐3 KO mouse to chronic low Pi diets was impaired in the case of NaPi‐2c but not in NaPi‐2a. In response to a low Pi diet NHERF‐3 KO mice showed robust upregulation of NaPi‐2a but decreased adaptive response of NaPi‐2c. These results indicate that differential affinity of the NaPi transporters for NHERF‐1 and −3 proteins could partially explain their differential regulation. Interaction between NaPi‐2c and NHERF‐3 seems to play an important role in the physiological regulation of NaPi‐2c.
Hybrid lactoferrins containing transferrin C1 or C2 subdomains bind asialoglycoprotein receptors
Authors: McAbee Douglas D. , Seth Sid , Jung Chun‐Ling , Wong Henry , Schryvers Anthony
Published: June 2021

The Fe‐binding proteins lactoferrin (Lf) and transferrin (Tf) are structurally similar, each Fe‐binding lobe (N, C) composed of two sub‐domains (N1/N2; C1/C2). Lf, but not Tf, binds the major subunit of the asialoglycoprotein receptor (RHL1) in a galactose‐independent manner. To identify C‐lobe regions involved in RHL1 binding, we generated recombinant Lf‐Tf hybrid proteins in which Tf C1 or C2 subdomains were positioned within a Lf background. cDNAs of human Lf and Tf encoding Lf:TfC1 and Lf:TfC2 were constructed and expressed in Sf9 insect cells. Binding constants of purified native and recombinant Lfs for RHL1 were determined by surface plasmon resonance. Binding of desialylated ligands and native and recombinant Lf proteins to immobilized RHL1 required Ca2+ and neutral pH. Tf did not bind RHL1 under any conditions. Average dissociation constants measured for the various proteins were as follows: native Lf: 138 ± 44 nM; r‐Lf: 87 ± 50 nM; Lf:TfC1: 31 ± 28 nM; Lf:TfC2: 150 ± 76 nM; and asialofetuin: 21 ± 16 nM. Binding constants for native and recombinant Lfs did not differ statistically. Thus, Tf C1 or C2 sub‐domains in a Lf background showed no loss of RHL1‐binding activity. We conclude that the RHL1 binding determinants on Lf do not reside solely in one of the C‐lobe subdomains. (Support: Research Corporation, NIH DK‐61984).
Dynamics of Protein Complexes
Authors: Washburn Michael
Published: June 2021

Protein complexes may change their stoichiometry, content, or modifications based on stimuli like changes in the cell cycle or due to a drug treatment. In addition, depending on which protein in a complex is used as a bait protein for affinity purification, different forms of a protein complex may be detected and identified. These changes in complex content and form have functional implications. Carrying out studies regarding the dynamics of protein complexes has required the development of advanced quantitative proteomics technologies. We have refined the proteomics approach named multidimensional protein identification technology (MudPIT) and developed the label free quantitative proteomics approach termed the normalized spectral abundance factor (NSAF) for the analysis of multiprotein complexes. With a primary focus on transcriptional regulatory complexes, we have applied these approaches to the study of the Rpd3 histone deacetylase chromatin remodeling complexes, Mediator, and RNA Polymerase II. This presentation will describe the technology we use to characterize these complexes and will demonstrate the changes in these complexes that occur depending on the cellular context.
Investigating The Impact of Two Functional Polymorphisms of Lipoprotein Lipase (LPL) Gene In the Occurrence of Hypertension
Authors: Khan Mariam , Al‐Lahham Yara , Salah Amna , Habibullah Samar , Esmail Najma
Published: June 2021

Background

Lipoprotein lipase (LPL) is a rate limiting enzyme responsible for the hydrolysis of triacylglycerol‐rich lipoproteins releasing monoglycerides and free fatty acids, which are taken up by skeletal muscles, adipose tissue.

Aim

To investigate the effect of genetic polymorphisms of LPL S447X and Hind III on blood pressure and its related phenotype.

Methods

S447X variant genotype and Hind III (+) variant of LPL were determined by polymerase reaction restriction fragment length polymorphism (PCR‐RFLP) assay in 50 hypertensive patients and 50 normotensive as a control group. Anthropometric measurements and serum lipoproteins were also determined in both groups. Analysis of data was done by using SPSS program.

Results

The frequency of (SS) and (H+/H+) genotype were 78% and 62% respectively in the hypertensive group compared to 66% and 38% in normotensive group. Carrier of either (SS) or (H+/H+) genotype were at higher risk of developing hypertension (OR=1.8, 95% CI=0.8–4.4) and (OR=2.4, 95% CI =1.1–5.5) respectively when compared with carrier of other genotypes. The risk for hypertension was increased when the individuals were carrier of (SS) and (H+/H+) haplotype (OR = 6 and 95% CI =2.5–14.2). Furthermore carrier of either (SS) or (H+/H+) genotype were at higher risk of developing atherogenic phenotype manifested by central obesity and/or abdominal obesity and dyslipdemia.

Conclusion

It was found that carriers of (SS) genotype and/or (H+H+) genotype were at high risk of developing hypertension and its related atherogenic phenotypes.

Source of research support: Dubai Medical College for Girls
A Novel Mechanism for 13‐cis Retinoic Acid mediated Angiotesin Type 1 Receptor Downregulation
Authors: Snyder Russell Odell , Thekkumkara Thomas
Published: June 2021

Transcriptional repression through cis and trans acting factors enabling an alternate approach to control of angiotensin type 1 receptor (AT1R) dysfunction has not been studied. We hypothesized that in liver cells 13‐cis‐retinoic acid (13cRA), an antioxidant, by PPARγ; activation enhances insulin sensitive glucose mediated downregulation of the AT1R. In this study, we demonstrated that 13cRA treatment (25μM) downregulated the AT1R protein (40%) and mRNA (40%) in a dose and time dependent manner. However, the AT1R downregulation was independent of insulin sensitive glucose uptake. Reporter gene assays show that the promoter activity was inhibited by 13cRA. Sequence analysis revealed a region 2766 bp upstream of the start site similar to the retinoic acid response element in other genes. Studies using MAP‐Kinase inhibitor PD 98059 prevented 13cRA mediated AT1R downregulation. Furthermore, treatment of Trichostatin A reversed 13cRA mediated AT1R repression, suggesting that the catalytic and nuclear bioavailability of histone deacetylases may play a role in 13cRA induced AT1R repression. In summary, our results indicate for the first time that 13cRA has a glucose independent mechanism for transcriptional inhibition of AT1R. The observed effects by 13cRA suggest its therapeutic potential as an anti‐hypertensive agent in nondiabetic as well as diabetic hypertension. (Supported by NIH Grant DK072140)
Acrolein Inhalation Suppresses Recruitment and Mobilization of Endothelial Progenitor Cells
Authors: Wheat Laura Anne , Hellmann Jason , Habertzettl Petra , Conklin Daniel J. , O'Toole Timothy E. , Bhatnagar Aruni
Published: June 2021

Exposure to cigarette smoke decreases the level of endothelial progenitor cells (EPCs) in peripheral blood (PB). We investigated the cardiovascular effects of inhaled acrolein, an unsaturated aldehyde present in high levels in tobacco smoke (up to 150 ppm). Adult male C57BL/6 mice were exposed to room air or acrolein (0.5, 1 or 5 ppm) for 2h, 6h, 1d, 2d or 4d (6h/d) and the EPC level in PB was measured by flow cytometry. To examine the proliferative capacity of EPCs, bone marrow cells (BMCs) were cultured on fibronectin‐coated slides. Brief exposure to acrolein (5 ppm, 6h) or 4 day (1 ppm) exposure led to a 42% decrease in PB Flk+/Sca+cells (air‐exposed: 8.9±1.7/μl, n=4; acrolein‐exposed: 4.9±0.5/μl, n=4, p<0.05). BMCs isolated from mice exposed to acrolein for 1d (5 ppm) or 4d (1 ppm) formed more colonies than BMCs of air‐exposed mice (air‐exposed: 7±1 colonies; acrolein 14±1 colonies at 1d in culture, p<0.001; air‐exposed 3±0.47 colonies; acrolein‐exposed 9±2 colonies after 4 days in culture, p<0.001). Combined VEGF‐A (100 μg/kg, ip, 4d) and AMD3100 (5 mg/kg; ip, last day only) treatment increased PB Flk+/Sca+ cells (air‐exposed: 5.7/μ1±1.8, n=4; acrolein‐exposed: 11.06/μl±2.2, n=4, p<0.05) in air‐exposed mice but not in acrolein‐exposed mice (1 ppm, 4d). These data indicate that PB EPCs are a sensitive target of airborne pollutants and that acrolein at concentrations encountered in tobacco smoke suppresses PB EPC level yet increases the proliferative capacity of BMCs. Acrolein‐induced depletion of circulating EPCs appears in part to be due to a defect in EPC mobilization and recruitment.

COBRA: P20RR024489; PPG: P01ES011860
Mst3 plays an important role in the hypoxia‐induced apoptosis of human trophoblasts
Authors: Wu Hung‐Yi , Lin Chia‐Ying , Pan Shien‐Tung , Yuan Chiun‐Jye
Published: June 2021

Mammalian Ste20‐like protein kinase 3 (Mst3), a key player in the apoptosis of cell lines, was recently found to be highly expressed in human trophoblast in response to hypoxia. Mst3 is postulated to be activated by hypoxia and to trigger the apoptosis of human trophoblasts. In this study, this hypothesis was confirmed by the observation that hypoxia‐induced apoptosis of 3A‐sub‐E, a human trophoblast cell line, was markedly suppressed by selective knockdown of Mst3. Hypoxia was shown to induce the expression of Mst3 and the subsequent apoptotic events via the activation of nitric oxide synthase (NOS). The hypoxia‐induced Mst3 expression and the apoptosis in human trophoblasts were markedly suppressed by the inhibitor of NOS. Oxidative stress was demonstrated to be induced by NOS and to participate in the upregulation of Mst3. The hypoxia‐induced apoptosis of trophoblasts can be effectively suppressed by DL‐£\‐lipoic acid, a ROS scavenger, and apocynin, a specific inhibitor for NADPH oxidase. C‐Jun N‐terminal kinase‐1 was shown to be activated by oxidative stress and induce the upregulation of Mst3. In conclusion, Mst3 was shown to play a role in the apoptosis of placental trophoblasts in response to hypoxia. Oxidative stress was found to be elevated by hypoxia, which further induced the expression of Mst3 that triggers trophoblast apoptosis.
Calcium hypochlorite induces apoptosis in cultured human skin keratinocytes
Authors: Sarkisian Simon Antranik , Parisi Joseph , Fredette Rebekah , Lu Shan , Wan Yinsheng
Published: June 2021

Calcium hypochlorite compositions are primarily used for disinfectant and general sanitation purposes in swimming pools. It has not been shown that calcium hypochlorite induces apoptosis in human skin cells. Therefore, we undertook this study to investigate whether and how calcium hypochlorite induces skin cell apoptosis using human skin keratinocytes cell line HaCaT cells. After treatment of HaCaT cells with different doses of calcium hypochlorite (Pulsar® Plus Briquettes), cell proliferation was measured using an MTT Dye assay. Detection of specific proteins from apoptosis pathways were analyzed using Western blot at different time intervals. Increased calcium hypochlorite concentrations lead to increased apoptosis in cultured HaCaT cells. It is also shown that prolonged exposure to calcium hypochlorite result in an increased rate of apoptosis. Although it is known that calcium hypochlorite is an effective disinfectant in swimming pools, little is known about its effects on human skin cells. Our results thus suggest that prolonged exposure at specific concentrations of calcium hypochlorite lead to varying rates of apoptosis in human keratinocytes.
Effects of Folic acid on stem cell proliferation and differentiation derived from posterior neural tube of Pax3 mutant (Splotch) mouse
Authors: Ichi Shunsuke , Shen Yueh‐Wei , Boshnjaku Vanda , Mania‐Farnell Barbara , Tomita Tadanori , McLone David G , Mayanil Chandra S.K
Published: June 2021

Periconceptional maternal folic acid (FA) supplementation has been shown to be extremely effective in reducing the occurrence of neural tube defects (NTDs). However, little is known about the anti‐teratogenic mechanism of FA in neural tube anomalies. The proliferation or differentiation of neural stem cells (NSCs) can be influenced by various maternal factors, leading to alteration of the cell‐fate of the neural crest. Here we try to elucidate how folic acid can influence on stem cell proliferation and differentiation in Pax3 mutant (Splotch) mouse embryo. By using neurosphere culture derived from posterior neural tube of E10.5 mouse embryos, wild type (WT) and Splotch homozygote (Sp−/−), we observed colony forming ability that could be extremely increased by folic acid treatment on Sp−/− that caused neural tube defect in 100% whereas only few colonies could be seen without treatment. Those rescued sphere colonies showed definite immunoreactivity to stem cell, e.g. Oct3/4, Sox2, CD133, nestin and alkaline phosphatase suggesting they were pluripotent progenitor‐like cell. After plating those spheres on Matrigel, cells that migrated out from spheres and differentiated could express GFAP, Brn3a, β‐‡V‐tubulin, O4 and synaptogenesis markers, e.g. Snap25 or synaptotagmin. Thus this study suggested that folic acid could play an important role on stem cell proliferation and differentiation.
Novel Gene C17orf37 as Therapeutic Target in Breast Cancer
Authors: Kpetemey Marilyne Fleurrete , Dasgupta Subhamoy , Vishwanatha Jamboor
Published: June 2021

Breast cancer is the most common type of cancer in women and the second most common cause of cancer death in women in the U.S. The National Cancer Institute estimates that about 40,170 women will die from breast cancer in 2009. While most diagnosed women survive the primary tumor, mortality rates remain extremely high due to disease metastasis. In breast cancer patients’ Her‐2/neu oncogene is frequently amplified along with neighboring genes in same amplicon. Although antibody‐based Herceptin therapy specifically targeting Her‐2/neu has been an encouraging advance in targeted treatments of solid tumors, the numbers of patients responsive to such treatment are however few. Novel gene C17orf37 which is co‐ amplified with Her‐2/neu has been found to be significantly high in breast cancer patients compared to normal breast cancer. C17orf37 expression also correlates positively with grade and stage of cancer. This suggests that C17orf37 may be an important mediator of cancer cell metastasis in breast cancer patients particularly in Herceptin resistance patients. We hypothesize that the over expression of C17orf37 contributes to the development of breast cancer and regulates its progression to more invasive phase. Our currents studies indicate C17orf37 plays a crucial role in breast cancer cell proliferation and may be an important therapeutic target either alone or in combination with Herceptin.
Runx2 regulates diverse gene expression programs to (de‐) sensitize the osteogenic and/or mitogenic responses of osteoblast progenitors and osteosarcoma cells
Authors: Teplyuk Nadiya M. , van der Deen Margaretha , Galindo Mario , Teplyuk Viktor I. , Stein Janet L. , Lian Jane B. , Stein Gary S. , van Wijnen Andre J.
Published: June 2021

The osteogenic and cell cycle regulatory functions of Runt‐related transcription factor 2 (Runx2) are perturbed in osteosarcoma cells. We investigated which target genes of Runx2 control proliferation of normal osteoblasts and osteosarcoma cells by Affymetrix expression profiling and ChIP‐on‐chip analysis using NimbleGen arrays. Target gene analysis shows that Runx2 modulates normal osteoblast proliferation by regulating distinct programs of genes involved in G protein‐coupled receptor signaling, sterol/steroid metabolism, the fibroblast growth factor/proteoglycan signaling axis, RNA helicases and heat shock proteins. Strikingly, our data reveal that the gene expression programs controlled by Runx2 in osteoprogenitors are fundamentally different from those in osteosarcomas. Thus cancer‐related perturbations in normal osteoblast growth and differentiation switch the biological function of Runx2 from a growth suppressor to a putative oncoprotein that supports the tumorigenic and/or metastatic potential of osteosarcoma cells.
Maturation and Effects of In‐Utero Brain Ischemia on IL‐1 and IL‐6 Protein Expression in Ovine White Matter and Cerebral Cortex
Authors: Sadowska Grazyna Barbara , Threlkeld Steven W , Flangini Alexia , Surendra Sharma , Stonestreet Barbara S
Published: June 2021

We examined the effects of maturation and ischemia‐reperfusion (IR) injury on changes in IL‐1β and IL‐6 protein expression in the ovine brain.

Ovine fetuses were studied after 30 min of bilateral carotid artery occlusion and 72 h of reperfusion. Fetal and maternal control groups had no ischemia. IL‐1β and IL‐6 protein expression was compared between cerebral cortex (CC) and white mater (WM) in the same study groups using Western immunoblot.

IR resulted in a 26% decrease in IL‐1β in CC and a 173% increase in WM as compared to the control fetuses. IL‐1β was 34% higher in WM than CC after IR. IL‐6 expression did not change in CC or WM after ischemia. IL‐1β was 31% higher in maternal than fetal control CC and 61% higher in maternal than fetal control WM. IL‐1β was 59% higher in fetal control CC than WM and 25% higher in maternal CC than WM. IL‐6 expression was significantly higher in maternal CC as compared to fetal control; there were no differences between fetal control and maternal WM. However, IL‐6 was 36% higher in control fetal WM compared to CC.

IR results in down regulation of IL‐1β expression in fetal CC and up regulation in WM, and does not affect IL‐6 expression in either region. There is a differential response between IL‐1β and IL‐6 after ischemia in fetal CC and WM. There are also regional differences in IL‐1β and IL‐6 expression in normal fetal and maternal brain.
C/EBP beta and delta act as downstream targets of High Mobility Group Box 1 signaling
Authors: Mccauslin Christine Seitz , Abu‐Bonsrah Nancy A. , Senseney Erica L. , Staiger Jennifer L.
Published: June 2021

The inflammatory response represents a significant component of brain related disease responsible for neuronal degeneration. However, in some cases, inflammation is also a necessary first step during innate immune responses involved in mediating tissue repair. HMGB‐1 is a nuclear, non‐histone, DNA binding protein that is normally involved in chromatin stabilization and transcriptional regulation. It also has a well established role in mediating pro‐inflammatory events following passive release from necrotic neurons and glia. Similarly, C/EBPβ and C/EBPδ are nuclear transcription factors known to promote the expression of pro‐inflammatory genes following ischemic insult; and, both C/EBP β and C/EBP δ have been shown to mediate tissue repair following inflammatory stimuli in various cell types. Here, we report the results of our investigations into defining the role of C/EBP proteins in mediating the downstream signaling events that promote inflammation in response to passive HMGB‐1 release. Primary neurons and glia, subjected to hypoxia, were examined by Western analysis to determine the levels of C/EBPβ and C/EBPδ. In response to hypoxia, both C/EBP proteins were observed to be upregulated. In addition, cells treated with purified HMGB1 protein also showed an increase in C/EBP expression. Taken together, these data suggest that C/EBP proteins may function as downstream targets of HMGB1 signaling to promote HMGB1 mediated cellular responses to tissue injury.
Kinase Inhibitor Specificity and Targeting Human Disease
Authors: English Jessie M
Published: June 2021

Because of their central role in cell signaling, protein kinases are often co‐opted and aberrantly activated in human disease. The ATP binding site of protein kinases affords an attractive small molecule binding site, but is highly conserved across the kinome. In the last decade protein kinases have been established as attractive therapeutic targets. However, the above two features also present challenges for targeting protein kinases with small molecule inhibitors. Obtaining small molecule inhibitors that are highly selective against the kinome can be difficult, but progress has been made. However dialing in or out desired specificity profiles is still problematic. In addition predicting the implications of kinase inhibition on both efficacy and safety can be difficult due to the pleitropic nature of kinase signaling. Kinase inhibitors currently in clinical trials and on the market vary significantly in their selectivity profiles. This presentation will provide an overview of approaches for identifying selective kinase inhibitors as well as the implications for drug discovery efforts and therapeutic utility of highly selective vs. multikinase inhibitors.
Regulation of cell adhesion and motility: a novel function of arrestin proteins
Authors: Cleghorn Whitney , Bulus Nada , Chen Dong , Zhang Xi , Zent Roy , Gurevich Vsevolod
Published: June 2021

Arrestins bind G protein‐coupled receptors and more than 100 non‐receptor partners, regulating various signaling pathways and cellular functions. The interactions of many proteins (e.g., Src, JNK3, ERK½, Mdm2, etc.) with receptor‐bound arrestin localize these molecules to receptor‐rich membranes. Our recent finding that arrestins bind microtubules and recruit signaling proteins to the cytoskeleton prompted us to investigate whether arrestins affect cell adhesion, motility, and morphology. Here we show that arrestin double knock‐out (DKO) and arrestin2 knock‐out (A2 KO) cells stained with Rhodamine‐Phalloidin look drastically different than arrestin3 knock‐out (A3 KO) and wild type (WT) cells. DKO cells appear to spread in an integrin‐independent manner, in contrast to its single knock‐out and WT counterparts. Additionally, DKO cells show a significant deficit in cell adhesion to extracellular matrix protein fibronectin, whereas A2 KO cells show a dramatic increase. WT and A3 KO cells are similar. DKO cells have dramatically decreased active Rho compared to A3 KO and WT cells. Thus, arrestins differentially regulate cell adhesion and spreading.

NIH Grants GM77561, GM081756 (VVG), VA Merit Award (RZ), training grant EY07135 (WMC).
Gene‐orientation‐dependent DNA methylation patterns in the mouse prolactin (Prl) family gene cluster locus
Authors: Hayakawa Koji , Hattori Naoko , Nakanishi Momo , Ohgane Jun , Tanaka Satoshi , Yagi Shintaro , Shiota Kunio
Published: June 2021

DNA methylation is an epigenetic modification involved in gene silencing and genome stability. In mice, Prl family genes form gene cluster in about 1‐Mbp region. The Prl family genes other than Prl can be divided into two groups based on their expression patterns. One group is expressed only in placenta (group I), and the other group is expressed in placenta and a few other tissues (group II). This suggests that each group has gained specific regulation system for expression pattern. In our previous study, rat PL‐I, a member of Prl family, has been shown to have a tissue‐dependent and differentially methylated region (T‐DMR) hypomethylated in placenta. To explore epigenetic regulation of all the Prl family genes, we analyzed methylation status of them in placenta, liver and brain. We found that all the Prl family genes have T‐DMRs. Moreover, group I genes tended to have T‐DMRs hypomethylated specifically in placenta, and group II genes tended to be hypomethylated in placenta but showed variable methylation level in other tissues. Two groups of DNA methylation patterns were associated with the gene orientation in the cluster. The present study suggests that tissue‐specific expression patterns of Prl family genes are regulated by DNA methylation in association with the gene orientaion.

This work was supported by KAKENHI (21248039) from MEXT, japan.