bluuebunny/crossref_metadata_2025 huggingface dataset:
HFE: An Iron Uptake Regulation Molecule
Authors: Dongre Bryan , Chen Yuhan , Fu Justin , Gerner Zach , Moore Shariq , Nabar Nick , Nabar Vick , Prasad Nikil , Speagle Joshua , Vangala Sai , Thompson Louise , Ferrante Andrea
Published: June 2021

Accumulation of excess iron results in a common hereditary disease, Hereditary Haemochromatosis (HH). There are various genetic mutations that lead to different forms of the disease. HH‐I is a form of this disease in which iron accumulates in hepatocytes and intestinal epithelial cells and is associated with a mutation in the HFE (high iron protein) gene. The Brookfield Central SMART Team (Students Modeling A Research Topic) developed a model of HFE using 3D printing technology. The HFE gene encodes for a non‐classical MHC class I protein. In physiological conditions, HFE is expressed and translocated to the cell surface where it may interact with a transferrin receptor (Tfr). The binding of the α1/β2 domains of HFE to the Tfr allows for controlled release of iron bound to transferrin‐transferrin receptor complex. A mutation (845G>A) causes the replacement of a cysteine with a tyrosine (C282Y). This replacement prevents the α3 subunit of HFE from folding properly and from interacting with β2 microglobulin, abrogating the translocation of the HFE‐microglobulin complex to the cell membrane and promoting its rapid degradation. This defect hinders the regulatory capability of HFE. Current treatments include phlebotomy to prevent organ damage from accumulated iron. Further study to increase understanding of the regulatory mechanism may lead to improved treatment design. Supported by a grant from NIH‐NCRR‐SEPA.
3D Physical Models of Acetylcholinesterase
Authors: Acheampong Mary , Dueño Daviana , Glover Bobby , Henry Alafia , Mata Randol , VanBrakle Marisa , Granberry Allison , Sussman Joel , Westblade Lars
Published: June 2021

Acetylcholinesterase (AChE) is essential for the hydrolysis of the neurotransmitter acetylcholine (ACh) in cholinergic synapses. Irreversible inhibition of AChE can lead to increased levels of ACh and ultimately death. Conversely, suppressed levels of ACh may lead to memory deficits associated with Alzheimer's disease. AChE has a deep (~20 Å) and narrow (~5 Å) gorge lined with 14 aromatic residues, with its active site at the bottom of the gorge. Initially, ACh binds the peripheral anionic site of AChE and is funneled down the gorge to the active site by interactions between the aromatic rings of the 14 aromatic residues and the quaternary ammonium ion of ACh. At the active site, ACh is oriented for hydrolysis by the active site by interactions between the catalytic anionic site and the quaternary ammonium ion of ACh. The Fasciculin‐II (FAS‐II) toxin, from the green mamba snake venom, inhibits AChE by binding and blocking the active‐site gorge. The Hostos‐Lincoln Academy SMART (Students Modeling A Research Topic) Team and MSOE have designed and made two physical models by 3D printing technology: Torpedo californica TcAChE in complex with a modeled ACh ligand and TcAChE in complex with FAS‐II. Supported by a grant from HHMI Pre‐College Program.
Role of CCR4 inCD4+CD25+Regulatory T cell on Asthma
Authors: Kwon Young Joo , Kim Young Eun , Park Soojin , Kim Yong , Lee Hyo Eun , Cho Young Eun , Shin Minkyu , Hong Moochang , Bae Hyunsu
Published: June 2021

Asthma is a chronic disease characterized by airway inflammation, airway hyperresponsiveness (AHR) and reversible airway obstruction. This study was conducted to examine the relative contribution of the chemokine receptors CCR4 in regulatory T cells on a murine model of allergic asthma and to determine chemotactic response and specific expression of CCR4 by CD4+CD25+ regulatory T cells. After ovalbumin (OVA) sensitization, we used adoptive transfer experiments to compare the effect of wild‐type CCR4 Treg cells with CCR4‐deficient Treg cells and then IL‐4,5,13, serum IgE were evaluated by ELISA. Additionally, stained lung tissue with H&E(Hematoxylin & Eosin), PAS(Periodic acid‐Schiff ) and IHC(Immuniohistochemistry) was observed. Sensitization with OVA mice led to increased levels of IL‐4, 5, 13 in BAL fluid and in serum IgE. Also, damage to epithelium, and hyperplasia of goblet cells, smooth muscle and fibroblasts were examined whereas the adoptive transfer of wild‐type CD4+CD25+ Treg cells to asthmatic recipient markedly down‐regulated the inflammation. In contrast, the transfer of CCR4‐deficient Treg cells was unable to significantly decrease the cytokine expression and structural remodeling of the airway. These result suggest that CCR4 is required for enhancing the role of CD4+CD25+ regulatory T cells and Tregs may attract CCR4 preferentially to suppress allergic asthma.
Investigating heme uptake in mycobacteria
Authors: Harmston Christine A , Iniguez Angie , McMath Lisa , Owens Cedric , Chim Nick , Goulding Celia
Published: June 2021

Iron is an essential element for all forms of life. Pathogens, such as Mycobacterium tuberculosis, must acquire iron from their host in order to survive. While M. tuberculosis has been shown to uptake host transferrin iron via a siderophore‐mediated mechanism, our laboratory has identified a unique heme uptake pathway in mycobacteria. Here, M. smegmatis is used as a model system to determine the genomic region proposed to be responsible for heme uptake. Thus far, we have deleted a particular region containing seven genes and this mutant only grew in iron supplemented media and not heme. We then complemented our heme uptake mutant with the corresponding genes in M. tuberculosis, and showed that the complementation restored growth in heme supplemented media. Hence, we have identified the genomic region responsible for heme uptake in M. tuberculosis. Additionally, we will present biochemical analyses of the putative hemophore and extracellular domains of MmpL11, a proposed heme transporter, and the possible pathway of transfer of heme from protein to another.
Proteomic evaluation of pesticide‐resistant breast cancer cell lines
Authors: Hale Julie , Yamamoto Fiona , Bush Jason A.
Published: June 2021

Considerable evidence links cumulative and sustained exposure to estrogens as a key promoter of breast tumor proliferation. Chemicals with estrogenic activity can bind to the estrogen receptor (ER) affecting signaling and estrogen‐responsive genes. Organochlorines are a class of chemical pesticides that act as xenoestrogens to disrupt normal endocrine function. Methoxychlor and Toxaphene are two organochlorine pesticides that have been widely used in California. This study investigates the response to these pesticides in an ER‐dependent manner by utilizing the breast cancer cell lines, MCF7 (ER+) and MDA‐MB‐231 (ER−), and a proteomic‐based approach to evaluate molecular pathway involvement. Cytotoxicity studies demonstrate that MCF7 cells are more sensitive to both pesticides supporting the premise that organochlorines may be acting as endocrine disruptors. Furthermore, pesticide‐resistant clones of MCF7 and MDA‐MB‐231 were established and compared to sensitive cells in proteomic experiments focused on the mitochondrial subproteome due to its role in cellular detoxification and apoptosis. We report here the elevated expression of a small subset of mitochondrial proteins in response to long‐term organochlorine exposure. These results provide a basis for understanding specific pesticide‐induced molecular mechanisms and their possible relationship to ER status in breast cancer.
EHD1 and EHD2 interact with Fer1L5 to mediate myoblast fusion
Authors: Posey Avery , Pytel Peter , Heretis Konstantina , Demonbreun Alexis , George Manju , Rainey Mark , Band Hamid , McNally Elizabeth M
Published: June 2021

Myoferlin and dysferlin regulate myoblast fusion and muscle membrane resealing, respectively. Correspondingly, myoferlin is most highly expressed in singly nucleated myoblasts while dysferlin expression is highest in mature, multinucleated myotubes. The mammalian ferlins are calcium‐sensing, C2 domain‐containing proteins involved in vesicle trafficking. Myoferlin mediates endocytic recycling and participates in trafficking the insulin like growth factor receptor. We now characterized a novel member of the ferlin family, Fer1L5, because of its high homology to dysferlin and myoferlin. We found that fer1l5 protein is abundantly expressed in nascent myotubes containing only 2–4 nuclei. We also found that fer1l5 protein binds directly to the endocytic recycling proteins, EHD1 and EHD2, and that this interaction is mediated by the second C2 domain in fer1l5. Loss of EHD1 and/or EHD2 inhibits myoblast fusion, and EHD2 is required for normal translocation of fer1L5 to the plasma membrane. Abolishing the nucleotide binding ability of the EHD2 ATPase domain recapitulates the fer1L5 translocation defect. The characterization of fer1L5 and its interaction with EHD1 and EHD2 underscores the complex requirement of ferlin proteins and mediators of endocytic recycling for membrane trafficking events.
MOLECULAR ANALYSIS OF NORMAL AND HYPOPLASTIC CHICK EMBRYONIC VENTRICLES
Authors: Pesevski Zivorad , deAlmeida Angela , Mrug Michael , Fresco Victor , Barth Jeremy , Cui Xiangqin , Krejci Eliska , Sedmera David
Published: June 2021

The term hypoplastic left heart syndrome (HLHS), describes a spectrum of cardiac abnormalities characterized by marked hypoplasia of the left ventricle We employed the Affymetrix gene chip technology to gain a better insight into molecular characteristics of embryonic and early fetal myocardial differentiation. RNA isolated from normal left (LV) and right (RV) chick ventricles on ED 6, 8, and 10 was checked for quality using Agilent capillary electrophoresis, and after cDNA synthesis hybridized on the chick whole genome chip. In addition, RNA from E8 LV and RV of hearts with developed left ventricular hypoplasia after left atrial ligation was treated in the same fashion. Comparisons were made of LV vs. RV at different stages of development, as well as between control and treated groups. Interestingly, the number of genes upregulated in the LV was much lower than those higher in the RV. This suggests that differentiation of the working myocardium lies more in a restriction of gene expression program, with specific upregulation of select few genes, such as MYH11, MYR8, GBJ6:gap junction protein, beta 6, others from contractile proteins/myocyte differentiation group of genes, FGF13, NF1 from proliferation/Growth factors genes. With this data we want to explain roles and involvement of some genes of the ventricular myocardium at different stages of development as well as their connection with congenital heart disease.
Cytotoxicity and Cell Cycle Perturbation of Arsenic and Mercury Exposure on MCF‐7 cells
Authors: Malik Fatiha Laila , Abou‐Zeid Nadia , Ishaque Ali B
Published: June 2021

Breast cancer is one of the leading causes of death in women in the United States. There are several factors that are known to contribute to the onset of this disease. We hypothesize that environmental exposure to heavy metals, such as mercury and arsenic, is a contributing factor. In this study, the cytotoxicity of As and Hg was studied in exponentially growing and asynchronous MCF‐7 human breast cancer cells. Cytoxicity was determined with real time cell electronic sensing (Rt‐CES). LC50 of each metal and the combination of metals was determined after 24, 48, 72, and 96 hours. The results show that the LC50 of As decreased tenfold from 24 hours through 96 hours of exposure: 1.8 x10‐3 to 1.8 x10‐4 mg/ml. The LC50 for Hg exposure showed a relatively slighter change: 7.2x10‐2 to 4.5 x10‐2 mg/ml. The LC50 for the mixture decreased from 3.3 x10‐4 mg/ml after 24 hours exposure to 3.0x10‐5mg/ml after 96 hours. Cell cycle analysis revealed significant differences in the percentage of cells in S and G2 phases in all samples, while the percentage of cells in G1 phase did not show any significant difference. As and Hg, individually and in combination, cause marked perturbation of the cell cycle kinetics of MCF‐7 human breast cancer cells by arresting the transit of cells at S phase. Further studies are needed to determine the mechanisms leading to the observed perturbations. Supported by NIH R25GM063775.
High‐throughput screening for hypothetical proteins in Salmonella typhimurium by affinity proteoimcs
Authors: Jung Che‐Hun , Pan SangO
Published: June 2021

Recently human genome project has identified a large number of open reading frames in many species, whose functions are not known. Those genes are annotated as hypothetical, unknown, or uncharacterized proteins. Quite often there is no experimental evidence even for the expression of the hypothetical proteins, although their existence has been predicted by bioinformatics tools. Nucleotides serve as structural units of RNA and DNA and also as important cofactors in cellular signaling pathways and metabolism. In this study several affinity ligands such as ADP, ATP, GTP, and NAD(P)+ were utilized for isolation of nucleotide(s)‐binding proteins in Salmonella typhimurium. Those binding proteins were identified by MALDI‐TOF MS and shotgun analysis. Fourteen proteins, as ATP‐binding proteins, were identified as hypothetical proteins. Six proteins among GTP‐binding proteins were also hypothetical proteins. Furthermore, 17 NAD(P)+‐binding proteins were hypothetical. These results suggest that affinity proteomics will be very effective for high‐throughput screening for hypothetical proteins. [This work has been supported by Ministry of Education and Science, Korea.]
Genomic and proteomic changes during the development of a tomato‐Verticillium pathosystem.
Authors: NAZAR Ross N. , Lee Barbara , Shittu Hakeem , Kurosky Alex , Robb Jane
Published: June 2021

Biology of plant‐microbe interplay is complex and can lead to susceptible, resistant or even tolerant host response states. To better define these at a molecular level we assessed global changes in gene expression at both transcriptional and translational levels in tomato infected by, Verticillium dahliae. DNA microarray (TVR chip) studies of the 3 states reveal complex but distinct patterns of change in plant defense related mRNAs. Many genes that, initially, are strongly induced in the susceptible response are then down regulated at 10 d.p.i. while transcription is less dramatically affected in resistance or tolerance. Similarly, proteomic analyses based on 2D gel comparisons and mass spectrometry showed distinct patterns of protein change. Using Progenesis SameSpots software, 1621 protein spots were mapped. In the three types of interaction, 575 proteins showed significant differences in intensity ≥ 1.5 for 35 pairwise comparisons and 220 spots indicate differences ≥ 2.0. Some proteins were strongly induced but levels of many were reduced in the compatible interaction consistent with "cost of defense". Differences progressed with time. Protein identification by MALD‐TOF indicated most known proteins were plant defense related or had unknown functions. Susceptibility was characterized by the most dramatic increases in defense related proteins but others were reduced relative to a resistant plant.
Enabling highly sensitive multiplexed western blot detection with WesternDot™ technology
Authors: Hart Courtenay R , Agnew Brian
Published: June 2021

Current chemiluminescent Western blotting technology requires sequential stripping and reprobing of the blot in order to detect more than one protein of interest. The multiple colors and long stokes shifts of quantum dot nanocrystals enable the simultaneous two‐color detection of two proteins of interest with minimal spectral overlap using a single UV or blue light excitation source on camera based imagers. WesternDot™ technology utilizes the brightest second generation quantum dots, Qdot® 625 and Qdot® 800, conjugated to streptavidin and goat anti‐mouse and goat anti‐rabbit secondary antibodies. Here we compare Qdot® 625 and Qdot® 800 conjugates to various chemiluminescent reagents. The results show that the Qdot® conjugate sensitivities and selectivities are comparable to chemiluminscent reagents, but also the work flow is faster and easier, as there is no requirement for messy chemical treatments or multiple film exposures after secondary antibody incubation. The Qdot® signal is also stable and blots can be reimaged over several weeks after processing. Our data also demonstrates that Qdot® conjugates can be effectively combined with chemiluminescent and colorometric reagents allowing for simultaneous multiplexed detection of two and three target proteins on the same blot.
Innate Immunity Signaling in Neonatal Rat Cardiac Myocytes
Authors: Xue Jianjing , Bauer John Anthony , Luce Wendy A
Published: June 2021

Neonates have a heightened inflammatory response to pathogens & are uniquely susceptible to sepsis‐related cardiac dysfunction. MAP‐kinase signaling plays a critical role in innate immunity/inflammation wherein MAPK phosphatase‐1 (MKP‐1) is a key negative regulator, but its roles in cardiac myocyte inflammatory signaling are not defined. Our hypothesis was that MKP‐1 deficiency amplifies inflammatory responses in neonatal rat cardiomyocytes, and is related to MAPK, Gαs & Gαq signaling pathways. H9c2 cells were transfected with siMKP‐1 or scrambled control (H9WT) and treated with 5μg/ml LPS. IL6 & TNFα mRNA and protein expression was detected by qRTPCR & ELISA. cAMP, PC‐PLC & activated PKC were detected by EIA. siMKP‐1 cells had a greater increase in TNFα (p<0.001) & IL6 (p<0.001) mRNA & protein expression at 1h & 4h after LPS vs. H9WT. MKP‐1 deficient cells also had greater increases in cAMP, PC‐PLC & PKC activity (P<0.001) after LPS. Inhibition of P38 completely blocked the increases in TNFα, IL6, cAMP & PC‐PLC, but significantly increased activated PKC (P<0.001) in siMKP‐1 cells. Neonatal rat cardiac myocytes generate inflammatory cytokines in direct response to LPS, which is enhanced by MKP‐1 deficiency, mediated by the MAP kinase P38 & associated with increased cAMP, PC‐PLC & PKC. Defining these cardiac myocyte signaling processes may lead to novel therapeutic targets for sepsis‐related cardiac dysfunction.
Coarctation of the Aorta Associated with Dissection of the Ascending Aorta in the 13 Year Old Boy
Authors: Miclaus Gratian Dragoslav , Zahoi Delia Elena , Ples Horia , Gaspar Marian
Published: June 2021

Cardiovascular emergencies represent one of the most challenging issues in daily practice. Use of MDCT allows to scan fast and with high accuracy critical and unstable patients.

In our Diagnostic Imaging Centre we have examined a young patient 13 years old who fall unconscious after a bath in the river. We have been asked to clarify if there is a dissection of the aorta and to asses the status of coronary ostia regarding dissection fold.

We have been able to precisely offer the extension of the dissection, and we find also coarctation of the aorta.

Patient was surgically treated with replacement of the ascending aorta and reimplantation of the coronary arteries, and in the second operating time coarctation was solved.

MDCT is fast and high quality investigation, in a very short time, with high resolution and possibility to offer anatomic details of the organs involved and surrounding tissues.
Host Specific Subversion Of The MAPK‐Dependent Apoptotic Response To B Virus Infection
Authors: Farah‐Abraham Rachael , Hilliard Julia
Published: June 2021

B virus (Macacine herpesvirus 1) is an alphaherpesvirus indigenous to Old World monkeys & is closely related to HSV‐1. B virus produces mild/no disease in its natural host. When transmitted to humans, it replicates at the site of injury & invades the CNS/travels directly to the CNS. In humans high morbidity & mortality are associated in 80% untreated cases. Five major signaling pathways can be activated after infection, inclusive of the MAPK pathway. Early stimulation of one/more of these pathways leads to the induction of cytokines & apoptosis. Apoptotic induction plays an important role in host‐pathogen interaction & adaptive immune responses. This study investigated host‐specific regulation of B virus induced apoptosis via p38 & JNK (MAPK) pathways in cells derived from the natural & foreign host. Our data suggest that macaque & human cells temporally differ in the induction of p38 & JNK. The overall early induction & downstream phosphorylation of factors associated with apoptotic events are significantly greater in cells derived from the foreign host. The use of specific inhibitors demonstrated that inhibition of p38 resulted in a decrease in virus replication & lowered levels of proteins associated with apoptosis. By investigating the role of B virus viral genes in the differential induction & regulation of the MAPK pathway early after infection, we can further elucidate virus‐induced host specific modulations.
Phosphorylation of paxillin by JNK and p38 is essential for full T lymphocyte activation
Authors: Lai Ming‐Zong , Chang An‐Yun , Lee Yu‐Chi , Tsou Wen‐I , Lin Ming‐Hsien
Published: June 2021

Paxillin is a focal adhesion complex‐assciated adaptor protein, and is regulated by phosphorylation at tyrosine and serine/threonine. Recent studies have revealed a critical role for phosphorylation of paxillin by JNK and p38 MAPK in epithelial cells and neurites. In this study, we examined the requirement of paxillin phosphorylation by p38 MAPK or JNK in T cells using paxillin with mutations at the respective phosphorylation sites, Ser‐85 and Ser‐178. Surprisingly, [S85A]‐paxillin or [S185A]‐paxillin did not interfere with T cell migration and integrin‐mediated T cell adhesion. Neither was T cell receptor‐stimulated activation affected by [S85A]‐paxillin or [S185A]‐paxillin. Interestingly, [S85A/S178A]‐paxillin, mutatant at phosphorylation sites for both p38 and JNK, effectively suppressed T cell activation, but not migration and adhesion. The profound inhibitory effect of [S85A/S178A]‐paxillin in T cell activation was also found in normal T lymphocytes using transgenic mice expressing [S85A/S178A]‐paxillin. Our results demonstrate an unexpected role of paxillin phosphorylation in T cell, whereas phosphorylation by JNK and p38 MAPK is dispensable for T cell motility and adhesion, it is essential for full T cell activation.
Characterization of a novel kinase involved in biomineralization of diatom silica
Authors: Sheppard Vonda Chantal , Hipp Dustin , Poulsen Nicole , Kroger Nils
Published: June 2021

Diatoms are unicellular, eukaryotic algae that produce cell walls made of intricately nanopatterned SiO2 (silica). Formation of diatom silica involves highly phosphorylated proteins (silaffins and silacidins), analogous to other biomineralization systems (e.g., human bone and teeth), which also depend on diverse sets of phosphoproteins. The phosphate moieties on biomineralization proteins are essential for their mineral formation, yet the kinases catalyzing their attachment have not been identified in any organism. Such kinases are expected to reside within in the secretory pathway (endoplasmic reticulum, Golgi apparatus) through which the biomineralization proteins pass on their route to the biomineral forming compartment (extracellular space or specific intracellular vesicle). Here we present data on the characterization of a novel kinase, tpSTK1, which is strongly upregulated during silica formation in the diatom T. pseudonana. TpSTK1 is an abundant component of the ER lumen, and is capable of phosphorylating silaffins but not silacidins. Bioinformatic analysis identified tpSTK1 homologs in other diatom species, but only weak homology to kinases from other organisms, suggesting that tpSTK1 belongs to a novel family of kinases. TpSTK1 is the first molecularly characterized kinase that appears to catalyze phosphorylation of biomineral forming proteins in vivo.
Stimulation of GTP Cyclohydrolase I by Phosphorylation Upon T Cell Activation
Authors: Chen Wei , Li Li , Brod Torben , Saeed Omar , Dikalov Serguei , Harrison David G
Published: June 2021

Recently, we found that phosphorylation of GTP cyclohydrolase‐1 (GTPCH‐1), the rate‐limiting enzyme for BH4 biosynthesis, increases its activity and BH4 levels in endothelial cells. To determine if this occurs in non‐endothelial cells, we stimulated T cells both in vitro and in vivo. Naïve T cells had nearly undetectable levels of BH4 and minimal expression of either endothelial or inducible NOS. Anti‐CD3 stimulation robustly induced T cell eNOS and iNOS and increased biopterin from near undetectable levels to 5.4 pMol/mg protein. Biopterin induction was similar in CD4 and CD8 cells. Western blots showed that this was associated with GTPCH‐1 phosphorylation at S72. Pharmacological inhibition of casein kinase II prevented GTPCH‐1 phosphorylation and blunted the increase in T cell BH4. Inhibition of GTPCH‐1 with diaminohdroxypyimidine (DAHP, 500 μm) prevented T cell BH4 accumulation and increased T cell superoxide production, which was dependent on uncoupling of both eNOS and iNOS. GTPCH‐1 inhibition also promoted TH2 polarization in memory CD4 cells. OVA immunization in vivo confirmed a marked increase in T cell BH4. Thus, T cell activation is associated with GTPCH‐1 phosphorylation, which increases GTPCH‐1 activity and biopterin production. In inflammatory settings where BH4 oxidation is favored, this could modulate T cell function. This study was supported by NIH grants PO1 HL58000 and RO1 HL39006.
Ultrasensitive western blot and AlphaScreen® SureFire® assays – complementary tools for studying cellular kinase signaling pathways
Authors: Campisano Michael , Titus David , Hurt Stephen
Published: June 2021

Western blotting is widely used for studies of cellular kinases in signaling pathways because it provides high specificity by combining separation with immunodetection. However these assays suffer from limited sensitivity and throughput. Here we demonstrate that an ultra‐sensitive western blot substrate (Western Lightning Ultra) increased detection sensitivity 5‐10 fold, enabling much more robust detection of phosphorylated targets in cellular extracts while significantly reducing consumption of primary antibodies. We further compared the detection of multiple phosphorylated targets using AlphaScreen SureFire assays for cellular kinase activity. Our data indicated that the AlphaScreen SureFire assays for several targets in the IP3 signaling pathway gave results comparable in sensitivity to ultra‐sensitive western blots, but with higher throughput and a much less time‐consuming protocol. We further demonstrated how this assay platform can be used to quantify phosphorylation levels of Akt as a percentage of total Akt protein, and to normalize phosphorylation data using GAPDH protein as a control for variations in cell number.
Inhibition of Translation via Phosphorylation by p21‐Activated Protein Kinase 2 (Pak2)
Authors: Xu Lingyun , Orton Kevin , Traugh Jolinda A.
Published: June 2021

Translation is down‐regulated in response to a variety of stresses. Under moderate stress, p21‐activated protein kinase (Pak2) is activated by Cdc42 and induces cytostasis, while caspase 3 cleaves and activates Pak2 under apoptotic conditions. Pak2 inhibits translation through phosphorylation of initiation factor 4G and the serine/threonine kinase Mnk1. The present study shows that Pak2 phosphorylates three ribosomal proteins, S6 and S10 in the small ribosomal subunit and L34 in the large ribosomal subunit. S10 and L34 are phosphorylated by Cdc42‐activated and caspase‐cleaved Pak2, while S6 is phosphorylated only by caspase‐cleaved Pak2 at one site on serine 235. The same site is phosphorylated during early apoptosis following treatment of 293T cells with H2O2. Addition of active Pak2 to a reticulocyte lysate results in the phosphorylation of S6 and S10, inhibiting protein synthesis. Reconstitution of reticulocyte lysate with ribosomes phosphorylated by caspase‐activated Pak2 in vitro, inhibits translation by 33%, as compared with lysate reconstituted with non‐phosphorylated ribosomes. Active and inactive Pak2 binds to ribosomes and 40S ribosomal subunits, but not to 60S subunits. In summary, Pak2 is shown to directly down‐regulate translation through phosphorylation of ribosomal proteins.
Lipid raft association with TLR4 and TLR2 in the lungs of foals
Authors: Kaur Navjot , Suri Sarabjeet , Townsend Hugh , Lohmann Katharina , Singh Baljit
Published: June 2021

Rhodococcus equi (R. equi) establishes long‐term pulmonary infection and causes pneumonia in foals because of inefficient TLR2 signaling and inability to produce IFN gamma. Lipid rafts act as signaling platforms for TLRs. Because there are no data on the lipid rafts in the horses and because of efforts to develop vaccines against R. equi, we developed protocol to isolate lipid rafts and examined its association of TLRs in lung tissues alveolar macrophages (AM) obtained from foals infected with R. equi with (N=7) or without vaccination (N=8). Lipid raft fractions were identified through the presence of flotillin‐1 and GM‐1. While TLR4 and TLR2 were localized in most of the fractions (1–9) in control foal AM collected, the TLR4 and TLR2 association was restricted to the lipid rafts AMs of vaccinated foals. Lung sections showed co‐localization of flotllin‐1 with TLR4 and TLR2 in the airway epithelium. Immunohistology showed increased expression of TLR4, TLR2 and TNF alpha in alveolar septa and in some cases in the vascular endothelium and airway epithelium in the lungs of both the infected groups compared to the untreated control foals. There were no differences in histopathology and gross pathology in the lungs between two groups. The data show that while there is some effect of vaccination on the association of TLR4 and TLR2 with the lipid rafts, the vaccine does not protect the foals against pathology caused by R. equi infection.
Effect of extracts from Leaves of Perilla frutescens treated with sucrose aqueous solution on antioxidant activities and glutathione level in HepG2 cell.
Authors: Yang Sung‐Yong , Hong Chung‐Oui , Koo Yun‐Chang , Nam Mi‐Hyun , Kim Ji Hoon , Wang Zeng , Ma Yuanyuan , Noh Sang‐Hee , Lee Kwang‐Won
Published: June 2021

The purpose of this study is to find a variant of Perilla frutescens leaves carrying strong antioxidant activities, and having more levels of caffeic acid and rosmarinic acid. We have investigated two species of perilla frutescens (Namchun, Bora) which were grown with 6% of sucrose solution and without. The perilla leaf extracts showed antioxidant activities in 2,2‐azinobis‐(3‐ethylbenzothiazoline‐6‐sulfonic acid) radical cation assay, 1,1‐diphenyl‐2‐picrylhydrazyl free radical‐scavenging assay, ferric‐reducing antioxidant power assay, total flavonoid content and total polyphenol content. The intracellular reduced glutathione (GSH) in HepG2 cells was determined by HPLC.

According to radar chart demonstrating characteristic feature in the antioxidant properties, Namchun has 22.46% of radar chart area and Bora has 23.04%. But, Namchun, grown with 6% sucrose aqueous solution has 80.1% of radar chart area and Bora, same condition, has 82.5%. So, Namchun and Bora, grown with 6% sucrose aqueous solution have higher antioxidant activity than non‐sucrose. GSH level were increased in HepG2 cells treated with Namchun compared with non‐treated. And treated with Namchun growing 6% sucrose aqueous solution were even higher than non‐sucrose in GSH level. Also, GSH level in HepG2 treated with Bora growing 6% sucrose aqueous solution were even higher than non‐sucrose.
Influenza A NS1 Helps Prevents Innate Immunity via Inhibition of TRIM25
Authors: Chavez Edwin , Cox Justin , Grosjean Ellie , Ibrahim Fady , Polk Kortnie , Sturdivant Michael , Taylor Kilah , Stubbs Tamica , Colton Shannon
Published: June 2021

One of the key components to vertebrate survival is the innate ability to produce interferons (IFN). Type 1 IFN are regulatory proteins expressed in response to infection caused by invading viruses. The synthesis of these cytokines is upregulated through several different cellular pathways. For instance, the RIG‐I pathway detects foreign viral RNA enabling the CARD domains of RIG‐I to be ubiquitinized by the enzyme, TRIM25. This initiates a signaling cascade which ultimately results in the expression of IFN. Once produced, IFN are released from the infected cell and bind to IFN‐alpha/beta receptors on the epithelial cells of the respiratory system. This consequently produces antiviral cytokines which degrade viral RNA. In defense to this mechanism, Influenza A virus carries an NS1 gene that has been shown to compromise the regulation of IFN. NS1 binds to TRIM25, preventing the enzyme from ubiquitinating RIG‐I and ultimately inhibiting cytokine production. NS1, although not necessary for construction of the virus itself, compromises the innate immune system, resulting in the flu. The E.E. Waddell SMART Team (Students Modeling A Research Topic), in collaboration with MSOE, has built accurate models of IFN and NS1 using 3D printing technology. Supported by a grant from NIH‐NCRR‐SEPA.
Metabolic Reconstruction of Thermotoga Maritima – Toward Complete Sequence‐Structure‐Function Understanding of a Model Organism
Authors: Wilson Ian A.
Published: June 2021

Genome sequencing from individual organisms has opened up new opportunities for deeper understanding of the metabolic systems in cells and in whole organisms. Knowledge of the three‐dimensional structures of the component proteins of such networks enables a reconstruction of these vital processes at the atomic level. We have developed a metabolic network reconstruction at the structural level for the hyperthermophilic bacterium, Thermotoga maritima, through combination of 120 experimental and 358 computational models to achieve 100% coverage of the proteins involved in central metabolism (1). We have been able to assess the number and diversity of folds that encompass this network and the relationship between enzymes that catalyze sequential connected reactions versus those that carry out similar types of reactions. We have been able to confirm functional assignments of the protein in the network and been able to predict the function of previously uncharacterized proteins to enable further development of the reconstruction. This work has been accomplished through a collaboration between the Joint Center for Structural Genomics (www.jcsg.org), the Joint Center for Molecular Modeling ( PI Adam Godzik, Burnham)), Andrei Osterman (Burnham) and Bernhard Palsson (USCD) and supported by the NIH NIGMS Protein Structure Initiative and grants NIH U54 GM074898, U01 GM076221, DE‐FG02‐08ER64686.
Crystal Structure of FolD Bifunctional Protein, a Methylenetetrahydrofolate Dehydrogenase/Cyclohydrolase from Campylobacter jejuni
Authors: Makowska‐Grzyska Magdalena Maria , Zhang Rongguang , Peterson Scott N , Joachimiak Andrzej
Published: June 2021

Tetrahydrofolate (THF) plays a crucial role as a molecule for the production and utilization of monocarbon units in various oxidation states. Enzymes involved in THF metabolism are of pharmaceutical interest, as their function is crucial for amino acid and DNA synthesis. The interconversion of two major one‐carbon donors, methylene and methenyl groups, occurs through the sequential activities of NAD(P)‐dependent methylenetetrahydrofolate dehydrogenase (MTHFD) and methenyltetrahydrofolate cyclohydrolase (MTHFC). These activities frequently coexist as a part of a bifunctional enzyme in prokaryotes. In higher organisms, MTHFR and MTHFC are often linked together with another protein, formyl‐THF synthetase (FTHFS) and operate as a trifunctional enzyme. We have determined the crystal structure of a FolD protein, a predicted MTHFR/MTHFC from Campylobacter jejuni at 2.20 Å resolution. The protein exists as a dimer and each subunit is composed of two α/β domains that assemble to form a wide, largely hydrophobic cleft. Comparison with the structure of the homologous enzyme domain from a trifunctional human protein allowed the identification of a highly conserved cluster of basic residues that most likely participate in the binding of a THF substrate. Exploring the differences between the active sites of bacterial and human enzymes might allow the development of inhibitors specific to the bacterial target. This research was supported by the NIH contract No. HHSN272200700058C, and by the U.S. DOE, Office of Biological and Environmental Research, contract DE‐AC02‐06CH11357.
Female anxiety: Role of estradiol and group I metabotropic glutamate receptors within the amygdala
Authors: De Jesus‐Burgos Maria Ines , Pérez‐Acevedo Nivia L
Published: June 2021

Females have a higher prevalence to develop anxiety disorders, suggesting that estrogen might be involved in female anxiety. Estrogen alters neural activity by affecting neurotransmitter release and the activity of metabotropic glutamate receptors (mGluRs). Whether estradiol interact with group I mGluRs within the basolateral amygdala (BLA) to modulate female anxiety, is unknown. We therefore studied the role of estradiol and group I mGluRs in the BLA, a key region involved in anxiety, by infusing the group I mGluR agonist, (RS)‐3,5‐dihydroxyphenylglycine (DHPG). We used ovariectomized female rats with (OVX‐EB) and without (OVX) estradiol replacement to test whether estradiol‐mGluR interaction modulate female anxiety. We analyzed generalized and anticipatory anxiety using the Elevated Plus Maze and Risk Assessment Behaviors (RABs). In generalized anxiety, DHPG (1.0 μM) increased the percentage time in the open arms, in OVX‐EB but not OVX female rats. In anticipatory anxiety, DHPG (0.1 and 1.0 μM) decreased the number of RABs in OVX‐EB female rats only. DHPG (10.0 μM) did not alter generalized and anticipatory female rat anxiety. We found that: the anxiolytic‐like effect of DHPG depends upon estradiol treatment, suggesting an interaction between estradiol‐mGluR signaling in anxiety. Supported by MBRS‐RISE (GM61838) to DJB, EARDA (NIH‐NIH‐1G11H046326) to PA.
Apoptotic effects of EGCG via AMPK/mTOR pathway and AMPK/CSK signaling on HT‐29 colon cancer cells and HepG2 hepato‐carcinoma cells
Authors: Park Song Yi , Lee Yun‐Kyoung , Choi Bu‐Young , Lee Won Sup , Kim Young‐Min , Park Ock Jin
Published: June 2021

AMPK (AMP‐activated protein kinase) as an anti‐cancer target has been received attention since it functions as an upstream regulator of proliferative proteins such as tuberous sclerosis complex (TSC), mammalian target of rapamycin (mTOR), p70S6 kinase, and elongation factor‐2. Upon activation, mTOR, a cell survival molecule, exhibits uncontrolled proliferation and dysregulation of apoptosis. CSK (C‐terminal Src kinase) is an inactivator of Src and Src‐family protein kinases, proto‐oncogenes that play a role in cell survival and cell proliferation. In this study, we examined the regulation of AMPK/mTOR pathway in HT‐29 colon cancer cells and AMPK/CSK signaling in HepG2 hepato‐carcinoma cells treated with EGCG. EGCG decreased cell viability, and increased apoptotic cell death in both types of cells. The stimulation of AMPK by EGCG resulted in the down‐regulation of mTOR in HT‐29 cells and up‐regulation of CSK activities through decreasing inactive form in HepG2 cells. The application of AMPK siRNA supported the evidence that AMPK acts as an upstream signal of mTOR in cells treated with EGCG. In HepG2 cells, AMPK was found to act as a positive regulator of CSK. [This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (NO. R01‐2008‐000‐20131‐0)]
Analysis of Branched‐Chain α‐Keto Acid Dehydrogenase Complex Activity in Rat Tissues Using α‐Keto[1‐13C]isocaproate as Substrate.
Authors: Matsumoto Hideki , Akita Keiichi , Sakai Ryosei , Kobayashi Tsuyoshi , Shimomura Yoshiharu
Published: June 2021

The branched‐chain α‐keto acid dehydrogenase complex (BCKDC) is the rate‐limiting enzyme in the catabolism of branched‐chain amino acids (BCAA). The radiochemical method has been used to measure the low level of the BCKDC activity in tissues such as skeletal muscle. In the present report, we have developed a highly sensitive, non‐radioisotope method for the measurement of BCKDC activity using α‐keto[1‐13C]isocaproate ([1‐13C]KIC) as substrate. The 13CO2 was released from [1‐13C]‐KIC in the reaction catalyzed by BCKDC. Amounts of 13CO2 were calculated from the molar ratio of 13CO2/12CO2 assayed by gas chromatography isotope ratio mass spectrometry. A distinct linear relationship between 13CO2 production rate and BCKDC activity was obtained when using purified BCKDC. The detection limit of 13CO2 in the novel assay method was at least 0.167 nmol. We measured BCKDC activities in several rat tissues including skeletal muscle such as low levels of BCKDC activity. The activities of BCKDC obtained are comparable to those reported previously. These results suggest that the novel BCKDC assay method using [1‐13C]KIC is useful for the measurement in tissues with low BCKDC activity such as skeletal muscle.
Comparison of Various Preparations of Gynostemma pentaphyllum (Jiaogulan) and its Main Active Constituents, Gypenoside Saponins, on Resting Metabolic Rate: Controlled Human Clinical Study
Authors: Amagase Harunobu , Handel Richard
Published: June 2021

We have compared efficacies of 3 different Gynostemma pentapyllum (jiaogulan) samples on resting metabolic rate (RMR) by hand‐held indirect calorimeter in healthy human adults (n=10). The samples were jiaogulan leaf extract (A), jiaogulan blend composed of extracted and freeze‐dried whole Gynostemma plant (B), and JULE of the OrientTM (JULE), an herbal blend consisting of B plus goji, astragalus, codonopsis, jujube, longan, luohanguo, rhodiola, schisandra, açai, amalaki, aronia, camu‐camu, elderberry, ginger, Japanese sophora, blueberry, bilberry, lychee, cranberry, cherry and reishi mushroom. Total saponins were adjusted at the same level in all samples. Each sample (60 ml) was separately consumed immediately before a nutritional beverage (360 kcal) after 12 h fast. B increased RMR by 178 kcal over baseline level (1,703 kcal) at 2 h post‐intake. JULE intake further increased RMR by 231 kcal significantly (P<0.05), about 14% of baseline level. A increased RMR by 134 kcal over baseline, which was same level with control (139 kcal), and was significantly lower than the effect of B or JULE. These results suggest that constituents other than saponins in jiaogulan may have stimulating effects on metabolism, that whole plant jiaogulan may have greater efficacies than leaf alone, and that the synergistic effect of various herbs in the JULE causes significant increase in the effect of jiaogulan on human metabolism.
Eating behaviors, fitness and body mass index in a multi‐ethnic college‐aged population
Authors: McEligot Archana Jaiswal , Alano Ryan E. , Johnson Sara E. , McMahan Shari , Cullin Jennifer , Nguyen Tina , Anozie Janet
Published: June 2021

We investigated the relationship between eating behaviors and fitness levels with body mass index (BMI) in a multiethnic [non‐Hispanic White (n = 75), Hispanic (n = 83) and Asian (n = 48)] college‐aged student population. Self‐reported eating behaviors included eating regular meals, missing meals and snacking between meals. Other measures included daily activity level (self‐reported), V02 max and blood pressure. Analysis of variance (ANOVA) was used to assess BMI differences between ethnicities, and multivariate regression (controlled for gender, age, ethnicity, eating behaviors, blood pressure, V02 max and daily activity level) was used to assess predictors of BMI. Significant differences (p < 0.05) in BMI were observed between non‐Hispanic whites (23.79 ± 4.63), Hispanics (24.40 ± 4.88) and Asians (22.04 ± 3.99). Hispanic ethnicity (compared with Asians) (β = 1.73; p = 0.02), snacking in between meals (β = −2.86; p = 0.001), physical activity level (β = 0.78; p = 0.05), V02 max (β = −0.16; p < 0.001) and systolic blood pressure (β = 0.12; p < 0.001) were significantly associated with BMI. Findings from the present study suggest that even at younger ages, the Hispanic population has higher BMI, and that weight management strategies should be targeted towards the younger Hispanic population, potentially reducing subsequent obesity. Further, increasing aerobic fitness (i.e. V02 max) and improving SBP can reduce BMI, and eating in between meals could also reduce BMI, however because younger college‐aged students differ in eating behaviors, further studies assessing meal patterns and snacking in relation to BMI should be conducted.
Drinking water intake among adolescents: associations with sociodemographic and behavioral variables— Florida, 2007
Authors: Park Sohyun , Sherry Bettylou , O'Toole Terrence
Published: June 2021

Little is known about whether water intake is associated with sociodemographic and behavioral disparities in adolescents. This cross‐sectional study examined associations between water intake and both sociodemographic and behavioral factors among adolescents. Analyses were based on the 2007 Youth Physical Activity and Nutrition Survey using a representative sample of 4,292 students in grades 6–8 in 86 Florida public middle schools; and analyses included logistic regression. The response rate was 70%. The outcome measure was low water intake (<3 glasses/day during the past 7 days). Overall, 64% reported low water intake. Based on multivariate logistic regression analyses, the significant predictors for low water intake were frequent consumption of snack/soda while watching TV/movies (most/every time vs. none, odds ratio (OR)=2.32; 95% confidence interval (CI): 1.64–3.29), no 100% juice intake (none vs. ≥ 3 times/day, OR=1.71, 95% CI:1.28–2.29), no team sports participation (none vs. ≥ 3 teams/year, OR=1.67, 95% CI:1.37–2.04), drinking soda (≤1 soda/day vs. none, OR=1.43, 95% CI:1.18–1.73), frequent fast food restaurant use (≥3 days/week vs. none, OR=1.37, 95% CI:1.05–1.79), and no milk intake (none vs. ≥ 2 glasses/day, OR=1.36, 95% CI:1.06–1.74). Targeted interventions to increase water intake in adolescents are needed based on findings from the present study.
The effect of home delivered DASH meals on the diets of older adults with cardiovascular disease
Authors: Racine Elizabeth , Troyer Jennifer , Grace Ngugi , McAuley William
Published: June 2021

Background

There is little information regarding whether free therapeutically designed meals have a beneficial impact on the diets of older adults with hyperlipidemia and/or hypertension.

Objective

The objective of this study is to determine the degree to which dietary change is influenced by providing one therapeutically designed meal per day.

Design

We conducted a one‐year randomized controlled trial among 298 people aged 60 or older with hyperlipidemia and/or hypertension where 50% of participants received seven therapeutic meals/week for 12 months. Those in need of dietary change at baseline (n=210) as measured by accordance to the Dietary Approach to Stop Hypertension (DASH) diet were examined. Change in intermediate DASH accordance, DASH accordance and the nutrients that comprise the DASH diet were measured using 24‐hour food recalls at baseline, 6 months, and 12 months. Chi‐square tests, t‐tests, and multiple regression were used to examine the association between receipt of meals and dietary change over time.

Results

Participants who received meals were 20% (p=0.001) more likely to reach intermediate DASH accordance at 6 months and 18% (p=0.007) more likely to meet saturated fat accordance at 12 months compared to those who did not receive meals. When stratified by race and income gains were marginally larger for whites and higher income individuals.

Conclusions

Delivery of seven therapeutic meals per week was found to increase compliance with dietary recommendations among non‐compliant older adults with cardiovascular disease.

This project was funded by the Administration on Aging (#90AM2665).