bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional specialization in the forelimbs of two digging mammals: the American badger (Taxidea taxus) and groundhog (Marmota monax)
Authors: Moore Alexis Lee , Butcher Michael
Published: June 2021

Limb muscles are often studied for their properties related to locomotion, but studies of architectural properties in limb muscles of animals specialized for other behaviors, such as digging, are less common. This study quantified muscle architectural properties in the forelimbs of badgers (Taxidea taxus) and groundhogs (Marmota monax), two digging species from phylogenetically different clades (Carnivora vs Rodentia), and estimated maximum force production and power output of their forelimb muscles. Architectural properties measured included: muscle mass, belly length, volume, physiological cross‐sectional area, fascicle length and pennation angle. Both species showed massive elbow extensors and digital flexors that together accounted for approximately 60% of intrinsic forelimb muscle mass. The elbow extensors displayed a low degree of pennation with longer fascicles, an architecture consistent with appreciable shortening capability and high power. The digital flexors showed relatively greater pennation and shorter fascicle lengths in addition to compartmentalization of muscle heads for both force production and range of contraction. These findings suggest both the elbow extensors and digital flexor muscles in badgers and groundhogs have the capacity for high force during scratch‐digging, while the elbow extensors are better suited for high power during rapid digging movements. URC #3‐11.

Grant Funding Source: URC, Youngstown State University
Presence and neurochemical coding of intrinsic VIP/PACAP‐positive enteric neurons in the adult zebrafish
Authors: Uyttebroek Leen , Harrisson Fernand , Hubens Guy , Shepherd Iain T , Timmermans Jean‐Pierre , Van Nassauw Luc
Published: June 2021

In a previous study, detecting the co‐expression of neurochemical markers, five different neuronal classes were identified in the zebrafish enteric nervous system. Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) did not yield immunoreactivity (IR) in neuronal cell bodies, only in nerve fibers. At present, we attempted using an in vitro colchicine pretreatment prior to fixation to detect VIP and PACAP in neuronal cell bodies to determine peptidergic subpopulations in the ENS of the adult zebrafish. The proportional distribution of VIP and PACAP was studied using multiple immunofluorescence. Co‐expression with other neurochemical markers was examined. Treatment of colchicine enhanced IR for VIP and PACAP as such that IR was not only observed in neuronal varicosities, but also in neuronal cell bodies. A complete colocalization for VIP and PACAP was observed. VIP/PACAP‐expressing neurons accounted for ±20% of the total neuronal population. Coexpression of VIP/PACAP was observed with calretinin and calbindin (±20%), nNOS (±70%) and serotonin (±20%). ChAT‐IR was not observed after pretreatment with colchicine. These results complement and extend previous data regarding neurochemical properties of the zebrafish ENS. Further immunostainings are necessary to establish which of the subpopulations previous identified coexpress VIP and PACAP.
Extraocular Muscles Morphometry and their Surgical Importance
Authors: Villarreal‐Silva Eliud Enrique , Hinojosa‐Amaya José Miguel , Bazaldúa‐Cruz Juan José , Martinez‐Fernández David Alejandro , Elizondo‐Omaña Rodrigo Enrique , Guzmán‐López Santos
Published: June 2021

Extraocular muscles (EOM) are important references in strabismus surgery and in placement of intraorbital devices. We conducted a descriptive, observational, cross‐sectional and non‐comparative study that includes an analysis of the EOM morphometry and possible anatomical variances in twenty orbits. We report the length, width, and points of insertion of the EOM. No anatomical variations were found. With regard to the rectus muscles, it was found that the superior rectus and lateral rectus are the longest muscles and that the width difference between the superior and inferior rectus is greater than that between the medial and lateral rectus and that the point of insertion of the rectus muscles has a variable morphology. It was observed that, in the oblique muscles, the width of the superior oblique is comparatively the smallest and the inferior oblique morphology coincides with and complements what has been reported in previous studies. Knowledge of the detailed morphology of EOM is fundamental in strabismus surgery and represents a key factor for the innovation of surgical techniques and orbital procedures.
Investigating the interaction between the alpha subunit of DNA polymerase III and UmuD
Authors: Silva Michelle C , Ronayne Erin , Beuning Penny J
Published: June 2021

The alpha subunit of DNA polymerase III (DNA pol III) is the main replicative polymerase in Escherichia coli. Because alpha cannot copy damaged DNA, replication is stalled in presence of DNA lesions. To compensate, the cell initiates the SOS response, inducing the expression of at least 57 genes. The products of these genes are involved in DNA damage tolerance mechanisms, one of which is translesion synthesis, a process by which lesions are bypassed by specialized DNA polymerases with the ability to copy damaged DNA. DNA pol V, one of these polymerases, is composed of two proteins: UmuD'2, the RecA/ssDNA‐facilitated cleavage product of UmuD2, and UmuC, the polymerase subunit. UmuD and UmuD’ both interact with the alpha, beta processivity clamp, and epsilon proofreading subunits of DNA polymerase III, likely regulating DNA pol III activity in response to DNA damage (Sutton et al PNAS 1999 v 96 p 12373–12378). We are currently investigating the interaction between alpha and UmuD2. Thermofluor data suggest that UmuD2 binds alpha in a specific conformation, while UmuD cleavage assays show that alpha competes with the RecA/ssDNA filament for UmuD2 binding. We are identifying the binding site for UmuD2 by using a number of alpha mutations and truncations. Research supported by NSF Career MCB‐0845033.
DNA double‐strand breaks induced by high NaCl occur predominantly in gene deserts
Authors: Dmitrieva Natalia I , Cui Kairong , Zhao Keji , Burg Maurice B
Published: June 2021

High extracellular NaCl increases DNA double strand breaks (DSBs) both in cell culture and in vivo. The exact nature of the DSBs, as well as their location, has not been entirely clear, nor has it been evident how cells survive, replicate and maintain genome integrity in environments like the renal inner medulla in which the DSBs persist because the cells are constantly exposed to high NaCl. When the high NaCl is reduced, the breaks are rapidly repaired, accompanied by formation of γH2AX foci. This histone modification occurs around DSBs, contributing to their repair. Here we show that γH2AX foci that occur during repair of high NaCl‐induced DNA breaks are non‐randomly distributed in the mouse genome. By chromatin immunoprecipitation using antiγH2AX antibody, followed by massive parallel sequencing (ChIP‐Seq), we find that during repair of DSBs induced by high NaCl, γH2AX is predominantly localized to regions of the genome devoid of genes (“gene deserts”), indicating that the high NaCl‐induced DSBs are located there. Localization to gene deserts helps explain why the DNA breaks are less harmful than are the random breaks induced by genotoxic agents such as UV radiation, ionizing radiation and oxidants. We propose that the universal presence of NaCl around animal cells has directly influenced the evolution of the structure of their genomes. Supported by the Intramural Program of NHLBI
Unexpected Properties of the Human LINE‐1 Retrotransposon ORF1p
Authors: Callahan Kathryn E , Hickman Alison B , Jones Charles E , Ghirlando Rodolfo , Furano Anthony V
Published: June 2021

LINE‐1 (L1) is the dominant autonomous retrotransposon in mammals generating ~40% of mammalian DNA over the last 100 million years of mammalian evolution. L1 encodes two proteins, ORF1p and ORF2p. ORF2p is the L1 replicase. However, the precise role ORF1p in L1 replication is unknown. Mouse ORF1p both binds nucleic acids and acts as a nucleic acid chaperone, an activity essential for L1 replication (Martin, S. 2006. J. Biomed. Biotechnol. 2006: ID#45621). We purified baculovirus expressed human ORF1p and found that it was similar to the mouse protein with respect to chaperone activity and its affinity for nucleic acids. However, we uncovered two unexpected properties: (1) Although ORF1p binds the mismatched dsDNA and ssDNA with the same affinity, this is not because it first melts the mismatched duplex and then binds the ssDNA. Rather, the protein binds and stabilizes the mismatched dsDNA before melting it, which only occurs when the protein is in molar excess. (2) ORF1p rapidly undergoes polymerization and depolymerization as a function of nucleic acid concentration. We discuss the implications of these findings with respect to the biochemical properties ORF1p in vitro and retrotransposition in vivo.
RNA helicase Mov10 is a potent inhibitor of HIV or retrovirus infectivity and retrotransposition of endogenous mammalian retroviruses
Authors: Furtak Vyacheslav , Unutmaz Derya
Published: June 2021

The objective of this research work is to characterize antiviral and anti‐retrotransposon activity of RNA helicase Mov10. We recently reported that ectopic expression of Mov10 restricted the production of HIV‐1 and other lentiviruses as well as the gammaretrovirus, murine leukemia virus. 293T cells were transfected with retrovirus coding plasmid and Mov10 or control plasmid followed by infectivity test. HIV produced in the presence of high levels of Mov10 is restricted at the pre‐reverse transcription stage in target cells. To determine if Mov10 can also modulate transposition of retroviral elements in human genome, we transfected HeLa cells with retrotransposon coding plasmid that contained neomycin resistance cassette with or without Mov10 expressing plasmid. We then quantitated retrotransposon integration frequency by counting G418‐resistant cellular colonies in the presence or absence of Mov10 transfection. Retrotransposition of LINE‐1, MusD and IAP was significantly reduced by Mov10 in a dose dependent manner. These retroelements also showed sensitivity to restriction by other proteins found within the mRNA processing bodies (P‐bodies) such as Ago‐2 and Rent1. These findings suggest a potentially important role for cytoplasmic RNA processing machinery in retroviral infectivity, production and control of mobile genetic elements.

This work was supported by NIH Grant R21 AI078839 to D.U.
Complex regulation of a carbonic anhydrase gene cluster
Authors: Lee JongJoo , Song Sang‐Hyun , Dean Ann
Published: June 2021

The widely expressed nuclear factor LDB1, in a complex with erythroid DNA binding proteins GATA‐1 and SCL, occupies the beta‐globin locus control region and gene and is required for chromatin loop formation between these distant elements, which underlies LCR enhancer function. LDB1 is a widely expressed protein, suggesting it may facilitate long range enhancer interactions in multiple loci. We selected the carbonic anhydrase genes for further study of potential long range enhancer looping by LDB1 because CA1 and CA2 are strongly down‐regulated in MEL cells in which LDB1 had been reduced by shRNA. Using the ChIP assay, we validated LDB1 binding sites at the CA1 promoter and at two strong sites located ‐8 and ‐9 Kb upstream of CA2 in MEL cells that we considered to be a potential long range enhancer site. Unexpectedly, the enhancer blocking insulator protein CTCF occupied a site within the locus, potentially confounding enhancer function. Interestingly, 3C in erythroid cells showed long range interactions between the CTCF site, the potential LDB1 enhancer site and the promoters of the CA1 and CA2 genes. These results suggest an organizational role for CTCF in long range gene activation by an enhancer.
Polymorphisms in the Flt1 gene and their relation to expression of the secreted Flt1 variant
Authors: Ajlouni Burouj , Huckle William R.
Published: June 2021

Vascular endothelial growth factor (VEGF) is a potent angiogenic agent. VEGF activates its biologic responses through two cell‐surface receptors, Flt1 and Flk1. In addition to the transmembrane form of Flt1, the Flt1 gene also encodes a secreted, truncated form of the receptor (sFlt1) translated from an mRNA in which a portion of intron 13 is preserved. sFlt1 retains high affinity for VEGF and thereby inhibits its angiogenic activity. Intron 13 contains important cis elements involved in sFlt1 mRNA formation. Here, we test the hypothesis that polymorphisms in the human Flt1 gene, particularly SNPs at sites suspected to contain splicing or cleavage‐polyadenylation signals, influence Flt1 pre‐mRNA processing and rates of Flt1 and sFlt1 expression. The NCBI SNP database contained 23 SNPs in the region of interest, one each in exons 13 and 14. Our independent human SNP screen confirmed several of the reported SNPs. The web‐based ESEfinder software predicted that the exon 13/14 SNPs had reduced potential for recruitment of splicing components. To test effects of exonic SNPs on Flt1 pre‐mRNA processing, wild type and mutant Flt1 minigene plasmids were transfected into NIH/3T3 cells. Both exonic SNPs were associated with ~40% decreases in Flt1:sFlt1 mRNA ratios determined by real‐time PCR. These results support the notion that small changes in exon composition can have significant effects on splicing activity.
Examination of Kink‐Turn and Related Motifs
Authors: Garoutte Travis Justin , Grover Neena
Published: June 2021

The kink‐turn motif is a secondary structural element found in many different RNA. It is an important protein recognition element that interacts with magnesium ions to form a bent (“kinked”) structure. It is comprised of a trinucleotide bulge, RNN (R = purine; N = any nucleotide), flanked by both a canonical and non‐canonical stem. The canonical stem is closed by two G‐C base pairs and the non‐canonical stem by two mismatched G•A base pairs. We are examining the thermodynamic properties of constructs displaying this motif in 1 M KCl and in varying concentrations of divalent metal ions. Two of the examined constructs display greater stability in the presence of calcium ions than in the presence of magnesium ions. In contrast, other constructs tested display an increased stability in the presence of magnesium ions than in the presence of calcium ions. Further examination of the kink‐turn motif is necessary in understanding the molecular interactions between RNA and divalent metal ions.
Thermodynamic Examination of U6 ISL Bulged RNA
Authors: O'Connell Allison , Grover Neena
Published: June 2021

There is a highly conserved bulge in the internal stem‐loop (ISL) of the U6 subunit of the spliceosome. It is responsible for spliseosome activation and catalysis. The U6 ISL bulge structure is pH dependent. At a pH around 7.0 the unpaired U80 bulged nucleotide is stacked within the helix causing a bend of approximately 25 degrees. At a lower pH U80 is flipped out into the major groove allowing the RNA to take up a conformation close to A‐form helix. There are several single mutations in this bulge that have proven lethal in yeast, and a few single mutations that exacerbate the lethality. Ion binding, pH effects, and the stability of various sequences involving the bulge will be studied using thermodynamics.
The small molecule chemical chaperone 4‐Phenylbutyrate inhibits epithelial‐to‐mesenchymal transition in human renal proximal tubular epithelial cells
Authors: Brimble Elise , Carlisle Rachel E , Austin Richard C , Dickhout Jeffrey G
Published: June 2021

We have shown that thapsigargin (Tg), an endoplasmic reticulum (ER) stress inducer and activator of the unfolded protein response (UPR), causes epithelial‐to‐mesenchymal transition (EMT), the process by which epithelial cells transform to myofibroblasts. Myofibroblasts contribute to renal fibrosis and the progression of chronic kidney disease through their accumulative production of extracellular matrix. We hypothesize that EMT can be repressed through the use of low molecular weight chemical chaperones, such as 4‐Phenylbutyrate (4‐PBA) and tauroursodeoxycholic acid (TUDCA), in human renal proximal tubular epithelial cells (hRPTECs). 4‐PBA and TUDCA were found to inhibit hRPTEC death from Tg and TGFβ1 treatments as determined by LDH and TUNEL assays. Immunofluorescent labeling of myofibroblast markers showed that both 4‐PBA and TUDCA inhibited F‐actin cytoskeletal rearrangements and repressed β‐Catenin nuclear translocation in TGFβ1‐induced EMT. 4‐PBA inhibited the expression of ER stress marker GRP94 and myofibroblast marker α‐smooth muscle actin. TUDCA inhibited the expression of ER stress marker GRP78 and the focal adhesion complex component vinculin. Both 4‐PBA and TUDCA demonstrated partial inhibitory activity against Tg‐ and TGFβ1‐induced EMT in HK‐2 cells. 4‐PBA was found to inhibit TGFβ1‐induced EMT in primary hRPTECs. Funding was provided by St. Joseph's Healthcare Hamilton
P2X7 receptor stimulation raises lysosomal pH and triggers cytokine release from RPE cells
Authors: Guha Sonia , Baltazar Gabriel C , Tu Leigh‐Anne , Lim Jason C , Eysteinsson Thor , Laties Alan M , Mitchell Claire H
Published: June 2021

Accumulation of lipofuscin in retinal pigmented epithelial (RPE) cell, results from incomplete degradation of phagocytic and autophagic material by lysosomes. Both increased lipofuscin and enhanced cytokine release are associated with macular degeneration in these cells, although the link between the two is unclear. Since stimulation of the P2X7 receptor (P2X7R) activates IL‐1β release in some cell types, and triggers lysosomal exocytosis in others, we investigated the effect of P2X7R activation on lysosomal pH in, and cytokine release from, RPE cells. Surprisingly, stimulation of the P2X7R with agonist BzATP did not lead to a release of IL‐1β but did trigger release of both IL‐6 and MCP‐1. BzATP led to a rise in intracellular Ca2+, and extracellular Ca2+ was necessary for IL‐6 release. Lysosomal pH was elevated by BzATP and this alkalinization was inhibited by P2X7R antagoinsts A438079 and Brilliant Blue G. BzATP on its own did not significantly increase expression of IL‐6, MCP‐1 or the ectoATPase NTPDase1, but did increase expression of all three genes in cells concurrently exposed to oxidized photoreceptor outer segments. In conclusion, stimulation of the P2X7R on RPE cells elevated lysosomal pH and enhanced cytokine release. P2X7R stimulation also upregulated cytokine gene expression in cells subjected to an oxidative challenge.

Grant: EY013434
Involvement of macroautophagy and chaperone‐mediated autophagy in the irreversible process of posterior silk gland histolysis during metamorphosis of Bombyx mori
Authors: Shiba Hajime , Yabu Takeshi , Kawabe Ryousuke , Mano Nobuhiro , Nakanishi Truyuki
Published: June 2021

As an approach to clarify the physiological condition and events occurring during the silk gland histolysis, western blot analysis were performed in addition to measuring the glucose level in body fluid and the lysosomal cathepsin activity at different stages, i.e. 6th day after entering 5th instar (V6), prior to spinning (PS), during spinning (S), after cocoon formation (C). The levels of glucose in the body fluid and the phosphorylated p70S6Kinase, which were high at the V6 stage, decreased dramatically after entering metamorphosis at the PS stage, reached the lowest level at the S stage and increased slightly at the C stage. The phosphorylated form of AMPKalpha became detectable at the S stages and increased remarkably at the C stage. The atg8 protein was detected at the stages of V6, PS and S, indicating that macroautophagy occurred before entering the metamorphosis stage and ended before completion of cocoon formation. The lysosomal cathepsin B/L activity showed a dramatic increase at the C stage; it was low at the stages of V6, PS and S. Regarding the ubiqutinated proteins level, it increased remarkably at the stages of S and C. These results suggest that chaperon‐mediated autophagy is involved in the silk gland degradation as well. In conclusion, our results reveal that the shift of the physiological condition at the PS stage allows the catabolic process to be promoted irreversibly.
The flexible connection of the N‐terminal domain in ClpB supports substrate binding and controls the aggregate reactivation efficiency
Authors: Zhang Ting , Ploetz Elizabeth A , Nagy Maria , Smith Paul E , Zolkiewski Michal
Published: June 2021

ClpB reactivates aggregated proteins in cooperation with the DnaK chaperone system. ClpB contains two nucleotide‐binding domains (NBDs) and a smaller N‐terminal domain attached to the first NBD with a 15 aa‐long unstructured linker containing a GG motif. We investigated the role of the linker in aggregate reactivation. We produced 7 variants of ClpB with modified linker sequence. To increase the linker's conformational flexibility, we extended the GG motif up to GGGGGG. To decrease the linker's flexibility, we deleted the GG motif and converted it into GP and PP. We found that all linker variants showed a lower aggregate reactivation rate and a lower aggregate binding capability than wt ClpB. Molecular dynamics simulations showed that all modified linkers display different end‐to‐end distributions than the wt linker. For the linkers containing multiple glycines, a lower than expected degree of conformational flexibility may be due to the transient formation of a salt bridge‐stabilized alpha‐helical segment. We conclude that the linker region supports the chaperone activity of ClpB by controlling the efficiency of substrate binding. Moreover, the length and conformational flexibility of the linker region may have been optimized during evolution to achieve the most advantageous aggregate‐removal rates. Supported by the National Institutes of Health and the Johnson Center for Basic Cancer Research at KSU.
Genetic Mechanisms Coordinating Protein Synthesis and Folding
Authors: Barral Jose M , Siller Efrain , Spencer Paige , Anderson John F
Published: June 2021

Since the process of translation is directional, nascent proteins emerge from the ribosome as incomplete chains that are susceptible to misfolding. Ribosome movement along an mRNA is influenced by tRNA availability and the physical‐chemical properties of the anticodon‐codon interaction. Experiments from our laboratory suggest that polypeptide elongation rates and local folding events of the nascent protein are intimately coupled. Our current goal is to understand how genetically encoded information regulates protein synthesis rates and folding pathways. Differences in tRNA availability and protein folding efficiencies between bacteria and eukaryotes are being exploited to study these relationships. In order to accelerate or decelerate translation speed, sequences are engineered that encode identical proteins but contain variations along wobble positions of synonymous codons. We find that harmonization of translation rates based on tRNA availability of the organism of origin of the polypeptide with that of the production host leads to robust enhancement of folding efficiencies. These studies provide insight into how the genetic code, although nearly universal in its amino acid encoding properties, is utilized differently between bacteria and eukaryotes to regulate local protein folding events.

This work was supported by The Welch Foundation and The Pew Charitable Trusts.
Role of charge distribution in a flexible loop on bioluminescence color of firefly luciferase: a lesson from nature
Authors: Hosseinkhani Saman , Moradi Ali
Published: June 2021

The presence of Arg353 in PhRE luciferase, which corresponds to the deleted residue in green emitter luciferases, is an important distinctive structural feature of it. Insertion and substitution of positively‐charged amino acids in a flexible loop (352TPEGDDKP359) of firefly luciferase brought about with change of bioluminescence color. Insertion of Arg356 in a green‐emitter luciferase (Lampyris turkestanicus), that is corresponding to Arg353 in P. hirtus, changed the emitted light from green to red. In order to further clarification of the effect of this position in the light shift, residues with positive side‐chain (Arg356 and Lys356) were inserted in Photinus pyralis luciferase which changed the light color to red, while insertion of a residue with negative side‐chain (Glu356) had little effect and a neutral residue (Gln356) had not any effect on color. On the other hand, by insertion and substitution of positive‐charged residues, in the same loop of Lampyris turkestanicus luciferase, different mutation (E354R/Arg356, E354k/Arg356, E354R, E354K) lead to changes of the bioluminescence color. Bioluminescence emission spectra indicate that substitution of E354 by R along with insertion of Arg356 produces a luciferase that emit red light with a single peak bioluminescence spectrum.
Identification of a Glutathione Transferase Superfamily gene in Leishmania tarentolae
Authors: Osei‐Owusu Nana Yaw , Hearne Jennifer L , Currington Rashalai , Pitula Joseph
Published: June 2021

Leishmaniasis is a tropical disease that is caused by the parasitic protozoa Leishmania, a member of the Trypanosomatidea family. Unlike other eukaryotes, members of the Trypanosomatidea family lack a complete heme biosynthetic pathway; therefore, they must acquire heme exogenously. The mechanisms of heme scavenging and storage remain to be fully elucidated. Here, it is revealed that one mechanism of these functions may be accomplished through the coordination of heme with glutathione (GSH) via membrane associated proteins in eicosanoid and glutathione metabolism (MAPEG), members of the glutathione transferase (GST) superfamily. It is known that Macaca fascicularis microsomal prostaglandin E synthase type 2 (mPGES‐2), a MAPEG GST, binds GSH which has the potential to form a coordination bond with heme. Therefore, it is reasonable to hypothesize that there is an enzyme similar to mPGES‐2 which scavenges free heme in promastigote Leishmania for use during its maturation. Identification of a GST superfamily member gene target gene in the L. major genome was accomplished using the Basic Local Alignment Search Tool at GeneDB. Because Leishmania lack a complete heme biosynthetic pathway, the exploitation of its heme dependency is a key target for drug discovery. Identification of this gene provides the first evidence of a GST superfamily member in Leishmania.

Supported by TEDCO‐MRASC #W81XWH‐07‐2‐0055
Characterization of an unusual Trypanosoma brucei beta‐hydroxybutyrate dehydrogenase
Authors: Shah Tina D , Hickey Meghan , Palenchar Jennifer B
Published: June 2021

We report the characterization of an unusual β‐hydroxybutyrate dehydrogenase (TbβHBDH) from Trypanosoma brucei, the unicellular eukaryotic parasite responsible for causing African Sleeping Sickness. βHBDH catalyzes the reversible NADH‐dependent conversion of acetoacetate to hydroxybutyrate. Unlike its eukaryotic homologs, TbβHBDH does not appear to be a membrane protein and has greater sequence similarity to its soluble bacterial counterparts. The enzyme was cloned from T. brucei genomic DNA, and active, recombinant enzyme was purified from E. coli. Kinetic studies were carried out to determine the KM, Vmax, and Ki values for substrates and inhibitors. Cofactor preference was examined for TbβHBDH and it was found that TbβHBDH utilizes both NAD(H) and NADP(H) equally, distinguishing the parasite enzyme from other characterized βHBDHs. Furthermore, the enzyme appears to bind NAD(P)+ in a cooperative fashion. Through size exclusion chromatography, the apparent molecular weight of TbβHBDH is 96.5 kDa. RNA interference (RNAi) studies were carried out to begin to determine the in vivo role of TbβHBDH. Upon knockdown of TbβHBDH, verified through Western blot analysis, a noticeable reduction in parasite growth was observed; suggesting βHBDH has an important physiological role in T. brucei. This work is funded by the Research Corporation for Science Advancement.
Response of colon cancer cells to selenium and cis‐platinum exposure
Authors: Stockert Amy , Amend Kara , Stechschulte Melissa , Aulthouse Amy , Kinder David
Published: June 2021

Colon cancer is normally refractive to the chemotherapy drug cis‐platinum, (Cis‐Pt). Selenium (Se) containing glutathione peroxidase has been studied because of its role in reduction of reactive oxygen species which would improve the efficacy of Cis‐pt. The influence of Se on the effect of Cis‐PT on the HT‐29 colon cancer cell line was tested using a three dimensional agarose culture model. This method allowed independent analysis of mitosis and cell viability. Single cells were suspended in low temperature agarose and grown for 1 week. Se at low doses did not affect cell viability or mitosis when compared to vehicle controls. To simulate the in vivo scenario, colonies were allowed to form prior to Cis‐Pt exposure. Experiments were conducted to determine the time course of exposure to Se and Cis‐Pt. Some cultures were untreated or pretreated with Se (0.33 μg/ml) at day 0 followed by or in conjunction with Cis‐Pt (3 and 6 μg/ml) on day 4. On day 7 cultures were evaluated for cell viability, using trypan blue exclusion, and mitotic activity by counting single cells and colonies alive and dead. Differences between treatment groups were analyzed using ANOVA at 95% confidence level. Cell growth conditions allowed evaluation of glutathione peroxidase acitivity under each treatment condition following analysis of mitotic and cell viability. Glutathione peroxidase activity was compared to total protein in each cell culture. Initial results indicate that cultures treated with Se in conjunction with Cis‐Pt exhibited higher glutathione peroxidase activity coinciding with increased Cis‐Pt efficacy. These data suggest that supplementation with Se will increase efficacy of Cis‐Pt in colon cancer, specifically in the HT‐29 cell line, thus providing a more effective use of existing treatments. Support for this project was provided by The Raabe College of Pharmacy and the Department of Biological and Allied Health Sciences, Ohio Northern University.
Characterization of EF‐hand motifs and Investigation of Domain‐Domain Interaction in NADPH Oxidase 5 and Duox Oxidase by Fluorescence Spectrometry and Isothermal Titration Calorimetry
Authors: Wei Chin‐Chuan , Reynolds Nicole , Wetherell Kristen , Boyle Tiffany , Reimer Michael
Published: June 2021

The study of reactive oxygen species (ROS) generated by non‐phagocyte NADPH oxidase 5 (NOX5) and dual oxidase (DUOX) is of growing importance for vascular physiology and disease development. Their ROS activities appear to be regulated by their self‐contained EF‐hand domains (EFDs). Previously, we demonstrated that Ca2+ induces conformational change on NOX5‐EFD and suggested that its N‐ and C‐terminal halves behave differently, including conformational change, kinetics of binding, and chemical stability. Here we extended the study by using separated half domain proteins and characterized their Ca2+ and Mg2+ binding using fluorescence and isothermal titration calorimetry. The key residues for Ca2+ binding in each EF‐hand motif of the half domain proteins were mutated to determine the impacts on Ca2+ and Mg2+ binding. Since domain‐domain interaction is essential for ROS production, interaction of NOX5 EFD to flavoprotein was only observed in the presence of Ca2+. Furthermore, EFD enhances the cytochrome C reduction of flavoprotein. For DUOX‐EFD, the preliminary data suggests that a dramatic conformational change is not essential based on fluorescence data. Currently, we are performing experiments to identify the peptide sequence that interacts with its EFD and to investigate possible dimer formation that might play roles in DUOX ROS production. This work is supported in part by Cottrell College Science Award from Research Corporation.
Some effects of resveratrol and SRT1720 on PKA‐signaling and AMP‐kinase in adipocytes may be related to inhibition of Cyclic Nucleotide Phosphodiesterases (PDEs)
Authors: Khan Faiyaz , Park Sung‐Jun , Chung Jay , Manganiello Vincent
Published: June 2021

In animal models, caloric restriction (CR) decreases the incidence of age‐associated disorders such as cardiovascular disease, diabetes, and cancer. SIRT1 activators, i.e., resveratrol (RES) and SRT1720 (SRT), mimic effects of CR in lower organisms and mice. In 3T3‐L1 adipocytes, resveratrol and SRT inhibited PDE activities in membrane (IC50, ~40 and 7.5 uM respectively), and cytosol (IC50, ~90 and 10 uM, respectively). Cilostamide (10 uM), RES (12.5 uM), SRT (2 uM), and rolipram (30 uM), increased phosphorylation of Ser133‐CREB, Ser431‐LKB, Thr172‐AMPK, Ser79‐ACC as well as other unidentified PKA substrates. Rp‐8‐Br‐cAMPs, PKA inhibitor, blocked phosphorylation of these signaling molecules by cilostamide and rolipram. Rolipram had a relatively stronger effect on phosphorylation of PKA substrates and CREB than cilostamide, RES and SRT, but had smaller effect on the phosphorylation of LKB, AMPK, and ACC, suggesting the presence of a distinct cAMP pool involved in activation of AMPK. In adipocytes incubated for 90 min with CL‐316243 (B3‐agonist), 10 uM RES or 2 uM SRT increased phosphorylation of Ser563‐HSL, resulting in a significant increase in lipolysis (glycerol release). These and other results provide evidence that at least some effects of resveratrol and SRT‐1720 may be related to their inhibition of PDEs and, thereby, alteration of intracellular cAMP concentrations.
Caloric Restriction Decreases Telomerase Activity and Enhances BIBR 1532 Inhibition Response on MDA‐MB 231 Cell Line
Authors: Hilal George Y , Alaaeddine Nada , Wardi Layal , Assad Chafika
Published: June 2021

The metabolism of cancer cells is modulated by the nutrients content in their environment. Caloric restriction (CR) decreases proliferation, promotes differentiation, and transformation to quiescent cells. The immortality of cancerous cells is largely assured by the telomerase, an interesting target for inhibition by BIBR 1532. One obstacle faces the outcome of this inhibition is the long lag phase due to abnormal telomere length. In this study, we investigated the effect of CR on telomerase activity and its response to BIBR 1532. Breast cancer MDA‐MB 231 cells were cultured in DMEM with 0, 1 or 4.5 g/l of glucose. The telomerase activity was measured by quantitative Real‐Time PCR, the two telomerase subunits were semi quantified by RT‐PCR. Proliferation test and mitochondrial metabolism were assessed by tetrazolium salt oxidation and cell counts. CR decreased telomerase activity by 60% and enhanced cells response to 2.5 μM of BIBR 1532 without any modification in the expression of its two subunits hTERT (Reverse Transcriptase) and hTR (RNA Template) mRNA. BIBR1532 significantly decreased mitochondrial metabolism in dose dependent manner in cells cultured with 1 and 4.5 g/L of glucose. Taken together, our results demonstrated that CR decreases telomerase activity and mitochondrial metabolism which are directly modulated by BIBR 1532. This work was supported by the research council of Saint‐Joseph University.
Monocarboxylate transporters (MCTs) and lactate dehydrogenases (LDHs) as putative therapeutic targets in acute myeloid leukaemia (AML) Role of microenvironment in the metabolic switch
Authors: Serpa Jacinta , Domingues Germana , Gameiro Paula , Silva Maria Gomes , Dias Sérgio
Published: June 2021

Cellular respiration is the major pathway for ATP production in mammalian cells, and glucose transporters are commonly overexpressed in human tumours, enhancing glucose uptake. In hypoxia, cell metabolism switches to anaerobic pathways, transforming pyruvate to lactate; this event was described in several cancer cells. Nevertheless, for reasons yet unclear, cancer cells shift to glycolysis even in the presence of oxygen (aerobic glycolysis), the Warburg effect. Our main goal was to study the expression of expression of monocarboxylates transporters 1 and 4 (MCT1 and 4) and lactate dehydrogenase A, B and C genes (LDHA, B and C) in bone marrow samples from patients with in acute myeloid leukaemia (AML) and a human AML cell line (HL60). We observed in bone marrow samples from AML patients that the expression of MCT1 and LDH A genes are directly correlated with the percentage of leukemic blasts. The HL60 cell line when exposed to sodium lactate (10mM) changes the expression of a panel of genes involved in metabolism. The expression of MCT1 is decreased whereas MCT4, LDHB and C, glucose transporter 1 (GLUT1) and phosphoenolpyruvate carboxy kinase (PEPCK) are overexpressed, after at least 120h of exposure to sodium lactate. Our results indicate that AML cells have plasticity in order to adapt to microenvironment. The modulation of MCTs and/or LDH genes expression can be an efficient therapeutic approach.
Cancer cells can use acid gradient across the plasma membrane to produce ATP
Authors: Dhar Gautam , Sen Suvajit , Chaudhuri Gautam
Published: June 2021

Cancer cells have enhanced glycolysis and less mitochondria mediated ATP synthesis. This leads to excess lactic acid production and acidification of the external matrix. Glycolysis generates 15–20 folds less ATP as compared to the mitochondria. Whether glycolysis can be a sufficient source of ATP for proliferating cancer cell, and why such switch occurs has always been debated. Acid gradient across membrane is an energy that can be utilized by ATP synthase to generate ATP. Published proteomics and microscopic data indicated the presence of ATP synthase subunits in the plasma membrane of cancer cells. Here we present evidence that cancer cells can utilize the acid gradient to produce large amount of ATP. ATP production starts at around pH 7.1‐6.8 depending on the cancer cell type, increases exponentially with lowering pH till pH 6.1‐6.3. This range resembles the reported pH for cancer cell environment. ATP production followed a dependence on the external proton concentration whose order depends on cancer cell type. ATP synthesis is very rapid at about 107‐108ATP molecules/cell/s and depletes 3–5 protons from the external medium per ATP molecule, a value that closely resembles the ones published for other ATP synthases. Our work demonstrate that acidification of the external matrix through enhanced glycolysis is a strategy that enable cancer cells to harness the acid gradient for more efficient ATP production.
Further Evidence for AMP Deaminase as the target of Metformin Action in L6 Skeletal Muscle Cells
Authors: Parakhia Rahulkumar A. , Ochs Raymond S.
Published: June 2021

Metformin is known to activate the AMP kinase (AMPK), although the mechanism for the activation has not been resolved. We suggest that the locus of action is inhibition of the enzyme AMP deaminase (AMPD), which would subsequently raise AMP concentration and activate AMPK. In support of this hypothesis, we observed direct inhibition of AMPD (and the mechanistically related adenosine deaminase) by metformin in vitro, and replication of metabolic effects of metformin by the commercially available AMPD inhibitor EHNA (erythrohydroxynonyl adenine), as well as newly synthesized indazole compounds we have named SKTT‐1 and SKTT‐2. These inhibitors replicate metformin stimulation of glucose uptake, fatty acid oxidation, and inhibition of ammonia accumulation. Furthermore, siRNA knockdown of AMPD prevented metformin stimulation of glucose transport. Knockdown of LKB1 in L6 cells did not impair metformin action. This is a paradox, as phosphorylation of AMPK is essential for its activation, and LKB1 is the major upstream kinase of AMPK. We therefore explored the possible participation of another AMPK kinase, CAMKK, using a selective inhibitor of that enzyme, STO‐609. While STO‐609 did not itself alter metformin stimulation of glucose uptake, in cells that had been treated with siRNA to knockdown LKB1, the inhibitor effectively prevented AMPK activation by metformin. We conclude that either upstream kinase can activate AMPK, and that our findings are consistent with AMPD as the direct site of action for metformin.
Metabolomic Responses of E. coli to Oxidative Stress
Authors: Solimeo Renee , Palenchar Peter
Published: June 2021

Oxidative stress can be induced by different chemicals. Hydrogen peroxide is a strong and direct oxidizer of biological molecules, copper can participate in chemicals in biological systems that can produce strong oxidizing agents, while diamide is a weak, but direct oxidizing agent that can only oxdize thiols to disulfides. Oxidative stress is known to be an important in a large number of biological processes, including metabolism, aging, and defense response, but still little is known about the metabolic responses to oxidative stress at the metabolite level. Even less is known about responses to specific oxidative affects. We will present LC‐MS data regarding levels of metabolic respones at the metabolite level of E. coli in the presence of oxidative stress inducing molecules.
Genistein Affects Dickkopf‐related Protein 1 (DKK1) Expression Level through Histone Modification in SW480 Human Colon Cancer Cell Line
Authors: Wang Huan , Pan Yuan‐Xiang , Chen Hong
Published: June 2021

Genistein is a plant‐derived isoflavone and can block uncontrolled tumor cell growth by inhibiting cell division and survival. This study is to test the hypothesis that the anticancer effect of genistein in colon cancer is through inhibition of the WNT signaling pathway by promoting an antagonist Dkk1 of the pathway. Human cancer cell line SW480 was treated with 0, 1, 5, 15, 25, 50, and 75 μM genistein for 2 days. Flow cytometry of cell cycle analysis showed that genistein induced a concentration‐dependent G2 phase arrest in SW480. Genistein also reduced cell proliferation by WST1 assay. Both Dkk1 mRNA and protein expression are increased by genistein at higher concentrations. DNA methylation at Dkk1 promoter region in SW480 was not affected by genistein treatment. On the other hand, chromatin immunoprecipitation showed that genistein induced histone H3 acetylation at Dkk1 promoter region, which indicates that increased histone acetylation is associated with increased Dkk1 expression. Treatment with histone deacetylase inhibitor trichostatin A confirmed that increasing histone acetylation can lead to the induction of Dkk1 mRNA expression. In conclusion, genistein treatment decreases cell growth and proliferation in colon cancer cell line SW480. The effect is associated with increased Dkk1 expression through the induction of histone acetylation at the promoter region of the gene.
GENE CLONING, PURIFICATION, AND CHARACTERIZATION OF A COLD‐ADAPTED LIPASE FROM ACINETOBACTER SP. V28‐28
Authors: Kim Young‐Ok , Nam Bo‐Hye , Kong Hee Jeong , Kim Dong Gyun , Kim Woo‐Jin , Kim Kyung‐Kil , Kim Bong‐Suk , Lee Sang‐Jun
Published: June 2021

A bacterial strain that produced extracellular lipase was isolated from seawater and identified as Acinetobacter sp. V28‐28. In the present study, the corresponding gene was cloned using shotgun method. The amino acid sequence deduced from the nucleotide sequence (1,017 bp) corresponded to a protein of 338 amino acid residues with a molecular weight of 37,186. The lipase had 87 and 72% identities with the lipases of Acinetobacter junii SH205 and Acinetobacter calcoaceticus RUH2202, respectively. The lipase contained a putative leader sequence, as well as the conserved catalytic triad (Ser, His, Asp), consensus pentapeptide GXSXG, and oxyanion hole sequence (HG). The protein V28‐28 was produced in both a soluble and insoluble form when the Escherichia coli cells harboring the gene were cultured at 18?. The optimum pH and temperature for the enzyme activity was pH 9.0 and 40?, respectively. 50% of the activity observed at 40? was still remaining at 5?. The stability of the purified enzyme at low temperatures indicates that it is a cold‐adapted enzyme. The enzyme showed the highest activity toward the ?‐nitrophenyl ester of caprylate (C8) and metal ion such as Zn2+ inhibited enzymatic activity. Partial characterization indicated that the lipase from Acinetobacter sp. V28‐28 is a new cold‐adapted enzyme.
Effect of High Fat Diet On Reproductive Cycle
Authors: Ng Ka Yan , Yong Jennifer , Chakraborty Tandra R.
Published: June 2021

The prevalence of obesity is on its increase largely because of the interaction between dietary intake, genes, life style and environment. The consequences of obesity include type 2 diabetes, cardiovascular disease, cancers and insulin resistance. In addition, it leads to earlier puberty, menarche in girls and infertility. There is a known effect of two hormones, estrogen and leptin on energy homeostasis and reproduction. This study is aimed to study the effect of high fat induced obesity on reproductive cycles on mice model. When mice is fed with very high fat diet (VHFD) there is an increase in the weight gain within the first week. Estrous cycle in mice goes through four stages; proestrous, estrous, metaestrous, and diestrous. Preliminary experiments on estrous cycle showed that the number of cells in the VHFD treated animals were less than the animals in normal diet indicating disruption in the reproductive cycle. Further, leptin and estrogen will be analyzed in both models to understand the interaction of hormones on VHFD treated animals.
Positive association between obese postmenopausal women and breast cancer markers
Authors: Alokail Majed S , Al‐Daghri Nasser M , Alkharfy Khalid M , Draz Hossam M , Al‐Attas Omar S
Published: June 2021

The link between obesity and premenopausal women breast cancer development has been attributed, in part, to the fact that estrogen, a risk factor for breast cancer, is synthesized in adipose tissue. This prompted us to investigate the relationships between obesity and the presence of breast cancer markers. Hence, the purpose of this study is to pave a way for development of a blood based gene expression test for early detection of breast cancer in obese premenopausal women. We have determined the relative gene expression of adiponectin, P53, parathyroid hormone‐related peptide (PTHrP), interleukin‐6 (IL‐6), tumour growth factor‐beta (TGF‐â) in 62 samples of female subjects. The results showed Significantly increased in gene expression levels for P53, PTHrP and IL‐6, and significantly decreased levels of adiponectin were found in obese group, while the levels of TNF‐alpha and TGF‐alpha were unaltered. This data could help in explaining the mechanism behind the association between obesity and breast cancer development in premenopausal women. In conclusion, the strong positive relation of PTHrP, and P53 with Obesity in premenopausal women raises several questions for further experimentation concerning their exact role and physiological significance.
Inhibitory effects on adipocyte proliferation and differentiation by Rubus coreanus and Morus alba extracts in 3T3‐L1 cells
Authors: Kim Gun‐Hee , Park Hee‐Sook
Published: June 2021

Obesity is a serious health problem in industrialized countries because it is implicated in various diseases. In Current research, obesity of the human focus on intracellular triglyceride accumulation in adipocyte. These topics are discussed in relationship to the development of obesity. This study was performed to investigate the effects of Rubus coreanus and Morus alba on proliferation and adipocyte differentiation in 3T3‐L1 cells. Among the five fraction of Morus alba, hexan, chloroform, ethylacetate‐soluble fractions at concentrations of 100¥ìg/ml inhibited adipogenesis by 21.6, 23.9 and 38.5%, respectively. All of the fractions of Rubus coreanus extract attenuated intracellular lipid accumulation. Specially, ethylacetate‐soluble fraction of Rubus coreanus has the highest inhibitory effect on adipocyte differentiation as 86.8¥ìg/ml by IC50 value. On the other hands, Rubus coreanus and Morus alba ethanolic extracts inhibited preadipocyte proliferation and consequent differentiation within the first 3days of treatment. Rubus coreanus has the strongest anti‐proliferation effects on preadipocytes without cytotoxicity. These data indicate that the anti‐adipogenic effects of Rubus coreanus and Morus alba may derived from anti‐proliferation. From results, Rubus coreanus and Morus alba may prove to be valuable natural plant in the treatment of obesity.