bluuebunny/crossref_metadata_2025 huggingface dataset:
Calcium/calmodulin‐dependent protein kinase II links hepatic cytosolic calcium release and apoptosis after burn injury
Authors: Brooks Natasha Chevon , Boehning Darren , Jeschke Marc
Published: June 2021

Liver integrity is essential to survival post burn as impaired liver function worsens prognosis in burn patients. Hepatocytes isolated after thermal injury have increased cytosolic calcium which is associated with depletion of endoplasmic reticulum (ER) calcium stores which is associated with upregulation of proteins involved in the ER unfolded protein response (UPR) pathway and activation of Jun N‐terminal kinase (JNK). Recently, it has been shown that UPR induced increases in cytosolic calcium due to depletion of ER calcium stores can activate calcium/calmodulin‐dependent protein kinase II (CaMKII). Further, CaMKII was shown to play a pro‐apoptotic role via JNK‐mediated upregulation of Fas receptor. We therefore hypothesize that activation of CaMKII provides a mechanistic link between increases in cytosolic calcium and hepatic apoptosis after burn injury. As a model system, we employed the use of the 60% total burn surface area burn model on rats that recapitulates human clinical findings. We have found that CaMKII is activated one to three hours post burn. Activation of CaMKII is associated with increased JNK activity, upregulation of Fas signaling, and subsequent Fas‐mediated apoptosis. We conclude that burn‐induced increases in hepatic cytosolic calcium activate CaMKII and lead to Fas‐mediated apoptosis.

This work is supported by NIH/NIGMS grant GM081685 and Shiners grants 8660 and 8640
Nitric Oxide is Involved in Metamorphic Processes in the Anuran Tadpole, Xenopus laevis
Authors: Johnson Jennifer A. , Gardner E. Eileen , Menon Jaishri
Published: June 2021

In order to adapt to a different habitat, under the influence of thyroid hormones, amphibian skin undergoes morphological, biochemical and physiological changes and the tail completely regresses during metamorphosis. Body epidermis remains non‐keratinized throughout the larval life and keratinizes during metamorphosis, resulting in adult frogs with a cornified epidermal cells covering the body. The significance of nitric oxide (NO) in many biological processes is well known. Signaling mediated by nitric oxide can contribute to stress‐related responses where these molecules can act as regulators of metamorphic transitions. However, there is scanty information on how NO acts as a signaling molecule during cell death and differentiation in amphibian tissues. Presently, we investigated the role of nitric oxide synthase (NOS) in skin and tail using Western blot analysis and immunohistochemistry during metamorphosis of tadpoles, Xenopus laevis. Our results indicate involvement of only neuronal NOS (NOS I) in skin whereas in tail inducible NOS (NOS II) is also present. In skin, neuronal NOS is present during the functional maturation phase during metamorphosis. In contrast, in tail both NOS I and II are found to be present before the beginning of tail regression. Implications of these findings are discussed in terms of putative functional importance of differential isoform expression in the context of developmental processes in this system.

This research was supported by William Paterson University.
PGA2‐induced Heme Oxygenase‐1 Expression Requires both p38 MAPK and de novo Protein Synthesis in U2OS cells.
Authors: Ko Kyoung‐Won , Choe Yun‐Jung , Jeong Seong‐Whan , Kim Ho‐Shik
Published: June 2021

Previous studies have shown that PGA2 induces antioxidant enzyme heme oxygenase‐1 (HO‐1) expression, but the mechanism involved has not been fully understood. In this study, we suggest the signal transduction pathway of PGA2‐induced HO‐1 expression in human osteosarcoma (U2OS) cells.

HO‐1 expression was induced by PGA2 (20 ug/ml) treatment for 8 hr in U2OS cells. Parallel to HO‐1 expression, p38 MAPK and p44/42 MAPK were phosphorylated by PGA2. PGA2‐mediated HO‐1 expression was completely reduced by p38 MAPK inhibitor (SB203580) as well as cycloheximide, while inhibition of p44/42 MAPK using U0126 and PD98059 had no effect. To elucidate a role of p38 MAPK in the induction of HO‐1 by PGA2, constitutively active form of MKK6 (CA‐MKK6), the upstream activator of p38 MAPK, was co‐transfected with p38 MAPK into U2OS cells. In these cells, p38 MAPK was phosphorylated by CA‐MKK6 but HO‐1 was not induced, indicating that p38 MAPK activation is not sufficient for HO‐1 expression. Taken together, our findings suggest that PGA2 induces expression of HO‐1 via cooperative action of p38 MAPK and de novo synthesis of (an) unidentified protein(s). This research was supported by MRC from cell death disease research center funded by KOSEF.
Effect of Pathway‐Interconnectors in SEB Induced Apoptosos Related Events in Human PBMCs
Authors: Lillge Cayla , Klein Becka , Zindel Kristin , Mendis Chanaka
Published: June 2021

This study follows a molecular route to better understand the apoptosis related events that are key to the patho‐mechanism of Staphylococcal Enterotoxin B (SEB) in human peripheral blood mononumclear cells (PBMC). The pathway interconnector, JNK, was identified and may have crucial inhibitory effects on the unwanted SEB induced apoptosis. Our attempt was to inhibit JNK by SP600125, evaluate those attempts by altering a set of known apoptosis related genes, and analyze expression patterns by conducting RT‐PCR. The studied genes include Caspase 1, Caspase 3, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Caspase 10, Heperanse precursor (HEP), SOD, and USP. Further analysis and confirmation was conducted by carrying out a set of protein expression analysis. We believe JNK has the ability to sustain longer lasting inhibitory effects and that it may benefit other experimental models that focus on disease prevention and diagnostics.

Dr. Marti Jett ‐ ASBMB
Oxidative Stress Injury To PC12 Cells Under Glycating Conditions
Authors: Habib Nawaf , Mungre Sue
Published: June 2021

A major complication in diabetes is loss of peripheral nerve function, termed as diabetic neuropathy. Glycation of cellular proteins by reducing sugars contributes to neuronal death and loss of nerve function. Hyperglycemia present in diabetes leads to increased production of methylglyoxal (MG), a strong glycating agent, forming advanced glycation end products (AGEs). The accumulation of AGEs has been implicated in several long‐term diabetic complications. One of the mechanisms involved is oxidative stress. We used PC12 cells treated with MG to determine the mechanism(s) involved in apoptosis of cells under glycating conditions. MG at 0.8 mM caused 70% cell death. The effect was observed as early as 6 hours of treatment. Caspase activity was increased in treated cells, indicating that MG caused apoptosis of cells. Treatment with curcumin, a compound from turmeric known for its antioxidant properties, inhibited the effect of MG and 80% of treated cells remained viable. Treatment with glutathione showed a similar, although less effective protection (60% survival). These results suggested that MG may cause oxidative stress to cells. We are currently studying the expression of iNOS and glutathione metabolizing enzymes. Our studies suggest that restoring redox balance in the cell may be an effective way of reducing cell death observed under glycating conditions.
Structure‐Activity Comparison of the Cytotoxic Properties of Diethylmaleate and Chemical Analogs
Authors: West James D , Stamm Chelsea E
Published: June 2021

Molecules that contain α,β‐unsaturated carbonyls have a wide variety of uses in industry, molecular medicine, and biochemical research. Ester derivatives of maleic acid are commonly utilized to modify sulfhydryl groups within proteins due to their reactive (i.e., electrophilic) properties. In order to understand the effects of these maleic acid derivatives and structural analogs on cells, we have compared the cytotoxicities of diethylmaleate with molecules containing variations in the length of aliphatic chain linked to the ester bond as well as the type and orientation of the α, β‐bond that reacts with sulfhydryl groups. While each molecule that contains an electrophilic center causes apoptotic cell death in human RKO colorectal carcinoma cells, our results reveal significant differences in the cytotoxic potency of some molecules. Particularly, molecules that contain longer aliphatic chains linked to the ester bond are more potent at inducing cell death, as are molecules composed of a trans‐double bond (i.e., fumarate ester derivatives). Although the chemical rationale for the cellular effects of these molecules is still unclear, these results suggest that bond type, orientation, and lipophilicity have a pronounced influence on the cytotoxic potencies of electrophilic molecules containing α, β‐unsaturated carbonyls.
Signaling through the Aspergillus nidulans orthologue of PKC mediates septum formation
Authors: Ogburn Erinn , Hill Terry W. , Jackson‐Hayes Loretta , Loprete Darlene M. , Chavez Britanny , Groover Chassidy , Pluta Michael
Published: June 2021

We have shown that the Aspergillus nidulans orthologue of protein kinase C (PkcA) participates in regulating cell wall integrity (CWI) and localizes at sites of cell wall synthesis, including growing hyphal tips and septa. To better understand the mechanisms by which PkcA localizes to tips and septa, we have observed the formation of cortical rings at sites of septation by fluorescently tagged PkcA in hyphae defective in expression of other proteins necessary for septum formation. In addition, we have co‐imaged PkcA and other septation proteins bearing complementary fluorescent tags. Here we report that localization of PkcA to septa lies “downstream” of the functions performed by MobA (Mob1p orthologue), TpmA (tropomyosin), SepA (formin), SepD, SepG, and proteins encoded by two other not‐yet‐cloned Sep loci. In the absence of function of these proteins, PkcA cortical rings were not observed. PkcA localization lies “upstream” of MyoB (myosin II orthologue), the A. nidulans orthologue of Bud4p (in yeast, a bud site selection marker), and a protein encoded by a third not‐yet‐cloned Sep locus. PkcA cortical rings still form in the absence of function of these proteins, though septa do not develop. SepA, TpmA, MyoB, and MobA all appear to colocalize with PkcA during normal septum formation. Studies with other septation‐related proteins are ongoing.
Replication‐dependent interactions of RECQ4 with MCM holocomplex
Authors: Liu Yilun
Published: June 2021

Mutations in RECQ4, a member of the RecQ family of DNA helicases, have been linked to the progeroid disease Rothmund‐Thomson Syndrome (RTS). Attempts to understand the complex phenotypes observed in recq4 deficient cells suggest a potential involvement in DNA repair and replication, yet the molecular basis of the function of RECQ4 in these processes remains unknown. Here, we report the identification of a highly purified chromatin bound RECQ4 complex from human cell extracts. We found that essential replisome factors MCM10, MCM2‐7 helicase, CDC45 and GINS are the primary interaction partner proteins of human RECQ4. Importantly, complex formation and the association of RECQ4 with the replication origin are cell cycle regulated. Furthermore, we demonstrate that MCM10 is essential for the integrity of the RECQ4‐MCM replicative helicase complex. MCM10 interacts directly with RECQ4 and regulates its DNA unwinding activity, and that this interaction may be modulated by cyclin‐dependent kinase (CDK) phosphorylation. Thus, these studies show that RECQ4 is an integral component of the MCM replicative helicase complex participating in DNA replication in human cells.
CHARACTERIZING SEX DIFFERENCES IN miRNA EXPRESSION IN LEFT VENTRICULAR HYPERTROPHY IN MICE
Authors: Davidson Janeth Ordonez , VanderPloeg Daniel , Waikel Rebekah L
Published: June 2021

Recent research has shown that microRNAs (miRNAs) are highly regulated during cardiac hypertrophy. Some miRNAs such as miR21, 195, and 208 promote hypertrophy, whereas other miRNAs such as miR1 and 133a inhibit hypertrophy. To date no comparison of sex dependent cardiac miRNAs has been described. Estrogen has been shown to play a protective role in left ventricular hypertrophy and heart failure in both humans and rodent models. To determine sex specific regulation of miRNAs during cardiac hypertrophy we compared levels of miRNAs in a surgical mouse model for cardiac hypertrophy. Male and female C57BL6 mice underwent surgery to band their aortas, transaortic constriction. Banding results in pressure overload, which leads to left ventricle hypertrophy. Five weeks post surgery hearts were weighed and total RNA was extracted. The female hearts exhibited a lesser degree of hypertrophy in response to pressure overload. Real‐time PCR Analysis has revealed sex specific patterns of miRNA expression in left ventricular hypertrophy. An understanding of sex specific regulation of miRNAs in the heart could have a far‐reaching impact on the diagnosis and treatment of heart disease in both women and men.
Elucidating the regulation and dynamics of O‐GlcNAc during signal transduction
Authors: Carrillo Luz Damaris , Mahal Lara K
Published: June 2021

The ability of cells to respond to their microenvironment is controlled by a complex communication system. Cell signaling utilizes a series of post‐translational events to regulate and coordinate cellular activities. Although phosphorylation is thought to be the key regulator of these events, recent findings implicate the O‐GlcNAc modification as an additional control mechanism. Modulation of signal transduction requires compartmentalization of the kinases and phosphatases. Based on the evidence of subcellular localization of OGT isoforms, the diversity of O‐GlcNAcylated proteins upon stimulation, and its role during insulin signaling, it can be hypothesized that O‐GlcNAc is involved and regulates signal transduction in a compartmentalized manner.

To investigate the spatio‐temporal dynamics of O‐GlcNAc in cell signaling, we have generated a series of genetically encoded O‐GlcNAc reporters based on fluorescence resonance energy transfer (FRET). These reporters and localized variants have allowed compartment specific visualization of O‐GlcNAc activity in the nucleus, cytoplasm and plasma membrane. Herein we describe these reporters and their use to examine O‐GlcNAc dynamics in signaling using serum stimulation and environmentally relevant concentrations of arsenite. Acute exposure to arsenite through drinking water has become an environmental health concern worldwide. Our results imply a complex regulation of O‐GlcNAc on a fast timescale that is tied to more canonical kinase pathways.
Post‐transcriptional regulation of gene expression in response to iron deficiency in S. cerevisiae
Authors: Vergara Sandra Viviana , Thiele Dennis J
Published: June 2021

The yeast proteins Cth1 and Cth2 belong to the Tristetraprolin (TTP) family of AU‐rich Element (ARE)‐containing mRNA‐destabilizing proteins. In response to iron (Fe)‐deficiency, Cth1 and Cth2 coordinately promote the degradation of mRNAs encoding many proteins involved in highly Fe‐dependent metabolic pathways. As a consequence, they promote a genome wide remodeling of metabolism that permits the utilization of the limited Fe in essential processes, while lowering the metabolic Fe‐demand of other non‐essential pathways.

Our recent data indicate that while Cth2 expression gradually but robustly increases during Fe‐deficiency over time, Cth1 is more rapidly and only transiently expressed soon after the Fe‐deficient conditions are imposed. Furthermore, our data indicate that the CTH1 and CTH2 transcripts are themselves subject to ARE‐mediated degradation by the Cth1 and Cth2 proteins, creating an auto‐ and cross‐regulatory circuit responsible for differences in their expression. We believe that the tight regulation of Cth1 and Cth2 is a cellular strategy to adjust to either mild or transient versus severe or sustained forms of Fe‐deficiency. Our current work seeks to understand how auto‐ and cross‐regulation of Cth1 and Cth2 contributes to the cellular adaptation to Fe‐deficiency. This work is supported by NIH grant GM41840 (D.J.T) and NIH Pre‐Doctoral Fellowship 1F31‐DK081304 (S.V.V).
Role of RNA‐binding protein HuR and CUGBP1 in LPS‐induced Interleukin‐6 Expression in Macrophages
Authors: Zha Weibin , Wang Guangji , Pecora Beth S. , Studer Elaine , Hylemon Phillip B , Pandak Williams M , Zhou Huiping
Published: June 2021

Interleukin 6 (IL6) is an important proinflammatory cytokine involved in the pathogenesis of many inflammatory diseases. RNA‐binding proteins, HuR and CUGBP1, regulate the mRNA stability and protein translation through direct binding to the AU‐rich domain or CUG repeats in the 3 untranslated region (3 UTR) of target genes. However, the exact role of HuR and CUGBP1 in LPS‐induced IL6 expression remains unknown. The current study examined the potential role of HuR and CUGBP1 in posttranscriptional regulation of IL6 expression during LPS‐mediated inflammatory response in macrophages.

Methods

Mouse macrophages were used in this study. The IL6 mRNA and protein levels were determined by real time RT‐PCR and ELISA, respectively. Protein levels of HuR and CUGBP1 were determined by Western blot analysis. The interaction between RNA binding proteins and IL6 mRNA was identified by pull‐down assay and immunoprecipitation.

Results

LPS increased cytosolic HuR and CUGBP1 protein levels and the binding of HuR and CUGBP1 to 3 UTR of IL6 mRNA. LPS‐mediated IL6 expression was promoted by HuR overexpression and CUGBP1 silencing, but inhibited by HuR silencing and CUGBP1 overexpression.

Conclusion

HuR and CUGBP1 compete for binding to the 3 UTR of IL6 mRNA and differentially regulate IL6 expression at the posttranscriptional level during LPS‐mediated inflammatory response. This work is supported by NIH and Jeffress grant.
Alternative Editing of Cytochrome C Oxidase III mRNA in Trypanosome Mitochondria Generates Protein Diversity
Authors: Sykes Steven Edric , Hajduk Stephen Louis
Published: June 2021

Alternative editing of the cytochrome c oxidase III pre‐mRNA in Trypanosoma brucei (T. brucei) produces a novel mitochondrial membrane protein, alternatively edited protein‐1 (AEP‐1). Immunofluorescence microscopy reveals that this unique protein associates with two distinct regions intracellularly. AEP‐1 is distributed throughout the mitochondria and is also observable as a discrete condensed structure associated with the detergent resistant tripartite attachment complex adjacent to the mitochondrial DNA. Biochemical fractionation and Blue Native PAGE (BNPAGE) analysis has also shown that AEP‐1 associates with the detergent soluble mitochondrial membranes. To further investigate the role of AEP‐1 localized to the mitochondrial membrane, intact mitochondria were isolated using a hypotonic lysis method and the organelles were treated with a tandem nonionic detergent treatment to separate the mitochondrial matrix from the membrane fraction. Separation of this membrane fraction by BNPAGE and Western Blot analysis revealed the association of AEP‐1 with a high molecular weight membrane complex. The AEP‐1 complex was purified further using anion exchange chromatography and the resulting BNPAGE band was subjected to ESI‐ion trap‐tandem mass spectrometry. Sequencing revealed several proteins including the oxoglutarate dehydrogenase (E1) and the dihydrolipoyl succinyltransferase (E2) subunits of the 2‐oxoglutarate dehydrogenase complex (2ODC). The AEP‐1 complex did not retain the same NAD+reductive capacity as the bonafide 2ODC. These data suggest that AEP‐1 associates with proteins that may have bifunctional roles in T. brucei mitochondria.
Global identification of the Gene‐Specific Association of the Exon‐Exon Junction Complex in Drosophila
Authors: Haque Nazmul , Blanchette Marco
Published: June 2021

Exon Junction Complex (EJC) is a large multi‐protein complex that assembles 20‐24nt upstream of exon‐exon junction following splicing of pre‐mRNAs. EJC is involved in several aspects of mRNA metabolism, including mRNA export, translation and Nonsense Meditated mRNA Decay (NMD). We have recently found in Drosophila that EJCs assemble on specific spliced mRNAs, which prompted us to globally characterize the mRNAs bound by EJCs in Drosophila S2 cells.

RNAs bound to eIF4AIII and Y14, two of the core components of EJC, were immunopurified from stable S2 cell lines expressing affinity‐tag version of the proteins and used to hybridize Drosophila whole genome tiling array. We have identified that only ~10% of expressed mRNAs associate with an EJC. Surprisingly, our global analysis have reveal that some mRNAs are bound only by eIF4AIII or Y14, suggesting that these proteins might be part of complexes distinct from EJCs. In addition, global gene expression analysis in eIF4AIII‐depleted S2 cells by RNAi identified an over‐represented population of mRNAs bound by eIF4AIII with reduced expression while no significant overlap was observed in the up‐regulated mRNAs, suggesting a role for eiF4AIII in stabilizing these mRNAs. Bio‐computational analyses are underway to identify cis‐acting elements that could trigger gene‐specific EJC deposition.

This project is supported by Stowers Institute for Medical Research
Kinetics of substrate binding to human Cytochrome P450 3A4: A study with Fluorol‐7GA, a fluorescent allosteric ligand
Authors: Rumfeldt Jessica Anne Osborne , Halpert James R , Davydov Dmitri R
Published: June 2021

Stopped‐flow fluorescence and rapid scanning absorbance techniques were used to explore the mechanism of interactions of cytochrome P450 3A4 with Fluorol‐7GA, a fluorescent allosteric ligand. When monitored by rapid scanning absorbance spectroscopy, mixing of the enzyme and substrate resulted in a single exponential increase in the high spin fraction of the heme protein with a rate constant of ~10 s−1. A lack of effect of the substrate concentration on the rate constant indicates that the observed process does not reflect the formation of the enzyme‐substrate complex per se but rather some transition in the pre‐formed complex that results in the displacement of the spin equilibrium. Stopped‐flow experiments, which monitored the intensity of fluorescence of Fluorol‐7GA, showed that the intensity decrease observed in steady state measurements takes place in the dead time of the instrument (≤2ms), and its kinetics are therefore too fast to be observed with our technique. Similar to the binding mechanism proposed from steady state measurements, the different results observed by stopped‐flow absorbance and fluorescence indicate a multistep binding process, namely initial very fast association followed by a first order structural transition that affects the spin state of the heme protein. (Supported by NIH grant GM054995).
Catalysis by Microsomal Cytochrome P450 2B4 Proceeds via a “Stable Hydroperoxo” Intermediate Identified by Freeze Quench EPR
Authors: Waskell Lucy , Pearl Naw May , Im Sang‐Choul , Kunz Ryan , Ragsdale Stephen W.
Published: June 2021

Under single turnover conditions, catalysis by cytochrome P450 2B4 (P450) is 10 to 100‐fold faster in the presence of cytochrome b5 (b5) than in the presence of P450 reductase (CPR). Both redox partners reduce oxyferrous P450 at the same rate, indicating that a step downstream of reduction accounts for the faster rate by b5. We hypothesized that catalysis is faster in the presence of b5 because it causes an allosteric change in the distal heme pocket that facilitates delivery of the second proton to the hydroperoxo (Fe+3OOH)− intermediate. This results in heterolytic O2 bond cleavage and formation of the active oxidizing species. Conversely, we postulated that catalysis in the presence of CPR is slower because the second proton is delivered more slowly to the hydroperoxo intermediate. This predicts that the hydroperoxo intermediate will accumulate in P450 when the oxyferrous enzyme is reduced by CPR. To test this hypothesis, a prereduced P450‐CPR complex was rapidly mixed with O2 saturated buffer. The EPR spectra of the rapidly freeze‐quenched samples demonstrated an end‐on ferric hydroperoxo intermediate, with g values of 2.32, 2.20, and 1.948, which is consistent with g values observed with hydroperoxo species of P450 camphor and P450 2B4. In summary, catalysis by P450 in the presence of CPR but not b5 proceeds via a stable hydroperoxo intermediate under ambient conditions.
Expression, Purification, and Characterization of a New Sulfhydryl Oxidase from Trypanosoma brucei
Authors: Styer Amy , Kodali Vamsi , Daithankar Vidyadhar , Thorpe Colin
Published: June 2021

A sulfhydryl oxidase from Trypanosoma brucei was expressed, purified and studied enzymologically for the first time. The protein is homologous to ALR (augmenter of liver regeneration), an essential flavin‐linked enzyme which catalyzes disulfide bond formation in the mitochondrial intermembrane space (IMS) of eukaryotes. Trypanosomes lack a gene for Mia40, a necessary redox partner with ALR in yeast and mammals. Future research will determine how trypanosomes compensate for the lack of Mia40, and if this sulfhydryl oxidase has the same biological localization and function as ALR.

We have shown that six of seven cysteines in the 33 kDa trypanosomal ALR are present as disulfide bonds. In oxygen electrode assays, the enzyme catalyzed disulfide‐bond formation in the model substrate dithiothreitol (DTT), but not in the monothiols glutathione and cysteine. Molecular oxygen was a much better terminal electron acceptor for trypanosome ALR than for human ALR (TbALR Km = 15±1μM; some 15‐fold lower than the human enzyme). Further studies are aimed at characterizing the kinetic mechanism of this flavoenzyme by stopped flow spectrophotometry.

Understanding trypanosome redox biochemistry is important because Trypanosoma brucei, the causative agent of African Sleeping sickness, kills nearly fifty thousand people annually and current treatments are expensive and incur serious side reactions. This exploration of trypanosome mitochondrial IMS disulfide‐bond formation may lead to biomedical advances in the fight against trypanosome diseases.

Funded by a Beckman Scholars Fellowship to A.S. and NIH GM26643 to C.T.
Kinase Activity‐Independent Regulation of GRK in Zebrafish Early Development
Authors: Jiang Xi , Yang Peng , Ma Lan
Published: June 2021

G protein‐coupled receptor (GPCR) kinases (GRKs) are known as a family of serine/threonine kinases that function as key regulators of GPCRs. In our researches, we found that down‐regulation of GRK in zebrafish embryos with GRK morpholino results in developmental early arrest, and interestingly, that this arrest can be rescued by exogenous expression of a GRK kinase‐dead mutant, K220R. A physical interaction between GRK and cyclin B1 regulator PTCH1, stimulated by Hedgehog, rather than GRK‐mediated phosphorylation of downsteam targets, appears as the underlying mechanism. We identify residues 262‐379 as the PTCH1 binding region (BP). Interaction of GRK, K220R, and BP with PTCH1 reduces the association of PTCH1 with cyclin B1 and disrupts PTCH1‐mediated inhibition of cyclin B1 nuclear translocation; while the PTCH1‐binding deficient GRK mutant (D312‐379) does not. Cell cycle and cell proliferation assays show that overexpressing PTCH1 remarkably inhibited cell growth and this effect could be attenuated by GRK, K220R or BP, but not D312‐379. In vivo studies show that BP, as well as the nuclear‐localizing cyclin B1 mutant, is effective in rescuing the early arrest phenotype in GRK knockdown embryos, but D312‐379 is not. Our data thus reveal a novel kinase activity‐independent function for GRK and establish a role for GRK as a cell‐cycle regulator during early embryonic development.
Nuclear‐mediated function of Chmp1A in the regulation of ATM signaling activity for the control of human pancreatic tumor cell growth
Authors: Manohar Sumanth , Harlow Matthew , Mackie Christopher Ryan , Li Jing , Park Maiyon
Published: June 2021

Chmp1A is a member of the ESCRT‐III family that functions in the sorting of membrane proteins. Chmp1A was shown to regulate cell cycle progression and chromatin condensation. We recently reported that Chmp1A functions as a tumor suppressor by regulating P53 in pancreatic tumor cells. P53 is a substrate of ataxia‐telangiectasia mutated (ATM) kinase and the dynamics of ATM activation is closely related to chromatin modification. Thus, we hypothesize that Chmp1A, through its nuclear localization, regulates ATM signaling activity and subsequently pancreatic tumor growth. Our preliminary data indicates that Chmp1A overexpression led to an increase in phospho‐ATM. Immuno‐staining identified the co‐localization of ectopically induced Chmp1A with phospho‐ATM and P53 whose intensity closely reflected that of Chmp1A expression. ATM kinase assay indicates that Chmp1A overexpression increased ATM kinase activity as shown by an increase in the level of phospho‐P53 compared to control. In addition, ATM inhibitor‐treatment partly abolished Chmp1A mediated‐growth inhibition and P53 stabilization. We are testing the significance of nuclear localization signal (NLS) of Chmp1A by using various deletion constructs. We will discuss the significance of the NLS domain of Chmp1A on signaling activity of ATM and P53, and tumor cell growth.

This research is supported by NIH 5P20RR016477, WV‐INBRE and RR020180‐02, COBRE.
Guilt by association: functional annotation of TM0486 from Thermotoga maritima by identification of its bound ligands
Authors: Palanski Brad A , Snider Mark J
Published: June 2021

As the result of recent structural genomic initiatives, the Protein Data Bank contains approximately 3000 protein structures classified as having unknown function. The crystal structures of over 80 of these proteins show that they bind at least one ligand of unknown chemical identity. We analyze one such protein (TM0486 from Thermotoga maritima) and hypothesize that the isolation and determination of the identity of the unknown bound ligands will suggest a function for this protein. E. coli BL21 DE3 cells were transformed with expression plasmids, protein expression was optimized, and the His6‐tagged protein was purified by affinity chromatography. The protein was denatured by ethanol to release the ligands, and the precipitated protein was removed by centrifugal filtration. The filtrate was analyzed by electrospray liquid chromatography‐mass spectrometry. Preliminary chromatograms show several peaks with promising m/z ions that suggest that the ligands may be isolated. Once verified, additional analyses by mass spectrometry and nuclear magnetic resonance should reveal their identity and allow a functional hypothesis for TM0486 to be proposed and examined.
Formulation and Characterization of Antibody Coated Poly(lactic‐co‐glycolic acid) Nanoparticles to Target Metastatic Cancer
Authors: Thamake Sanjay I , Ranjan Amalendu , Raut Sangram L , Gryczynski Zygmunt , Vishwanatha Jamboor K
Published: June 2021

Chemotherapy is the only available option for treatment of metastatic cancers. However, increasing evidences of drug resistance and non‐specific toxicity of these agents limits their therapeutic outcomes. Nanoparticle mediated targeted therapies is one of the attractive therapeutic option to overcome these problems. We have formulated curcumin encapsulating poly(lactic‐co‐glycolic acid) (PLGA) nanoparticles (NPs) coated with annexin A2 antibody to target prostate cancer as a model for this project. We characterized these NPs for particle size, stability, efficiency of antibody attachment, surface charge etc. We performed cellular uptake studies in prostate cancer cells and quantified the amount of drug taken up by the cells. Our results showed that the average size of these NPs is 151nm and 179nm before and after attachment of antibody respectively. The cytometric analysis showed that approximately 87.5% of NPs were coated with annexin A2 antibody. The antibody coated NPs have nearly neutral surface charge and are readily taken up by the cell. When injected intravenously in athymic mice bearing xenografts of DU‐145 prostate cancer cells (annexin A2 positive), the annexin A2 antibody coated NPs preferentially localize to the tumor compared to uncoated NPs. In summary, we have formulated a novel targeted drug delivery system which will be a potent tool for therapeutic intervention of metastatic cancer.
Proximity of proregion anionic residues to the mature region maintains proCryptdin‐4 inhibition
Authors: FIGUEREDO SHAREL MENEZES , Gable Jonathan E , Kim Judy E , Ouellette Andre J
Published: June 2021

α‐Defensins are synthesized as inactive precursors that must undergo proteolytic conversion to mature bactericidal peptides. Acidic amino acids near the proCrp4 N terminus mediate inhibition of proCrp4 as shown by selective deletion and charge neutralizing mutagenesis (J. Biol. Chem. 284: 6826–6831, 2009). To study the inhibitory mechanism, we have compared the proximity of those anionic residues to the Crp4 component of native and mutant proCrp4 molecules using Förster resonance energy transfer (FRET). To provide FRET probes and enable FRET conformational studies in native and charge neutralized proCrp4, single Crp4 Trp substitutions were introduced in the α‐defensin moiety at V58W, F66W, and at the C terminus, and a Cys residue was added to proCrp4 N terminus (CN) for IAEDANS labeling. ProCrp variants had bactericidal activities comparable to the parent molecules and were processed correctly by MMP‐7. FRET efficiency and distances between the FRET pairs in native and charge‐neutralized proCrp4 were determined. The N and C‐terminal regions of inactive CN(V58W)‐proCrp4 are closest with a FRET pair distance of 11.5 Å. In the active, bactericidal CN‐(DE/G‐V58W)‐proCrp4 mutant, this distance increased to 14.3 Å. Thus, mutations that relieve proCrp4 inhibition do so without markedly repositioning the relation of the proregion N terminus to the Crp4 component of the precursor.

Supported by NIH grants DK044632 and AI059346..
Endocytic nanodiamond for the labeling and tracking of cancer cells
Authors: Chao Jui‐I , Liu Kuang‐Kai
Published: June 2021

Nanodiamond (ND) is carbon nanomaterial developing for biological applications. We found that ND particles were taken into cells by macropinocytosis and clathrin‐mediated endocytosis pathways. ND particles were equal separating into two daughter cells of cell division approximately. Finally, the cell retained a single ND cluster in cytoplasm after sub‐cultured for several generations. Interestingly, ND clusters were carried inside of cell but without inducing damages after long‐term cell culture. Together, these findings provide that endocytic ND particles are non‐cytotoxic in cell division, which can be applied for the labeling and tracking of cancer cells.
Cytotoxicity Screening of Fungal Endophytes Using Metastatic Met‐1 Cells
Authors: Lee Kyung‐Seon Sunny , Adelzadeh Lily , Lloyd Margaret , Sweeney Colleen , Adams James D , Cheung Anthony TW
Published: June 2021

Breast cancer is the second most common cause of death in US women. The mortality significantly results from metastasis from primary sites to other organs. Chemotherapy is a major component of breast cancer treatment. Natural products have provided resources for new anticancer agents. However, many drugs on the market are limited by drug resistance, making the search for new anticancer agents from novel resources highly desirable. In this regard, endophytes represent an intriguing new source. These are symbiotic microorganisms harboring within tissues of vascular plants without causing an apparent, immediate infection in their host. Fungal endophytes from medicinal plants used by American Indians in the central valley of California were grown in potato dextrose broth at 28°C for 1 to 2 weeks. The culture media were filtered to remove fungal mycelia and spores (spent media). Each spent medium was tested against Met‐1 cells (a mouse mammary tumor cell line) using the formazan formation assay. Among 15 endophyte cultures tested, 6 spent media exhibited over 30% cytotoxicity including 3 spent media that showed over 50% cytotoxicity against metastatic Met‐1 tumor cells. Future studies will examine their anti‐angiogenic and anti‐metastatic effects in the Met‐1 mammary tumor mouse model.
Mitotic arrest induced by propargylic enol ethers derivatives
Authors: Luci Carla Patricia Rios , León Leticia G. , Montero Juan C. , Tejedor David , Pandiella Atanasio , García‐Tellado Fernando , Padrón José M.
Published: June 2021

The generation of diversity‐oriented libraries is a valuable tool for the discovery of new anticancer agents. The objective was to evaluate the anti‐proliferative activity of a set of 27 propargylic enol ethers derivatives in cancer cells. The antiproliferative activity was performed against the human solid tumor cell lines SW1573, HBL‐100 and HeLa. Additionally, cell cycle phase distribution was investigated by flow cytometry and protein expression by western blotting. The most active compound gave GI50 values in the range of 0.24–2.8 μM in all the cell lines. Cell cycle studies showed the ability of the drug to induce a prominent G2/M arrest. The cell arrest occurred with little evidence of DNA damage, as determined by the lack of γ‐H2AX signal. The persistence of cyclin B1 in HeLa cells correlates with their entry into mitosis. High levels of histone H3 phosphorylated at serine 10 confirmed that cells accumulate in mitosis in a time‐dependent manner. Mitotic arrest was consistent with the activation of BubR1 which delayed the progression to anaphase. Our findings provide insight into the mechanism of chemotherapeutic action of this family of compounds.

Funding co‐financed by EU‐FEDER: MICIIN (CTQ2008‐06806‐C02–01/BQU, CTQ2008‐06806‐C02–02/BQU, BFU2006‐01813/BMC), MSC (RTICC RD06/0020/1046, RD06/0020/0041); ACIISI (PI 2007/021); FUNCIS

(PI 01/06, 35/06). LGL: MSC‐FIS contract. JMP: MEC‐FSE RyC contract.
Identification of the putative drug binding site for novel agonists of the integrin CD11b/CD18
Authors: Stoub Darren G. , Gupta Vineet , Faridi Mohd. , Maiguel Dony , Barth Constantinos , Schurer Stephan
Published: June 2021

CD11b/CD18, one of the α/β integrin heterodimers, is the predominant integrin found in neutrophils, macrophages and monocytes and is involved in important cellular functions, such as cell signaling and cell‐ECM adhesion. We report the discovery of a class of small molecules that contain a 5‐(furanyl‐2‐methylene)‐2,3‐dioxo‐thiazolidine motif. Using an in vitro cell‐adhesion assay and an in vivo murine inflammation assay, the compounds behave as agonists of integrin activation with maximum ex vivo EC50 values of 10.5 μM and high in vitro selectivity for CD11b/CD18 integrins. Using in silico and site‐directed mutagenesis experiments, we report the location of the drug binding site for these compounds in a hydrophobic cleft of the ligand‐binding αA domain, implying an allosteric mechanism on the integrin activation mechanism. These small molecule agonists of integrin function have potential as anti‐inflammatory drugs and could be utilized to treat renal ischemia and reperfusion injury.

The work was supported in part by two NIH grants and the Van Houten Memorial Fund.
Mechanisms of Successful Drug Action
Authors: Swinney David C , Anthony Jason
Published: June 2021

The mechanism of drug action (MoA) couples molecular interactions to a physiological response. MoA includes the molecular target and the mechanism that communicates the response. The MoA defines the molecular descriptor (such as KI, kon, koff, and conformational state) for a biochemically efficient mechanism and optimal therapeutic index. Target‐focused drug discovery that is driven solely by equilibrium binding, without consideration for MoA and the molecular descriptors for an optimal therapeutic index, is a common practice in drug discovery today. In order to better understand the role of MoA in drug discovery we analyzed the MoAs of New Molecular Entities (NMEs) approved by the US FDA between 1999 and 2008. Data was collected from the product labels approved by the FDA and reports describing the discovery process. We identified that approximately 20% of the NMEs were first in class and/or had novel MoAs. Target‐focused approaches accounted for less than half of the novel MoAs and these targets were generally biologically well validated with molecular descriptors that included irreversible and slow kinetics, state dependence, induced conformations and equilibrium binding. The majority of the novel MoAs were identified from compound‐focused drug discovery by compound screening in physiological assays and modification of natural substances.
ABO Blood Group Genotyping by PCR‐RFLP
Authors: Wheeler Travis Walter , Waikel Rebekah L
Published: June 2021

Traditional blood typing involves mixing a blood sample with antibodies A and B and observing any antigen – antibody reactions (agglutination). Blood borne diseases, ranging from HIV to Hepatitis make blood typing in a classroom setting risky. The purpose of this research is to design a DNA based ABO blood typing protocol that does not require the use of blood. DNA can easily be obtained through a cheek swab. Blood type can be determined by the presence or absence of glycosyl transferase genes, whose protein products add carbohydrate antigens to proteins and lipids on the surface of erythrocytes. Analysis of the known gene sequences of the ABO glycosyl transferase genes revealed detectable sequence differences found in exons 6 and 7. We amplified exons 6 and 7 of the ABO genes via the Polymerase Chain Reaction (PCR). We then digested the amplimers with sequence specific restriction endonuclease and separated resulting DNA bands by agarose gel electrophoresis (RFLP). Based on predicted DNA band sizes, we have been able to successfully determine blood types through PCR‐RFLP of genomic DNA. To our knowledge, this is the first PCR‐RFLP experimental test for blood type. This genotyping scheme is currently being used in undergraduate laboratory courses to assess learning objectives.
Phylloquinone, MK‐4 and MK‐7 contents of some commercially available oral supplements
Authors: Ferland Guylaine , Bartle William R. , Fournier Chantal , Baker Christine
Published: June 2021

Vitamin K (VK), which exists in two natural forms – phylloquinone (K1) and the menaquinone family (K2) ‐ is now associated with a number of physiological functions in addition to its role in heamostasis. Furthermore, there is growing evidence that VK supplementation could help stabilize coagulation times (INR) in some patients on warfarin therapy. In light of this, supplements for K1, MK‐4 and MK‐7 are now commercially avalaible. Here we report the concentration accuracy of such supplements. Tested products claimed to contain 100 mcg (n=2) or 5 mg (n=1; prescription drug) of K1, 120 mcg of MK‐4 (n=1) and 100 mcg of MK‐7 (n=1), respectively. Assessments (by HPLC) were conducted on 10–18 pills/supplement. Means±SD/pill and % CV were established in each case. Contents of the two 100 mcg K1 supplements were 72.8±6,0 and 93.9±2,6 mcg/pill, respectively and that of the 5 mg K1 supplement, 4.7±0.21 mg/pill. Values for the MK‐4 and MK‐7 supplements were 135.8±90.2 and 421.0±33.1 mcg/pill, respectively. Percent CV were 8.2, 2.8 and 4.5 for the respective K1 supplements, and 66.4 and 7.8 for the MK‐4 and MK‐7 supplements. Results form this study points to a lack of concordance between the VK contents and the label claim of some commercially available supplements. Given the increasing number of individuals consuming these supplements, greater control should be exerted to ensure the quality of the products. Supported by CIHR.
Investigating the Complex Formation between Boron and Riboflavin using Electrospray Ionization Mass Spectrometry and Fluorescence Spectroscopy
Authors: Kim Danny H , Faull Kym F , Eckhert Curtis D
Published: June 2021

Boron, found richly in green vegetables, fruits, peanuts and legumes, has high affinity for cis‐diol functional groups found in nucleotides, sugars, as well as in vitamins such as riboflavin. For the first time, electrospray ionization mass spectrometry in the negative ion mode was utilized to observe the complex formation between boron and riboflavin. In addition, fluorescence spectroscopy was used to investigate whether boron can affect the binding affinity between riboflavin and riboflavin binding protein from chicken egg white. The results of this study can help support the conclusions made from previous work in which a correlation between boron exposure and riboflavin deficiency in chicks and hens was demonstrated. The results of this work are relevant for the treatment for human riboflavin deficiency that causes dry skin, photo‐sensitivity and inflammation of the lining of mouth and tongue.
Improved HPLC method for vitamin C analysis in serum
Authors: Craft Neal E. , Park Heasoon
Published: June 2021

Vitamin C (VC) is necessary for development of normal connective tissue and human health. VC HPLC separations have remained nearly the same for 25 yrs. Recent advances in column technology have permitted some improvements in the methodology.

Objective

To improve serum VC separation, sensitivity and reproducibility w/o ion‐pairing agents or gradients.

Methods

Several HPLC columns, possible internal standards and two buffers at different concentrations were examined. Both UV and electrochemical (EC) detection was used.

Results

Most columns offered poor retention and peak shape of VC. Phenomenex Synergi Hydro‐RP column provided good retention (k=1.3) symmetry (A=1.1) and tailing (T=1.15). Tris(2‐carboxyethyl)phosphine (TCEP) was used as an alternate reducing agent to dithiothreitol. Hypoxanthine, selected as an internal standard, eluted isocratically at k=3.2 with UV absorbance at 245nm but no EC.

Discussion

The HPLC method modifications yield good retention and peak shape. TCEP provides a cost effective and superior reducing agent. Efficiency and reproducibility were improved by incorporation of an internal standard. The method is amenable to short narrow bore columns.
Cancer Chemotherapeutic Effects of Modified Sodium Silicate (Alkahydroxy/Alka V6)
Authors: Townsend Deana , White Lauren , Chesnut Troy , Duesler Sandra , Cisneros Ignacio , Richardson C. Reed , Vattem Dhiraj
Published: June 2021

A proprietary modified sodium silicate manufactured by Cisne enterprises Inc. (Odessa, TX) was evaluated for its ability to modulate various parameters relevant to establishment and progression of cancer. Antimutagenic effects were determined using Ames test. Prevention of colon cancer cell (HT‐29) attachment and growth was done using standard methods. Apoptotic induction was measured by DNA fragmentation (DNAF) assay. Malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) activity were measured using standard assays. Chemical structure determined by nuclear magnetic resonance (NMR) and infra‐red (IR) spectroscopy suggested that the product was a mixture of trimeric sodium silicate and sodium silicate pentahydrate. A dose‐dependent reduction in attachment (EC50 = 3450 ppm) and growth (EC50 = 4000ppm) of HT‐29 was observed. At low levels (250–25000 ppm) the product was able prevent various sodium azide induced mutations in Ames test. A dose dependent increase in DNAF suggested induction of apoptosis. A drop in MDA levels and increased in GHS, SOD and CAT activities suggested induction of antioxidant response. We conclude that the product may have cancer chemotherapeutic properties in vitro due to its unique structural and electrochemical properties. In vivo tests are imperative to determine true effectiveness. Funding source: Cisne Enterprises Inc.