bluuebunny/crossref_metadata_2025 huggingface dataset:
Unique mechanism of nitrile hydratase formation
Authors: Kobayashi Michihiko , Zhou Zhemin , Cui Tianwei , Shiraki Kentaro , Hashimoto Yoshiteru
Published: June 2021

We have studied microbial degradation of nitrile (R‐CN). Nitrile hydratase (NHase) catalyzes hydration of nitrile to amide. Rhodococcus rhodochrous J1 produces two NHases, depending on an inducer in the presence of cobalt; when it is cultured in the medium containing urea and cyclohexanecarboxamide, high‐ and low‐molecular‐mass NHases (H‐NHase and L‐NHase) are selectively induced, respectively. In both NHases, Co acts as an active center; but, the incorporation mechanism of Co into them remains unclear. Here, we discovered a novel post‐translational maturation in NHase. An open reading frame, nhlE, just downstream of L‐NHase gene (nhlBA), was found to be necessary for L‐NHase activation; nhlG, just downstream of H‐NHase gene (nhhBA), was also essential for H‐NHase activation. In contrast to the Co‐containing L‐NHase (holo‐L‐NHase containing Cys‐SO2− and Cys‐SO− metal ligands) derived from nhlBAE, the gene products derived from nhlBA were cobalt‐free L‐NHase (apo‐L‐NHase lacking oxidized Cys). We discovered an L‐NHase maturation mediator, NhlAE, consisting of NhlE and the Co‐ and oxidized Cys‐containing α‐subunit of L‐NHase. SDS‐PAGE and enzyme assay with site‐directed mutagenesis for the apo‐L‐NHases revealed that the incorporation of Co into L‐NHase was dependent on the exchange of the non‐modified Co‐free α‐subunit of Co‐free L‐NHase with the Co‐containing Cys‐modified α‐subunit of NhlAE.
REDOX PROPERTIES OF ARSENATE REDUCTASE FROM SYNECHOCYSTIS SP. PCC6803
Authors: Shipley Sheena LaShae , Kim Sang Gon , Kim Sung‐Kun
Published: June 2021

In the environment, arsenate is commonly encountered at biologically deleterious concentrations. Microbes combat the collateral importation of arsenate by a two‐step mechanism: first, arsenate reductase reduces the compound to arsenite, which the cell subsequently exports via an inducible, transmembrane transporter. The redox properties of the arsenate reductase and a glutaredoxin from the cyanobacterium Synechocystis sp. PCC6803 have been investigated. Using mBBr (monobromobimane) and AMS (4‐acetamido‐4′‐maleimidylstibene‐2,2′‐disulfonic acid), redox titrations indicated one active dithiol/disulfide coupling with midpoint potentials of ‐140 mV for the arsenate reductase and ‐160 mV for the glutaredoxin. The interaction between arsenate reductase and glutaredoxin are also observed by using gel filtration. These findings suggest that glutaredoxin's donation of an electron pair to the arsenate reductase is a thermodynamically favorable redox reaction. As such, glutaredoxin's reduction capabilities are essential for the activation and proper functioning of arsenate reductase, which in turn is critical for cellular survival.
IDENTIFICATION OF PROTEINS OF VIGNA SUBTERRANEA L.VERDC (FABACEA)
Authors: Okpuzor Joy , Ogbunugafor Henrietta A. , Okafor Uzoma , Sofidiya Magarette O.
Published: June 2021

This study attempts to highlight the importance of Bambara groundnut, a legume as a booster of the dietary protein stock available to the lower strata of the population in most developing countries especcially in African as well as suspected health benefits of some of the proteins in the management of cardiovascular diseases, diabetes, digestive tract disorders and weight control.

The indentification of the proteins of Vigna subterrenea L. Verdc. was carried out using MudPit and LC‐MALDI TOF‐TOF mass spectrometry. Some of the proteins we isolated include Seed storage protein B, Mung bean seed albumin Chain A, Crystal Structure Of Adzuki Bean 7s Globulin‐3, Dehydrin and 8S globulin alpha subunit. We also observed the sequence homology of most of the proteins with those of some other Vigna species, especially Vigna radiata, Vigna angularis and Vigna unguiculata. Our overall goal is to direct attention to Bambara groundnut and cause it to be removed from the list of under‐utilized and neglected crops and initiate a shift in governmental policies towards this direction.
NSC 622124 inhibits human Eg5 and other kinesins via interaction with the conserved microtubule‐binding site
Authors: Walker Richard A. , Learman Sarah , Kim Catherine , Stevens Nathaniel , Wojcik Edward , Kim Sunyoung
Published: June 2021

Kinesin‐5 proteins are essential for formation of a bipolar mitotic spindle in eukaryotic cells. Notably, the human Kinesin‐5 motor, HsEg5, is a target for an expanding group of small‐molecule inhibitors that hold promise both as tools to probe mechanochemical transduction and as anti‐cancer agents. Although most such compounds are selective for HsEg5, some exhibit activity against at least one kinesin from outside the Kinesin‐5 subfamily. Here we show NSC 622124, despite identification in an inhibitor screen targeting the HsEg5 monastrol‐binding site, does not compete with 14C‐monastrol for binding to HsEg5, and inhibits the basal and microtubule (MT)‐stimulated ATPase activity of the monastrol‐insensitive KLP61F. NSC 622124 competes with MTs, but not ATP, for interaction with HsEg5, and disrupts MT binding of other kinesins as well. Proteolytic degradation of an HsEg5▸NSC622124 complex revealed that segments of the α3 and α5 helices map to the inhibitor‐binding site. Overall, our results demonstrate that NSC 622124 targets the conserved MT‐binding site of kinesins. Further, NSC 622124 does not produce any enhancement of basal ATPase activity, and only acts as a negative regulator through interaction with a site traditionally viewed as a binding region for positive regulators. Our work emphasizes that MT‐dependent motor proteins may be controlled at multiple sites by both positive and negative effectors.
A 31P‐NMR Study to Determine the Percent Composition of Aqueous Dispersion of Soy Lecithin
Authors: Liotta Marcus Toland , Amburgey‐Peters Judith C. , Peters Scott
Published: June 2021

Phospholipids are organic compounds that are comprised of a glycerol backbone, two fatty acid acyl‐chains, and a phosphate head group with various substituents. In an aqueous environment, the hydrophobic acyl‐chains minimize their contact with water by aligning to form a bi‐layered system. As the bi‐layer converges upon itself, stable bi‐layered spheres, liposomes, form. Liposomes have been used for years as a delivery agent for medical and commercial applications. In the commercial field, soy lecithin is typically used as the mixed phospholipid source to prepare liposomes. However, the concentrations of the different phospholipids can vary between providers and batches.

31P NMR is to be sensitive enough to differentiate each of the specific phospholipids found in the soy lecithin mixture. Deuterated Chloroform (CDCl3) and Methanol (CH3OH) were used as the solvent system, with a mixture of Cesuim‐Ethylenediaminetetraacetic acid (Cs‐EDTA) added to further separate the signals. An internal standard of Triethyl phosphate (TEP) was used to quantify the individual concentrations of phospholipids. Utilizing 31P‐NMR, it is possible to analyze the phospholipid content of liposomes and follow the degradation of the phospholipids over time and under various conditions. This research was made possible by funding from Ingredient Innovations International and the Chemistry Department of Chemistry at The College of Wooster.
Nuclear Oxysterols, 25HC and 25HC3S, Regulate Nuclear Orphan Receptor Activities and Attenuate Intracellular Lipid Levels
Authors: Xu Leyuan , Ma Yongjie , Hylemon Phillip , Heuman Douglas , Pandak William , Ren Shunlin
Published: June 2021

We recently identified the sulfated oxysterol, 5‐cholesten‐3beta, 25‐diol 3‐sulfate (25HC3S), as an important regulatory molecule. When cholesterol delivered to mitochondria was increased by overexpression of the transport protein StarD1 in primary rat hepatocytes, 25‐Hydroxycholesterol (25HC) and 25HC3S were accumulated in the nucleus, suggesting a role in transcriptional regulation. Nuclear orphan receptors are key transcriptional regulators of hepatocyte lipid metabolism. We now have studied the effects of 25HC and 25HC3S on the activation of nuclear receptors, LXR, RXR, PPAR, and SREBPs. Addition of 25HC3S to hepatocytes in culture markedly decreased nuclear LXR but increased PPAR protein levels, followed by dose‐ and time‐dependent decreases in SREBP‐1 protein and mRNA levels. 25HC3S administration also led to a decrease in the expression of a number of SREBP‐1 responsive genes including acetyl CoA carboxylase‐1 (ACC‐1) and fatty acid synthase (FAS), key enzymes involved in fatty acid biosynthesis, and subsequently, decreased the intracellular free fatty acids. 25HC, a LXR ligand and the precursor of 25HC3S, had opposite effects: increase nuclear LXR protein as well as SREBP‐1 and FAS mRNA, and decrease nuclear PPAR protein levels. This work implies that sulfation of 25HC does more than simple inactivation of 25HC. It provides a new regulatory mechanism involved in lipid metabolism.
Differential efficiency of lysophospholipid markers for oxidative stress
Authors: Schober Celestina , Fuchs Beate , Schiller Jürgen
Published: June 2021

Inflammatory liver diseases are associated with marked oxidative stress. At inflammatory loci, hypochlorous acid (HOCl) is generated by myeloperoxidase. HOCl reacts with a variety of molecules and induces the formation of lysophosphatidylcholine (LPC) from polyunsaturated phosphatidylcholine (PC). Since liver tissue contains huge amounts of polyunsaturated PC the arising of enhanced LPC concentrations could be expected and was experimentally verified in various species. Surprisingly, further lysophospholipids (LPL) could not be detected, although human liver contains many further polyunsaturated phospholipid (PL) classes. Therefore, four different isolated PL classes were investigated regarding the extent of LPL generation under the influence of HOCl. Using MALDI‐TOF mass spectrometry (MS) PC could be identified as the only PL leading to LPC, whereas phosphatidylglycerol, phosphatidylserine and phosphatidylethanolamine led exclusively to chlorohydrins by the reaction of HOCl with the double bonds of their unsaturated fatty acyl residues. Thus, LPC represents a useful biomarker of oxidative stress and inflammation that can be easily determined by MS ‐ even in complex mixtures.
Changes in Jak3 Expression during the Differentiation of Intestinal epithelial cells*
Authors: Mishra Jayshree , Kumar Narendra
Published: June 2021

Changes in genetic expression play a central role during the differentiation of intestinal epithelial cells (IEC). Previously we have reported that Jak3 plays an essential role during mucosal wound repair an important ramification for patients with ulcerative colitis and Crohn's disease. Here we determine the physiological significance of Jak‐3 expression during enterocytic differentiation. Jak‐3 expression was determined in sub‐confluent, confluent, 2‐ and 3‐week post‐confluent HT‐29 Cl‐19a cells through western analysis using Jak‐3 antibody. Reduced Jak‐3 expression was observed in sub‐confluent cells and the expression was increased with differentiation. Interestingly an additional lower mol. wt. Jak3 was detected only at 2 weeks of post confluent cells but not at 3 weeks post confluence indicating the possibility of a non phosphorylated form. To determine the effect of Jak‐3 on the differentiation of HT‐29, Jak‐3 was over expressed in HT‐29 cells and its differentiation was monitored using the differentiation marker sucrose isomaltase. Results show that over expression of Jak‐3 potentiate the differentiation of IEC. Densitometric analysis of the ratio of Jak3 to â‐actin expression shows a 1.5 fold increase in Jak3 expression. Taken together these results indicate that Jak3 expression regulate the differentiation of IEC. *This work was supported a grant from CCFA (to NK)
Structural analysis of the first GAF domain from Mycobacterium tuberculosis Rv3132c (DosS) protein
Authors: CHO HA YEON , CHO HYO JE , KANG BEOM SIK
Published: June 2021

DosS‐DosR is a two‐component regulatory system for hypoxia sensing in Mycobacterium tuberculosis and DosS is a sensor histidine kinase. The N‐terminal sensory domain of DosS contains two GAF domains. Here, we determined the molecular structure of the first GAF domain (GAF‐A) by X‐ray crystallography in order to give a hint for the sensing mechanism. The crystal GAF‐A obtained by sitting‐drop vapor diffusion method in the condition of 0.2M calcium acetate, 18% polyethylene glycol 6000K. The GAF‐A consisted of a central five‐stranded anti‐parallel β‐sheets, and four α‐helices. The N‐and C‐terminal helices are at one side of the sheet and a mixture of long loops and two helices at the other side. A b‐type heme was found in a cavity between the β‐sheets and the loops. UV‐visible spectroscopic and X‐ray crystallographic analysis revealed that the heme of ferric form is a six‐coordinated state and upon its reduction, it is converted to a five‐coordinated ferrous form. Cyanide complex form of GAF‐A suggests its diatomic ligand binding.[Grant sponsor: 21C Frontier Microbial Genomics and Application Center Program, Ministry of Science and Technology, Republic of Korea]
Temporal patterns of cold‐induced membrane restructuring in fish cells.
Authors: Crowder Lauren Alyssia , McMullen Jacob C , Williams E Eugene
Published: June 2021

The cells of many animals compensate for altered environmental temperature by changing the phospholipid composition of their membranes. We monitored in fish cells the amounts of mRNA coding for some of the enzymes responsible for making these changes while the cells were exposed to reduced temperature. Chinook salmon embryo cells cultured at 20ºC were transferred to 10ºC for different times before total mRNA was extracted. Primers of our design were used to probe cDNA copies of the mRNA using PCR. PCR products were separated by agarose gel electrophoresis and digitally recorded. We found that mRNAs for the delta‐6 and delta‐9 desaturases were detectable at 20ºC and their quantities increased after 24 h, and again after 48 h, of cold exposure. The amount of message for glycerol‐3‐phosphate acyltransterase was low at 20ºC and remained low until it dramatically increased after 10 days at 10ºC. mRNAs for other enzymes showed other patterns of expression. Overall our data suggest that the metabolic adjustments that restructure membrane phospholipids in response to cold are activated at different times and persist for different intervals. It is likely they occur with a well defined temporal hierarchy. This work was supported by Undergraduate Research Grants from the Henson School of Science and Technology at Salisbury University and by a Guerrieri Summer Research Award.
Role of N‐glycans of the β2 subunit in assembly and trafficking of the α1β2 Na,K‐ATPase
Authors: Vagin Olga , Tokhtaeva Elmira , Sachs George
Published: June 2021

The non‐glycosylated αsubunit of the Na,K‐ATPase is unable to exit the ER unless it is co‐expressed with the N‐glycosylated ? subunit. We tested whether N‐glycans of the ?2 subunit are required for proper folding, assembly and quality control of the Na,K‐ATPase in the ER. The over‐expressed ?2 subunit was co‐localized with the endogenous α1 and ?1 subunits in the plasma membrane of MDCK cells and co‐immunoprecipitated with the α1 subunit from cell lysates. A minor fraction of the ?2 subunit was detected in the ER without the α1 subunit. The ?2 subunit, but not the α1 subunit, was co‐immunoprecipitated with the ER chaperone, calnexin. Mutation of more than three of the eight N‐glycosylation sites of the ?2 subunit prevented α1 ?2 assembly, augmented association of the ?2 subunit with calnexin, and resulted in complete retention of the ?2 subunit in the ER. In contrast, the endogenous α1 subunit was neither associated with calnexin nor retained in the ER. Thus, particular N‐glycans are required for calnexin‐mediated folding and quality control of the unassembled ?2 subunit, but not of the α1 ?2 complex. The proper folding of the ?2 subunit is a prerequisite for the α1 ?2 assembly, which, in turn, is necessary for the export of the ?2 subunit from the ER to the Golgi. Therefore, neither the α1 nor the β2 subunits are able to exit the ER unless they are assembled in the full enzyme.

Supported by NIH, DK077149 and DK58333.
Membrane bound prenylated C17orf37 is a critical regulator of cancer cell migration and invasion
Authors: Dasgupta Subhamoy , Gryczynski Zygmunt , Vishwanatha Jamboor K
Published: June 2021

C17orf37 is located in the hot spot locus of human chromosomal region 17q12‐21 next to ErbB2. Previously we have shown that C17orf37 expression is significantly higher in prostate cancer cells and tissue specimens compared to normal prostate cells. By gene knock down studies, we demonstrated that C17orf37 regulates prostate cancer cell migration and invasion by modulating key proteins involved in these processes, such as MMP‐9, uPA and VEGF. The migration‐defect phenotype in C17orf37 depleted prostate cancer cells were restored on C17orf37 overexpression, with concomitant increased expression of MMP‐9, uPA and VEGF proteins. Knockdown of C17orf37 abolished p‐Akt and reduced DNA binding activity of NF‐κB. Cellular localization studies using confocal and TIRF microscopy demonstrated predominant expression of C17orf37 protein in the membrane. C17orf37 protein has a C‐terminal prenylation motif 'CVIL' and using inhibitors of prenyl transferase enzymes we demonstrate that C17orf37 is preferentially geranylgeranylated by GGTase‐1 enzyme and this inhibition results in reduced membrane bound C17orf37. We identified annexin A2 as a novel interactor of C17orf37 and by FLIM based FRET assay we found that these two molecules physically interact at the membrane region. Our studies show C17orf37‐annexin A2 interaction is critical for tumor cell migration and invasion, by regulating downstream signaling molecules.

(Supported grants from the National Institutes of Health CA109593 and MD 001633)
Structural and functional analysis of GPR56 protein in human frontal cortex developmental disease BFPP: The role of disease‐associated mutations on receptor trafficking and functions
Authors: Chiang Nien‐Yi , Lin Hsi‐Hsien
Published: June 2021

G protein‐coupled receptor 56 (GPR56) plays a role in the development of neural progenitor cells and has been linked to the developmental malformations of human brain. Similar to other adhesion GPCRs, GPR56 contains a GPCR proteolytic site (GPS) motif. Genetic mutations in GPR56 cause a human brain cortical malformation called bilateral frontoparietal polymicrogyria (BFPP). However, it remains unclear how these mutations affect GPR56 function. We cloned the wild type GPR56 cDNA and disease‐associated point mutants, including R38W, Y88C, and C91S in the N terminal domain, two mutations (R565W, L640R) in the extracellular loop domain, and the two mutations in the GPS domain (C346S and W349S). We aim to investigate how the BFPP‐associated mutations affect the intracellular trafficking and cell surface expression of GPR56. We found that disease‐associated GPR56 missense mutations produce proteins with reduced intracellular trafficking and poor cell surface expression or impaired GPS cleavage that fail to traffic beyond the ER. Furthermore, we has produced recombinant GPR56 extracellular domain as a protein probe for ligand search on various cell lines. We have identified a potential novel cellular ligand for GPR56. NSC97‐2628‐B‐182‐030‐MY2
Myristoylation and its role in conformational changes associated with Galphai subunit activation
Authors: Preininger Anita M. , Meier Scott M , Funk Michael A , Busenlehner Laura S , Armstrong Richard N , Iverson Tina M. , Hamm Heidi E
Published: June 2021

G‐protein signaling from 7‐transmembrane GPCRs controls a vast array of physiological responses from external stimulii. While much is known about the structure and function of individual Galpha and Gbeta‐gamma subunits, as well as heterotrimeric G proteins, the majority of the structural information collected to date on Galpha subunits has been obtained using unmyristoylated Galpha proteins. Since the alpha subunits of Gi family members are irreversibly myristoylated in vivo at the amino terminus, and since myristoylation of Galphai subunits is not sufficient to tether activated subunits to cell membranes in vitro, new insights may be gained by studying the structure and function of these subunits in the context of the myristoylated form of the protein. Using a combination of structural and biochemical approaches, we probe the effects of myristoylation on the conformation of Galpha subunits to reveal new insights into processes associated with G‐protein activation. This work supported by: TMI, NARSAD; HEH, NIH Grant EY06062.
VEGF Regulates Gene Expression of Oxytocin during Pregnancy in the Cervix of Rodents
Authors: Willner Weston , Jones Fallon Anitra , Mowa Chishimba
Published: June 2021

Cervical remodeling is a physiological inflammatory process regulated by various factors including vascular factors. We have previously shown that VEGF regulates multiple genes involved in cervical remodeling using DNA microarray analysis, including oxytocin. Here, we validate the DNA microarray data using real time PCR. Consistent with DNA microarray data, we found that levels of oxytocin mRNA and its receptor were up‐regulated by VEGF treatment. We conclude that VEGF plays a role in regulating levels of VEGF in the cervix of rodents.
Discovery of novel peptide hormones using prohormone convertase screening
Authors: Ozawa Akihiko , Lee Ernestine , Hoshino Akina , Halenbeck Robert , Kothakota Srinivas , Lindberg Iris
Published: June 2021

Bioactive peptides play critical roles in diverse biological phenomena; the discovery of novel peptides thus has the potential to open entirely new fields of research. The successful sequencing of entire genomes of various organisms has yielded numerous novel candidate precursor genes; however, the identification of novel bioactive peptides from such genomic information has been hampered by their small size. In this study, we attempt to identify novel peptides using a combinatorial screening approach: a predictive algorithm followed by "restriction protease" screening.

The algorithm was trained using various properties associated with known peptide precursors. Approximately 150 genes were identified as coding for candidate peptide precursors.

In a preliminary screening, 21 genes were chosen from this list and expressed in tagged form in HEK293 cells. Proteolysis using prohormone convertases 1 and 2 and furin was performed in order to confirm cleavage of expressed proteins. Sixteen of 21 precursors were cleaved by one or more convertases; 5 proteins were not cleaved and may represent false bioinformatics hits. In situ hybridization of mouse brain with antisense cDNA from a cleaved precursor indicated limited expression within the mediobasal hypothalamus. These results demonstrate that our bioinformatics/convertase screening approach is a promising method for peptide hormone discovery. (DA05984)
Rho signaling is involved in HIV‐1‐mediated upregulation of P‐glycoprotein expression in human brain endothelial cells
Authors: Zhong Yu , Hennig Bernhard , Toborek Michal
Published: June 2021

Limited drug penetration into the brain is an obstacle in treatment of human immunodeficiency virus type‐1 (HIV)‐associated CNS diseases. Specific drugs used in HAART, particularly HIV‐1 protease inhibitors, are substrates for the efflux transport systems, such as P‐glycoprotein (P‐gp). P‐gp is a 170 kDa ATP‐dependent integral membrane protein and belongs to the family of ATP‐binding cassette (ABC) drug efflux transporters. We hypothesize that overexpression of P‐gp at the blood‐brain barrier level may contribute to enhanced removal of antiretroviral drugs from the brain and lead to the development of resistance to HAART. Treatment with HIV Tat protein markedly elevated GTP‐RhoA levels in human brain endothelial cells as assessed by the pull‐down assay, while the total level of RhoA was not changed. Moreover, Tat increased phosphorylation of Rho associated kinase (ROCK). Inhibition of the Rho signaling cascade by C3 exoenzyme or by Y27632 blocked Tat‐induced P‐gp overexpression. The present data indicate the critical role of the Rho signaling in upregulation of P‐gp in the brain in the context of HIV‐associated CNS diseases. It appears that the Rho cascade may be a potential target to prevent the development of HAART resistance in HIV‐infected patients. Supported by the NIH grants: MH 63022, MH 072567, NS 39254, and P42 ES 07380.
Calcium restriction inhibits cancer cell proliferation in vitro
Authors: Sridhar Rajagopalan , Cousins Valerie Marie , Zhang Renshu , Zhou Yanfei , Haddad Georges , Ashayeri Ebrahim
Published: June 2021

Prostate and breast cancer cells metastasize to the bone. Strontium‐89 (Sr‐89) is a radioisotope that is used for treating bone metastases. The effect of strontium on calcium uptake was studied in human fetal osteoblast (hFOB) as well as androgen independent PC‐3 human prostate and MDA‐MB231 human breast cancer cells using digitized fluorescence microscopy in cells preloaded with Fura‐2AM. The effect of calcium deprivation on cell growth was studied using the MTT assay. When compared to cultures grown in complete medium, calcium and serum deprivation for 24 hours decreased cell growth by 80, 58 and 52 percent in hFOB, MDA‐MB231 and PC‐3 cells respectively. Addition of strontium chloride (up to 2 mM) did not cause further growth inhibition in calcium and serum starved medium. Calcium deprivation caused a G1 block and decreased the proportion of cells in the S phase. PC‐3 cells cultured for 24 hours in complete calcium containing medium supplemented with fetal bovine serum had a cell cycle distribution of 56% G1, 18.4% G2 and 25.6% S phase cells. These parameters shifted to 74.5% G1, 12.8% G2 and 12.6 % S phase cells in calcium and serum starved medium. Although strontium did not affect cell growth, it inhibited uptake of calcium by PC‐3 and MDA‐MB231 cells. Uptake of calcium from the medium by cells was inhibited by the anti diarrhea drug loperamide, which is a calcium channel blocker. Interestingly, loperamide inhibited hFOB and MDA‐MB231 cell growth even in complete medium. Preferential inhibition of calcium uptake by cancer cells may be an option in cancer therapy.

(Supported in part by USMRMC grants DAMD17‐03‐1‐0759, DAMD17‐03‐1‐0123, 2 G12 RR003048 from RCMI, NCRR, NIH and R 25HL003679 from HLBI, NIH)
New Human Inducible Nitric Oxide Synthase Interacting Proteins
Authors: Rashid Mohammad B , Challawala Amama , Eissa Tony
Published: June 2021

Nitric oxide (NO) is a potent cell‐signaling molecule. NO can be synthesized by three different (isoforms) nitric oxide synthases (NOS). NO can be physiological in some tissues, but in some cases it may be toxic. Designing the isoform‐specific inhibitor(s) is a key issue so that it would not interfere with physiological NO production. Here, we are investigating human inducible nitric oxide synthase(hiNOS)‐interacting protein(s) that will give us a clue to design such inhibitors of hiNOS. We have employed BD‐Clontech made Yeast Two‐Hybrid System to screen a mouse testis cDNA library and identified a number of candidates for such interaction e.g. Dnaj, fem1b etc.. We are also screening a human testis cDNA library to see if our results look the same.
Subcellular Localization of Human Myocardial PDE3 Isoforms and Components of cAMP Signaling Pathways
Authors: Khan Faiyaz Ahmad , Krall Judith , Vandeput Fabrice , Shen Weixing , Manganiello Vincent , Movsesian Matthew
Published: June 2021

Caveolae‐enriched plasma membrane (PM) fractions from failing heart myocardium are highly enriched in b2‐ARs, b3‐ARs, Gα (i), adenylyl cyclase 5/6 and caveolin‐1, but not b1‐ARs or Gα (s). Approximately 30% of the total PDE3 activity is associated with PM and 70% with internal membrane fractions. PDE3A3 is the dominant PDE3A isoform in PM fractions, while all isoforms of PDE3A (A1‐3) and PDE3B1 are present in internal membranes. On gel filtration chromatography, PDE3 activity in solubilized membrane fractions was partitioned between distinct high molecular weight (HMW) and low molecular weight (LMW) peaks. The HMW peaks likely contain multiprotein complexes, including PDE3A1, PDE3A2, and PDE3B as well as several proteins involved in b‐adrenergic receptor‐mediated signaling, including b1‐AR, b2‐AR, AC5/6 ,PKA‐RII, caveolin‐1, and most of the AKAPs that we tested. PDE3 activity in cytosolic fractions consisted primarily of PDE3A2 and PDE3A3, and eluted only in LMW fractions. PDE3A and SERCA2 were coprecipitated from myocardial membranes using anti‐PDE3A antibodies or cAMP‐agarose beads. These results indicate that PDE3 isoforms in myocardial membranes are likely to incorporate into multiprotein complexes; one such complex might contain PDE3A and SERCA2,, suggesting a key role for PDE3A in the regulation of Ca+2 cycling by sarcoplasmic reticulum.
The Role of Heme in Diverse Kinase‐Mediated Cell Signaling Pathways.
Authors: Yao Xiao , Arvey Aaron , Balamurugan Parimaladevi , Shah Ajit , Leslie Christina , Zhang Li
Published: June 2021

Heme not only serves as a prosthetic group in many enzymes and globins, but it also directly controls many regulatory and signaling processes in living organisms. In mammals, heme has shown to bind to and to regulate the activity of diverse signal transducers and regulators, including MAP kinases, the heme‐regulated inhibitor kinase HRI, the transcriptional regulator Bach1, and the circadian clock coordinator Rev‐erbα. Most, if not all, of these heme‐regulated proteins contain one or more heme‐responsive motifs containing two invariable CP residues. To explore the potential roles of heme in regulating diverse cellular processes, we employed computational methods to identify human proteins that contain one or more CP motifs and may thus be regulated by heme. We found that about 10% of human proteins contain one or more CP motifs. Particularly, we found that 47 out of 159 human protein tyrosine kinases, including receptor tyrosine kinases, such as insulin and insulin‐like growth factor receptors, EGF receptors, and Ephrin receptors, contain one or more CP motifs. Experiments are underway to determine whether heme directly binds to and controls the activity of these kinases.
Determining specificities and affinities between BH3 peptides and BcL‐2 proteins using yeast surface display
Authors: Genilo Elyza Joy , Dutta Sanjib , Keating Amy
Published: June 2021

Apoptosis in every cell is determined by protein‐protein interactions between the BcL‐2 family, consisting of two subgroups: anti‐apoptotic proteins (BcL‐2, BcL‐xL, BcL‐W, McL‐1, and A‐1) and pro‐apoptotic proteins. A specific subgroup of the pro‐apoptotic proteins consist of "BH3 only" proteins. Our interest is to examine the specificities and affinities between the anti‐apoptotic and the "BH3 only" proteins. These interactions occur between the amphiphilic regions of the BH3 peptides with the hydrophobic indentations of the BcL‐2 proteins. A pool of BH3 peptides selected for specificity or promiscuity against BcL‐xL or McL‐1 were displayed on yeast cells and tested against several anti‐apoptotic proteins. To understand specificities, the BH3 peptides were tested against these receptors using a high‐throughput approach to determine which peptides were specific or promiscuous to the receptors. To understand affinity, a selected amount of specific BH3 clones were used for quantitative measurement of binding affinity. By observing specificities and versatilities in protein sequences, we can then manipulate the proteins to act as inhibitors or stimulators of favorable cellular outcomes.
The Characterization of Recombinant Riboflavin Binding Protein
Authors: Jones Robert T , Smith Sheila R , Benore Marilee
Published: June 2021

Riboflavin Binding Protein (RBP) is a phosphoglycoprotein that is responsible for binding and delivery of riboflavin during embryonic development in hens. There are three forms of RBP: serum RBP (sRBP), yolk RBP (yRBP) and albumen RBP (wRBP) made from one gene. RBP binds copper in vitro in a 1:1 ratio, and the elucidation of the binding site would be of significance. Previous evidence suggests copper binding is at a Histidine residue. The goal of this study was to create and characterize recombinant plasmids of RBP in order to determine if His is required for copper binding. This work is supported by a grant from the University of MI at Dearborn.
Copper Binding To Riboflavin Binding Protein‐ The Role Of Phosphoserine Residues
Authors: Brandt Eric , Smith Sheila R , Benore Marilee
Published: June 2021

Riboflavin Binding Protein (RBP) is a hen glyco‐phospho‐protein that transports the nutrient riboflavin to the oviduct and oocyte. Recent work in our labs indicates the protein also binds copper in a 1:1 ratio in vitro. The site of copper binding to Riboflavin Binding Protein is unclear, although it appears Histidine residues are involved. We are interested in determining if the phosphoserine residues participate in copper binding. It is possible, though unlikely, that the metal binds to one of the numerous phosphoserine residues or that the phosphoserine residues alter the binding affinity or ratio. RBP was dephosphorylated, dialyzed against copper, and the copper binding characterized using Atomic Absorption Spectroscopy. Evidence suggests that the dephosphorylated protein can still bind copper. This work is supported by a grant from the University of MI at Dearborn.
Expression in Pichia pastoris and Backbone Dynamics of Dendroaspin and Erabutoxin b: Motion Variability within Three‐fingered Toxins
Authors: Cheng Chun‐Ho , Chen Chu‐Chun , Chen Yi‐Chun , Chen Chiu‐Yueh , Chuang Woei‐Jer
Published: June 2021

Dendoaspin (DEN) and Erabutoxin b (EBTb) are three‐fingered toxins which possess a similar architecture with unrelated biological functions. They share a common structure consisting of a β‐sheet core that tightly cross‐linked by four conserved disulfide bonds. DEN inhibits platelet aggregation by blocking the binding of fibrinogen to the integrin αIIbβ3 of platelets. In contrast, EBTb is a potent inhibitor of nicotinic acetylcholine receptor. To study their function and dynamics relationships, we expressed DEN and EBTb in Pichia pastoris (P. pastoris). The recombinant DEN expressed in P. pastoris inhibited platelet aggregation with a KI of 149 nM. The recombinant EBTb expressed in P. pastoris inhibited carbachol‐induced muscle contraction with a KI of 42 nM. They are the same potency as those of native proteins. NMR analysis showed that DEN and EBTb expressed in P. pastoris processes the same structures as those of native proteins. The results provide the first direct evidence that highly disulfide‐bonded three‐fingered toxins can be expressed in P. pastoris with the correct fold. Analysis of the dynamics properties of DEN and EBTb showed that they exhibited flexibility on the loops II and III and motions on multiple time scales. The RGD‐containing loop of DEN was the most flexible region and the apex of the loops II and III of EBTb exhibited flexible motions on the ps/ns timescale. Comparison of the dynamics properties of DEN and EBTb with other members of three‐fingered toxins suggests that their dynamics deviations may play an important role in interacting with different target proteins.
Structure‐Dynamics‐Function Relationships of Disintegrins in Integrin Recognition
Authors: Chen Yi‐Chun , Chuang Woei‐Jer
Published: June 2021

Integrins are the major cell surface receptors for cells to interact with extracellular matrix proteins and play an important role in cytoskeleton reorganization and signal transduction. Disintegrins are potent integrin inhibitors found in snake venoms. Depending on the size and the number of cysteines, they can be classified into small, medium, long, and dimeric disintegrins. To study structure and functional relationships of short and medium disintegrins, we expressed rhodostomin (Rho), a 68‐residue disintegrin with six disulfide bonds, and echistatin (Ech), a 49‐residue disintegrin with four disulfide bonds, in Pichia pastoris as the model proteins. The analysis of cell adhesion assay showed that Rho mutant containing ARGDDM sequences in the RGD loop and NPHKGPAT sequence in the C‐terminus exhibited 14.4‐, 4.1‐, and 2.33‐folds decrease in inhibitory activity to integrins £\IIb£]3, £\v£]3 and £\5£]1, respectively. Backbone dynamics analysis showed that their similar dynamics properties on the RGD motif. In contrast, the C‐terminal region of Rho is more flexible of that of Ech. This suggests that the dynamics properties of the C‐terminus of disintegrins also play a crucial role in determining their affinity and selectivity to integrins.
Vitamin A Value of Provitamin A‐Rich Fruits and Vegetables in Mongolian Gerbils
Authors: Arscott Sara Andrea , Howe Julie A , Davis Christopher R , Tanumihardjo Sherry A
Published: June 2021

Fruits and vegetables are rich sources of provitamin A (pro‐VA) carotenoids. We evaluated the vitamin A (VA) bioefficacy of a whole food supplement (WFS) and its constituent green vegetables (Study 1, n=77) and a variety of fruits with varying ratios of pro‐VA carotenoids (Study 2, n=77) in VA depleted Mongolian gerbils. Diets were equalized to VA. Both studies utilized negative (‐VA, VA‐free diet) and positive (+VA, VA‐fee diet + VA in oil) controls. In Study 1, liver VA was greatest in the +VA group (0.82 ± 0.16 µmol/liver, P < 0.05), followed by brussels sprouts (0.50 ± 0.15 µmol/liver), Betanat® (β‐carotene from B. trispora) (0.50 ± 0.12 µmol/liver), and spinach (0.47 ± 0.09 µmol/liver) groups. These groups did not differ from baseline (0.58 ± 0.11 µmol/liver, P > 0.05). The WFS (0.44 ± 0.06 µmol/liver) and kale (0.43 ± 0.14 µmol/liver) groups had lower liver VA than the baseline group (P < 0.05), but did not differ from the brussels sprouts, Betanat®, and spinach groups. In Study 2, treatments included banana (2.2 nmol α‐carotene/g, 1.7 nmol β‐carotene/g), mango (2.8 nmol β‐carotene/g), papaya (3.2 nmol β‐cryptoxanthin/g, 1.2 nmol β‐carotene/g), orange (4.7 nmol β‐cryptoxanthin/g, 0.5 nmol β‐carotene/g), tangerine (4.6 nmol β‐cryptoxanthin/g, 0.5 nmol β‐carotene/g) diets. The pro‐VA activity of these fruits will be calculated. (Supported by Hatch WI Ag Exp St WIS04975 and Standard Process, Inc., Palmyra, WI)
Alpha ‐ linolenic acid (ALA)‐ rich flaxseed oil (FO) enhances the effectiveness of trastuzumab (TRAS) in reducing the growth of HER2 overexpressing human breast cancer tumors (BT‐474)
Authors: Mason Julie Kathleen , Truan Jennifer S , Thompson Lilian U , Chen Jianmin
Published: June 2021

TRAS is the primary drug for breast tumors that overexpress epidermal growth factor receptor 2 (HER2) but tumors develop resistance within 1 year of treatment. Our objective is to determine whether combining dietary ALA‐rich FO with TRAS treatment will enhance TRAS effectiveness. HER2 overexpressing breast tumor cells (BT‐474) were tested for proliferation and HER2 level in vitro after treatment with ALA (50µM), TRAS (10µg/ml) or their combination. Compared with untreated control, ALA, TRAS and combination caused a 35%, 54% and 61% reduction in proliferation and a 23% increase and 19% and 88% reduction in HER 2 expression (p<0.05), respectively. Ovariectomized mice with BT‐474 tumors were fed the basal diet (BD) (control) or treated with TRAS (2.5 or 5mg/kg) and fed either the BD or BD supplemented with 8% FO for 4 weeks. Compared to control, both TRAS and FO+TRAS treatments caused significant reductions in tumor growth over time (P<0.05). Control tumors increased in size by 63% while tumor size with FO+TRAS2.5 was reduced by 85% which is greater than that with TRAS2.5 (16%) (p<0.05) and not significantly different from that with TRAS5, alone or with FO. FO+TRAS2.5 caused greater reduction ( 85%) in cell proliferation (Ki67 labeling index) than with TRAS2.5 alone (59%; p<0.05). In conclusion, ALA and ALA‐rich FO enhanced TRAS effects and combined FO and low dose TRAS was just as effective as high dose TRAS.

Grant Funding Source

NSERC
Efficacy of curcumin in lung cancer hamster model
Authors: Geamanu Andreea , Goja Arvind , Zaheeruddin Shaistha , Gupta Smiti
Published: June 2021

Curcumin, the polyphenolic pigment from the rhizome Curcuma longa, is known for its antioxidant, anti‐inflammatory, antibacterial, and recently, anticarcinogenic properties. The aim of this study was to investigate efficacy of curcumin in lung cancer induced hamster model. 32 male Golden Syrian hamsters were divided into test (TO, T2 n= 10 each) and control (CO, C2 n=6 each) groups. Test groups received BOP (N‐nitrosobis (2‐oxopropyl) amine; 10mg/kg), a carcinogen, for 12 weeks, while the control animals received saline injections. Effect of curcumin was examined by providing 0 (C0, T0) or 2% (C2, T2) dietary curcumin. After 24 weeks tissues were harvested and lungs fixed for H&E and AE1/AE3 immunohistochemistry staining. COX‐2 and COMET assays were performed to determine the degree of inflammation and DNA damage in the tissues respectively. Histology showed significant differences in cancer development between the treatment groups with (T2) and without (T0) curcumin intervention. Cancer incidence (11.1% (T2) vs 45.5 %(T0)) and survival rate (72% (T2) vs. 33% (T0)) were significantly different. Loss in body weight, increase in Cox‐2 levels and DNA damage reinforced the beneficial role of curcumin in decreasing lung cancer in the hamster model. Overall, the study shows the chemopreventive effect of curcumin. Further investigations are needed to test curcumin as a therapeutic agent in the human population.

Grant Funding Source

Wayne State University
The Effects of Soy Germ and Tomato Powder Containing Diets on the Growth of Dunning R3327‐H Transplantable Tumors
Authors: Zuniga Krystle Erin , Lindshield Brian L , Erdman John W
Published: June 2021

A number of studies have supported the hypothesis that dietary intake of soy foods or tomato products are associated with a lower risk of prostate cancer. Soy isoflavones and lycopene are considered the primary bioactive components of soy and tomato, respectively. Soy germ, the hypocotyledon of the soybean, has a different isoflavone profile from the rest of the bean. The ratio of genistein:daidzein:glycitein in soybeans is typically 1.1:1:0.4 while it is 0.3:1:1 in soy germ. Our goal was to determine if soy germ, due to its altered isoflavone ratio, would be effective in reducing growth of Dunning R3327‐H transplantable tumors. In addition, we wanted to evaluate the potential interactive effects of tomato powder, which we have previously found to reduce tumor growth, and soy germ. Diets containing 10% tomato powder, 2% soy germ, neither, or a combination were fed to male Copenhagen rats (n=63) for approximately 24 weeks. Diets were initiated 1 month prior to receiving subcutaneous tumor implants. Tumor areas were measured weekly beginning 10 weeks after tumor implantation. Primary endpoints will include tumor area, tumor weight, urinary isoflavones, and tissue carotenoid levels.
A novel circulating factor promotes body fat loss early in the development of cachexia in the methylcholanthrene (MCA)‐induced sarcoma bearing rat
Authors: Byerley Lauri Olson , Lee Sang Ho , Culberson Cathy , Redmann Steve , Clemens Mark , Lively Mark
Published: June 2021

We provide evidence that a novel factor other than the lipid mobilizing factor, zinc alpha 2 glycoprotein (ZAG), promotes lipolysis in the MCA‐induced sarcoma‐bearing rat cachexia model. We hypothesize malignant growth promotes changes in circulating factor(s) that alter adipocyte biology prior to the clinical manifestations of cachexia. We compared tumor‐bearing ad lib fed rats (TB) to non‐tumor‐bearing ad lib fed rats or non‐tumor‐bearing pair‐fed rats at various time points throughout development of tumor induced cachexia. Prior to cachexia, the TB rats lost more than 10 + 0.7 % of their body fat before losing protein mass and decreasing their food intake (markers of cachexia). Fat loss occurred because adipocyte size, not number, was reduced. Increased turnover of palmitate and significantly higher serum triglyceride levels prior to cachexia were additional indicators of an early loss of lipid from the adipocytes. Circulating levels of known lipolytic hormones, norepinephrine and epinephrine, cytokines such as TNF‐á, and ZAG were not increased prior to the loss of fat mass. We provide evidence that a serum factor(s) promotes the loss of stored adipose lipid prior to the loss of lean body mass in this model and that the serum factor(s) is most likely a protein that is not ZAG. Supported by a grant from the American Institute for Cancer Research.
A fish oil/pectin diet suppresses radiation‐enhanced colon carcinogenesis via down‐regulation of the β‐catenin signaling pathway
Authors: Kim Hyemee , Turner Nancy D. , Taddeo Stella S. , Davidson Laurie A. , Wang Naisyin , Vannucci Marina , Chapkin Robert S. , Carroll Raymond J. , Lupton Joanne R.
Published: June 2021

In our previous study, a fish oil/pectin (FP) diet protected against radiation‐enhanced colon carcinogenesis by maintaining an elevated level of apoptosis relative to a corn oil/cellulose (CC) diet. The goal of this study was to determine the differences between the protective and promotive diets in the patterns of gene expression measured in exfoliated colonocytes from rats exposed to radiation (+/‐ 1 Gy, 1 GeV/nucleon Fe ions) and a chemical carcinogen (AOM, 2x, 15 mg/kg BW). Microarray data were analyzed using a mixed model ANOVA procedure, and genes exhibiting differential expression underwent Gene Ontology (GO) enrichment analyses. Radiation treatment affected expression of genes within the Wnt/β‐catenin pathways, which are involved in cell proliferation and apoptosis. FP feeding reduced expression of tyrosine kinase related genes, which are regulators of the Ras‐PI3K/Akt signaling pathway. Therefore, a FP containing diet may suppress β‐catenin signaling induced by the radiation treatment, thereby contributing to the maintenance of apoptosis.

Grant Funding Source

Supported by NSBRI (NASA NCC 9‐58) and NIH (CA57030 and CA59034).
Nutritional status and intake post nutritional supplementation in prostate cancer patients undergoing radiotherapy
Authors: Bartalone Rachel Leigh , Tyrpak Donna , Browne Richard W. , Wilding Gregory E. , Horvath Peter J. , Brown Jean K.
Published: June 2021

Patients undergoing radiation therapy show decreased appetite, declining nutritional intake and status. The use of dietary supplementation during therapy has been questioned. To examine this effect, 53 prostate cancer patients were assigned to 3 groups (Placebo, DRI ‐ Dietary Reference Intake levels of vitamins/minerals, or OM ‐ Orthomolecular levels of antioxidants were added to DRI ‐Vitamin C, E, Se, and Carotenoids). Blood, body measures, and 3 day diet/activity records were collected at baseline and 3 times during supplementation ‐ before, during, post therapy. Energy imbalance increased with Placebo due to increasing energy expenditure and decreasing energy intake (from a decrease in carbohydrate and sugar intake), but not with DRI and OM. Before supplementation, micronutrient intake was generally below DRI levels and stayed low with Placebo. After DRI and OM, 95% of subjects reached DRI intakes, except Vitamin D and A (50 and 75%, respectively). Only plasma vitamin C increased with DRI. After OM; plasma vitamin C, alpha‐tocopherol, beta‐carotene and lycopene increased (80, 110, 160, and 40%, respectively), while lutein did not increase significantly. Energy balance was more negative over therapy without supplementation, due to increased activity and a decreased carbohydrate intake. The negative energy balance increase may be due to lack of micronutrient intake combined with radiation therapy.

Grant Funding Source

NCI/NIH 5R21CA102391‐2