bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of lipoperoxidation processus of HIV‐infected patients
Authors: Rakhmanova Zebiniso P
Published: June 2021

It is well known, that problem on the nature and direction of changes of activity of enzimes of antiraducal defence at patological occurence of immunoreactivity of organisems, especially at HIV infection, still remains very important at present time and not investigated completely yet.

Development of “oxidazing stress” at HIV infections, occured by disfuntion of immune system, can be linked with unsufficient capacity of AOS of infected organisme.

We have examened 21 HIV infected paticient from 17 to 43 years of age. HIV infectin was detected by solidfase immunoenzimes analysis in AMI‐HIV‐IFA‐UNIF tests. The intencity of PLO analysis of this group HIV paticients were analysed by methods developped L.I.Andreeva et al., (1988). Determination of SOD has been done on V.T. Mkhitaran, T.E. Badalan and catalase activity was determned on the methods described by M.A.Koroluk (1988)

Obtained results showed that amount of MDA content of blood of HIV infected pacients was considerably higher as compared to non‐infected ones. The level of SOD of both infected and non‐infected paticient had no significant difference. Catalase activity was significant higher level in HIV‐infected pacients as compared to non‐infected ones.
Cleavage of S/O‐glucosides by β‐glucosidase from sweet almond
Authors: Shen Hong , Byers Larry D.
Published: June 2021

Sweet almond β‐glucosidase (EC ) is a highly efficient (GH family 1) exo‐glycohydrolase. The activity of this enzyme depends on a deprotonated carboxylate and a protonated carboxylic acid for the optimal activity. In this study we report on the hydrolysis of thioglycoside catalyzed by β‐glucosidase. While the Km values for the S‐ and O‐glycosides are similar, the kcat values are about 1,000‐times lower for the S‐glycosides. Linear free energy relationships between the binding and catalytic parameters and the aryl substituents on the phenyl glucosides reveal a striking similarity between the S‐ and O‐glycosides. For both substrates, catalysis (kcat/Km) shows a strong dependence on the pKa of leaving group (βlg= −1) suggesting that in the transition state there is nearly full development of a negative charge on the (thio)phenolic oxygen or sulfur. Substrate binding shows a dependence on both hydrophobicity and the electronic properties of aryls substituents. The brφnsted coefficient for substrate binding is β ≈ −0.45, suggesting a weakening of glucosidic bond when the E · S complex is formed.
The phosphatase laforin crosses evolutionary boundaries and links carbohydrate metabolism to neuronal disease
Authors: Gentry Matthew S. , Dowen Robert H. , Worby Carolyn A. , Mattoo Seema , Ecker Joseph R. , Dixon Jack E.
Published: June 2021

A hallmark of Lafora disease (LD), a fatal neurodegenerative disease, is the accumulation of insoluble carbohydrates called Lafora bodies (LBs). LD is caused by mutations in the gene encoding the phosphatase laforin, which reportedly exists solely in vertebrates.

We utilized a novel bioinformatics screen to identify laforin orthologs in five protists, Toxoplasma gondii, Eimeria tenella, Tetrahymena thermophila, Paramecium tetraurelia, and Cyanidioschyzon merolae. Strikingly, laforin is absent in all invertebrate organisms, and only exists in vertebrates and these five protists. These protists all evolved from a progenitor red‐alga and all synthesize an insoluble carbohydrate, whose composition closely resembles LBs. Furthermore, we show that Kingdom Plantae, which lacks laforin, possesses a protein with laforin‐like properties called SEX4. Mutations in the Arabidopsis SEX4 gene results in a starch excess phenotype. We demonstrate that human laforin fully complements the sex4 phenotype, and establish laforin and SEX4 as functional equivalents.

Thus, despite the broad evolutionary diversity between vertebrates, plants and protists, laforin and SEX4 conduct a heretofore overlooked conserved function: they regulate the metabolism of “insoluble glycogen”, starch and floridean starch. Finally, we show that this family of phosphatases dephosphorylates insoluble carbohydrates, providing a molecular explanation for the etiology of LD.
Identification of 14‐3‐3 Binding Proteins in Mouse Testis by Tandem Affinity Tag Purification
Authors: Puri Pawan , Snow Alan J , kline Douglas W , Vijayaraghavan Srinivasan
Published: June 2021

The 14‐3‐3 proteins interact with various cellular proteins in a phosphorylation dependent manner. The ability of 14‐3‐3 to bind phosphorylated proteins implicates 14‐3‐3 proteins in a variety of cell signaling pathways. 14‐3‐3 proteins play important regulatory role in mitosis and meiosis. Sperm are produced in testis by a complex process of spermatogenesis. While 14‐3‐3 is present in testis and sperm its role in male gametes is relatively unknown. Tandem affinity tagged (TAP) 14‐3‐3 was expressed in transgenic mice driven by an ubiquitin promoter (Angard et al 2006). TAP‐tagged 14‐3‐3 was purified from testis of the transgenic mice. A total of 150 proteins were found to be associated with 14‐3‐3. These include proteins involved in cell signaling, transcription, translation, splicing, metabolism and protein trafficking. Some of the testis 14‐3‐3 binding proteins were protein were: protein phosphatase1γ2, GSK‐3, testis specific gene 2, SH3 domain GRB2 like B1, gravin, WLK1, Y box protein 2, phosphatidyl ethanolamine binding protein, and piwi homolog 1. PP1γ2 and GSK‐3 binding with 14‐3‐3 was further confirmed by GST pull down assays. Mice lacking PP1 gene are infertile due to impaired spermiogenesis. Efforts are underway to characterize the 14‐3‐3 /PP1γ2 complex to elucidate its function in spermatogenesis. Our results suggest 14‐3‐3 may have important regulatory role in male germ cell maturation in the testis.
Microarray profiling reveals calcium‐binding proteins S100A8 and S100A9 as candidate markers for invasive human breast epithelial cells
Authors: Moon Aree , Yong Hae‐Young , Song Jae‐In , Cukovic Daniela , Salagrama Sridevi , Kaplan David , Putt David , Kim Hyesook , Dombkowski Alan , Kim Hyeong‐Reh Choi
Published: June 2021

The goal of the present study is to unveil the gene expression profile specific to the biological processes of human breast epithelial cell invasion and migration using an MCF10A model in which the H‐Ras or N‐Ras signaling pathway is constitutively activated. H‐Ras, but not N‐Ras, induces invasion/migration. H‐Ras‐mediated MCF10A invasion involves increased expression of MMP‐2 and ‐9. Analysis of whole human genome microarray revealed that 412 genes were differentially expressed among MCF10A, N‐Ras MCF10A and H‐Ras MCF10A cells. Two calcium‐binding proteins, S100A8 and S100A9, were prominently upregulated in an H‐Ras‐specific manner. Importantly, siRNA‐mediated knockdown of S100A8 or S100A9 expression significantly reduced H‐Ras‐mediated MCF10A cell invasion/migration. We further demonstrated that S100A8 was more closely associated with MMP‐9 expression mediated by ERKs pathway while S100A9 might play a major role in MMP‐2 upregulation which is dependent on p38MAPK pathway. Taken together, this study reveals S100A8 and S100A9 as candidate markers for metastatic potential of breast epithelial cells. Our gene profile data provide invaluable information which might be useful for identification of additional potential targets for prognosis and/or therapy of metastatic breast cancer [Supported by the NIEHS, NCI R41, USA and the Fostering Project of the Lab of Excellency, Korea].
Establishing luciferase expressing bioluminescent Leishmania major
Authors: Miller Stacy‐Ann Maria , Rowton Edgar D , Sumaida Dena , Hammamieh Rasha , Jett Marti
Published: June 2021

Leishmaniasis has been identified in 88, mostly tropical/subtropical countries around the world. There has been an estimated 12 million people infected, with 1.5 million new cases each year and 350 million people living in endemic regions. Leishmania parasites are transmitted by the Phlebotomus spp. sand fly. They are dimorphic and cycle between promastigotes and amastigotes. In this study we focused on the protozoan parasites of the genus Leishmania major (L. major). We aimed to produce significant bioluminescent signals from L. major recombinants that could be used in both in vitro and in vivo studies. We used the firefly luciferase gene/coding region with its catalytic reaction to the luciferin substrate, which was cut from pGL3 basic. This gene was integrated with the LEXSY expression vector which a marker gene selection for Hygromycin B by a series of restriction enzyme digests. The sub‐cloned vector was then linearized with SwaI and transfected into L. major parasites via electroporation. Gel electrophoresis and PCR were used to confirm ligation and linearization. With this bioluminescent L. major we will now be able to carry out in vivo studies in animal models using the Xenogen Ivis bioluminescence imager.
Role of lipoperoxidation and sulfhydryl oxidation on sensitization of mitochondrial complex III to oxidative damage by Fe2+
Authors: Cortés‐Rojo Christian , Calderón‐Cortés Elizabeth , Clemente‐Guerrero Mónica , Manzo‐Ávalos Salvador , Saavedra‐Molina Alfredo
Published: June 2021

Mitochondrial fatty acids (FA) are key oxidative stress (OE) targets. FA peroxidation propensity augments as its unsaturation degree (UD) increase. Thus, augment on UD of FA from mitochondrial membranes may exacerbate mitochondrial damage during OE. This aspect has physiological importance because increasing fashion in population to ingest polyunsaturated FA. To investigate the influence of UD over the damage to electron transport chain (ETC) by OE, as well localization and chemical nature of damage, we used S. cerevisiae mitochondria as model because its membrane FA content can be manipulated. In mitochondria with monounsaturated FA, lipoperoxidation levels were unaffected by 50 μM Fe2+, while only complex III activity was inhibited by 50%. Damage on complex III was attributed mainly to OH• attack because mannitol partially protected against activity impairment. In contrast, in mitochondria with linolenic acid (C18:3, 80% of total FA), Fe2+ increased ten‐fold lipoperoxidation levels and fully inhibits complex III, which was prevented in 75% and 23% by the anti‐lipoperoxidative agent BHT and β‐mercaptoethanol, respectively, indicating a dual damage on –SH groups and lipids. These results show that lipoperoxidation sensitize ETC towards oxidative damage, being complex III the most labile enzyme affected by –SH and lipid oxidation. Supported by Fondos Mixtos CONACYT‐ Gob. Edo. de Michoacán (64277, 64308), COECYT and CIC‐UMSNH (2.16) grants.
Tumor progression locus 2 (TPL2) is a novel target for regulating obesity associated liver inflammation and steatosis
Authors: Perfield James W , Tsichlis Philip N , Greenberg Andrew S
Published: June 2021

Tumor progression locus 2 (TPL2) is a MAP 3‐kinase that is required for toll receptor and tumor necrosis factor‐alpha (TNF‐α) stimulation of the ERK/MAP kinase cascade. TPL‐2 knockout (KO) mice exhibit a dramatic reduction in LPS induced TNF‐α production due to defective ERK‐1/2 activation. TNF‐α is increased in obesity, implicated in the upregulation of other inflammatory cytokines, and has been demonstrated to induce liver lipogenesis. The objective of this study was to determine if TPL2 is an important mediator of inflammation and liver steatosis associated with high fat feeding and obesity. Male wild‐type and TPL2KO mice were fed a high fat diet for 12 wks and the livers of these animals were investigated. TPL2KO mice were robustly protected from an increase in liver inflammation as mRNA expression of the inflammatory cytokines; TNF‐α, IL‐6, and IL‐1β were dramatically reduced by 60 to 80%. Despite no difference in body weight, TPL2KO mice tended to have smaller livers and decreased hepatic steatosis as determined by histological observation. In support of this observation, mRNA expression of lipogenic genes (FAS, ACC‐1, SREBP‐1c) was lower (>50%) in the livers of the TPL2KO mice. These data provide evidence that targeted disruption of TPL2 in the obese state results in decreased inflammation and hepatic steatosis. Ongoing studies will further define the role of TPL2 in mediating obesity associated inflammation. Supported in part by the Tufts‐NEMC Cancer Center Pilot Project Grant. USDA NIH
Promoting Content‐Independent Skills in the Biochemistry Active Learning Classroom
Authors: Loertscher Jennifer , Minderhout Vicky
Published: June 2021

As the scope of biochemistry becomes larger and more interdisciplinary, the question of what students should learn in a biochemistry course becomes a significant challenge. Although most biochemists agree that all students should know a set of biochemical fundamentals, decisions must always be made on what content to cover or omit when teaching a course. ASBMB has recognized that one solution is to make acquisition of content‐independent skills central in biochemistry courses. Such skills, including the ability to dissect a problem, the ability to collaborate with others, and the ability to think in an integrated manner, enable students to be life long learners. Biochemistry at Seattle University has been taught without lecture using a structured small group format for the past ten years. This format enables instructors to connect learning of biochemistry content to practice of essential skills. Skills are taught through the use of positively interdependent small groups, through activities explicitly designed to hone skills, and through assignment of periodic student self assessments. We have seen convincing evidence of skill acquisition using a variety of assessment methods including a student assessment of learning gains (SALG) survey. We are currently conducting an NSF‐funded effort to have our activities implemented and evaluated on a national level in preparation for wide dissemination.
Adiponectin membrane receptor 1 and 2
Authors: Faile Katie , James Alena , Parker Chasta
Published: June 2021

Metabolic syndrome is used to describe various health risk factors that can lead to obesity, Diabetes type 2, and cardiovascular disease. Adipose tissue plays a key role in metabolic syndrome in that it secretes many hormones and other metabolically functional molecules. One such hormone is Adiponectin (Acrp30), a 30 kDa protein hormone essential to the mediation of lipid and glucose metabolism. It has been shown that decreased levels of Acrp30 induce a higher risk for individuals to develop obesity, diabetes, and cardiovascular disease. Research on the structure and function of Acrp30 serves as an initial step in the development of novel treatment methods for obesity‐associated and vascular disorders. To complete these studies, a more in‐depth analysis of Acrp30's membrane receptors, AdipoR1 and AdipoR2, is needed. The purpose of this study is to produce high yields of recombinant Human AdipoR1 and AdipoR2 using Spodoptera frugiperda (Sf9) insect cells. Using Novagen's Insect Direct® system, AdipoR1 or AdipoR2 was transiently transfected into the Sf9 cells. The cells were then harvested and the receptors extracted using Pierce's Mem‐Per Extraction Kit. Both receptors have been successfully expressed and functionally purified. The project was supported by NIH Grant Number P20 RR‐16461 from the NCRR for support of the program entitled South Carolina IDeA Networks of Biomedical Research Excellence (SC‐INBRE).
“ROLE OF THE BETA‐ADRENERGIC RECEPTORS IN THE CARDIAC RESPONSE TO BETA‐BLOCKER THERAPY”
Authors: Anim Brandon N. , Mohapatra Bhagyalaxmi , Vatta Matteo , Murray Debra , Weinstock George , Gibbs Richard
Published: June 2021

Arrhythmias are electrical abnormalities leading to irregular heart rhythms. The central nervous system releases endogenous catecholamines, which bind to adrenergic receptors on the heart and may increase the risk of arrhythmias.

Patients with arrhythmias treated with â‐blocker therapy result in a significant reduction in the incidence of cardiac events, however, not all patients respond to â‐blocker therapy and the molecular basis is poorly understood in children. Previous studies in adults with heart failure identified S49G and G389R single nucleotide polymorphisms (SNPs) in â‐1ADR associated with the refractoriness using â‐blocker therapy.

We sought to investigate whether these SNPs affect â‐blocker therapy in children with arrhythmias. We setup a pilot study to test non‐responsive subjects, responsive individuals, patients with heart diseases without arrhythmias and general control subjects. Polymerase chain reactions were followed by direct DNA sequencing covering the coding sequence of both ADRB1/2 genes. The preliminary screening process identified the S49G and G389R SNPs in the ADBR1, while a novel ADBR2 variant was also identified. We will need to expand the patient cohorts to reach statistical significance. Finding â‐1/2ADR variants in subjects with arrhythmias will greatly help understand the role these receptors play in unsuccessful responsiveness to â‐blocker therapy.
Mutations in arrestin alpha‐helix I affect receptor binding
Authors: Gurevich Vsevolod V , Vishnivetskiy Sergey A , Francis Derek J , Hanson Susan M , Van Eps Ned , Hubbell Wayne L , Klug Candice S
Published: June 2021

Receptor‐binding elements of arrestin proteins were mapped to the concave surfaces of the two arrestin domains using a variety of methods. Therefore, it is generally believed that in the arrestin‐receptor complex these concave sides of the two arrestin domains face the plane of the membrane. We found that mutations in alpha‐helix I, localized on the side of the arrestin N‐domain opposite to the concave surface, significantly reduce visual arrestin binding to phosphorylated light‐activated rhodopsin and the stability of the arrestin‐rhodopsin complex. The analysis of accessibility of spin labels introduced in the helix to hydrophilic and hydrophobic reagents shows that the helix remains in a hydrophilic environment in receptor‐bound arrestin. Distance measurements by EPR in doubly spin labeled arrestin show that in the receptor‐bound and free arrestin states the position of the helix remains essentially the same. Our recent finding that each receptor molecule binds its own arrestin excludes the possibility that the concave sides of the two domains and the helix interact with two different receptor molecules. We propose two models of the arrestin‐receptor complex consistent with the role of alpha‐helix I in the interaction. NIH grants EY11500, GM 77561 (VVG), EY05216 (WLH), AI58024, GM70642 (CSK).
Characterization of Mosquito Iron Regulatory Protein 1 in Aedes aegypti mosquito
Authors: Borrego Stacey L. , Mayo Jonathan J. , Geiser Dawn L. , Winzerling Joy J.
Published: June 2021

The spread of insect‐transmitted diseases is prevalent due to the female blood‐feeding mosquitos’ ability to carry pathogens. The iron component of the blood meal is critical for mosquito oogenesis, and thus, the manipulation of iron metabolism is a potential option to develop mosquito control strategies and decrease the transmission of these diseases. A major protein in iron metabolism is iron regulatory protein 1 (IRP1). IRP1 controls translation of several proteins involved in iron metabolism. In mammals, phosphorylation occurs at two sites by protein kinase C (PKC) and alters IRP1 interaction with mRNA. Mosquito IRP1 contains one site homologous to the mammalian IRP1 as well as a second unique site. Phosphorylation at the unique site may explain the different behavior of mosquito IRP1. This study attempts to determine if mosquito IRP1 is phosphorylated. Aedes aegypti (yellow fever mosquito) CCL‐125 cells or Anopheles gambiae 4a3b cells were treated with phorbol‐12‐myristate‐13‐acetate (PMA) to stimulate PKC. We report our efforts to detect IRP1 phosphorylation in these cells using SDS‐PAGE, fluorescent staining and treatment of the cells in the presence of 32P‐orthophosphate followed by immuno‐precipitation. Experiments using mammalian cells served as controls. In the long term, this investigation will contribute to the solution of mosquito population control and reduction of infectious disease.
Identifying Protein Binding Partners of Phosphorylated Cortactin
Authors: Johnson Jennifer , Shaw Karen , Kruchten Anne E.
Published: June 2021

Cortactin is an actin binding protein that is overexpressed and phosphorylated in several cancers resulting in altered rates of metastases, however no definitive role for cortactin phosphorylation has been determined. Cortactin interacts with several cellular binding partners, although only a few interactions thus far have been reported to be affected by protein phosphorylation. We hypothesize that the phosphorylation of cortactin results in different binding partners and cellular functions. Two approaches to determine the cellular binding partners of cortactin phosphorylation mutants were used. The first approach evaluated cortactin binding proteins by his‐tagged pull downs using Ni‐NTA beads, cellular extracts, and cortactin followed by separation on SDS‐PAGE and sequencing. The second approach utilized overlay assays and western blotting to identify the protein partners and observe the effects of the phosphorylation treatment. In the future, the differential binding proteins identified through both of these techniques will be tested to determine their effects on cellular motility. This work is supported by a Faculty Start‐Up award from the M.J. Murdock Charitable Trust to A. E. Kruchten.
Histamine‐induced Cyclooxygenase‐2 Expression in Human Coronary Artery Endothelial Cells is Mediated Through MAP Kinases
Authors: KRISHNAN HARINIVAS H , Tan Xiaoyu , Stechschulte Daniel J , Dileepan Kottarappat N
Published: June 2021

Previous studies from our laboratory showed that treatment of human coronary artery endothelial cells (HCAEC) with histamine led to overexpression of cyclooxygenase‐2 (COX‐2) gene and protein (but not COX‐1), and increased production of its products PGE2 and PGI2 (Tan et al, J.Immunol, 179: 7899–7906, 2007). In this study, we investigated the involvement of MAP kinases in the signaling mechanisms leading to the histamine‐mediated COX‐2 overexpression in HCAEC. Treatment of HCAEC with histamine (10μg/ml) resulted in multi‐fold increases in the phosphorylation of p38 MAPK, ERK and JNK. Histamine‐induced phosphorylation of these proteins continued for 10–15 minutes post treatment, after which the phosphorylation levels returned to the basal levels. Incubation of HCAEC with pharmacological inhibitors of the individual MAP Kinases inhibited histamine‐induced COX‐2 gene expression as quantified by real time PCR. The inhibitors also abrogated phosphorylation of specific MAP Kinases confirming the involvement of MAP Kinase pathway in COX‐2 overexpression. In addition, inhibition of the MAP Kinases also inhibited HCAEC generation of prostacyclin (PGI2) confirming that histamine mediated phosphorylation of MAP Kinases is involved in the PGI2 production in HCAEC. These results indicate that histamine‐mediated overexpression of COX‐2 and the resultant production of PGI2 require p38, ERK, and JNK activation, and inhibition of any of these MAP kinases can decrease the expression of COX‐2 and the production of PGI2. (Supported by NIH R01‐HL070101 and Carey Arthritis Fund)
NMR structure and dynamics of the NADH nicotinamide ring bound to dihydrodipicolinate reductase
Authors: Ge Xia , Sem Daniel S.
Published: June 2021

The current development of NMR methods facilitates the study of the mechanism by which enzymes catalyze reactions. The focus of this study is the dynamics and geometry of cofactor NAD(H) upon its binding to a dehydrogenase. To facilitate NMR analysis, the NAD(H) carboxamide was labeled with 15N (15N‐CA). CPMG relaxation dispersion measurements for free [15N‐CA]‐NAD+ showed millisecond timescale conformation exchange, consistent with previous report of exchange between open/closed conformations. Interestingly, in the complex of E. coli dihydrodipicolinate reductase (DHPR) and [15N‐CA]‐NADH, the relaxation dispersion curve also indicated conformational exchange; upon addition of the DHPR substrate analog pyridine 2,6‐dicarboxylate, the exchanged is no longer observed, indicating restricted motion induced by substrate binding.

Survey of 288 X‐ray structures of bound NAD+ and we found that ∼10% of NAD+ carboxamide bonds are in a reversed orientation. We studied the carboxamide of NAD+ bound to DHPR with 15N filtered transferred NOEs and found that it bound with DHPR in the orientation with its C=O pointed in the direction of H− transfer, which is consistent with its X‐ray structure. This 15N‐carboxamide group of NAD(P)(H) permits determination of carboxamide geometry, as a complement to crystal structure analysis which often lacks resolution to determine this.
Analogs of the Sirtuin Metabolite as Chemical probes of Nudix hydrolases
Authors: Comstock Lindsay R , Denu John M
Published: June 2021

The sirtuin family of histone/protein deacetylases catalyzes an NAD+‐dependent deacetylation reaction that is coupled with the formation of a novel metabolite, O‐acetyl‐ADP‐ribose (OAADPr). Sirtuins are implicated in cellular processes ranging from silencing gene expression to lifespan extension, and the discovery of OAADPr has prompted the hypothesis that it may serve as a substrate for other linked enzymatic processes or as a second messenger. OAADPr has been demonstrated to interact with macroH2A1.1, regulate the TRPM2 ion channel, and is metabolized to AMP and acetylated ribose 5′‐phosphate by Nudix hydrolases. Due to the inherent instability of OAADPr to both spontaneous hydrolysis and cellular metabolizing enzymes, including the Nudix hydrolases, four non‐hydrolyzable analogs containing an N‐acetyl substitution have been synthesized. Here, the underlying specificity and activity of Nudix hydrolases towards OAADPr and these stable analogs was investigated. Kinetic constants (kcat/Km and Ki) were determined for human, yeast and E. coli Nudix hydrolases (NUDT9, YSA1, and Orf209). Though NUDT9 did not tolerate 2‐ or 3‐N‐acetyl substitution (500‐fold reduced kcat/Km), YSA1 and Orf209 demonstrated no significant discrimination for the acetylated substrate. Interestingly, a 2 to 3‐fold reduction in catalytic efficiency for the 3‐versus the 2‐N‐acetyl analog for both YSA1 and Orf209 was observed.
Selective biotinylation of inosine 5′‐monophosphate dehydrogenase in cell extracts with a small molecule inhibitor
Authors: Hoefler Chris , Hedstrom Liz
Published: June 2021

Recent investigations into the cellular functions of inosine 5′‐monophosphate dehydrogenase (IMPDH) have revealed roles for IMPDH in vivo that appear to be unrelated to its known catalytic activity. Of particular interest is its connection to the onset of autosomal dominant retinitis pigmentosa. To facilitate further investigation, it was necessary to design a system whereby IMPDH could be selectively labeled in cell extracts. A known small molecule inhibitor of IMPDH, 6‐chloropurine‐9‐β‐D‐ribofuranoside 5′‐monophosphate, was modified by introduction of an amino linker at the 2′‐OH. The modified inhibitor was then biotinylated using standard coupling chemistry. Studies with purified IMPDH were carried out to demonstrate the utility of the modified inhibitor. It was shown that IMPDH can be inhibited by the new compound in a time and concentration dependent manner. It was further shown that the inhibited IMPDH is also biotinylated by Western blot with avidin‐HRP conjugate. Finally, it was shown that IMPDH in bacterial cell extracts can be selectively labeled with the modified inhibitor and pulled out using avidin bound to sepharose beads. We can now use this compound to pull IMPDH out of native tissues and look for interactions with other proteins, nucleic acids, and macromolecular complexes. Supported by NIH EY017325 and GM054403.
The polymerase activity of a mycobacterial galactofuranosyltransferase suggests a novel mechanism for template‐independent processive polymerization
Authors: May John F , Splain Rebecca A , Brotschi Christine , Kiessling Laura L
Published: June 2021

Galactofuranose residues form the structurally crucial galactan polysaccharide in the cell‐wall core of Mycobacterium tuberculosis, the causative agent of tuberculosis. Elucidating galactan biogenesis will help efforts to fight drug‐resistant tuberculosis by providing new therapeutic targets. The mycobacterial galactofuranosyltransferase GlfT is thought to polymerize the galactan, but it is unknown whether GlfT has polymerase activity. Since galactan is built upon a lipid‐linked acceptor, we tested whether the lipid portion of the acceptor influences the activity of GlfT. We found that GlfT processively elongated synthetic acceptors that contained long lipids to produce polysaccharides as long as the naturally occurring galactan. We also found that the degree of polymerization by GlfT depended on acceptor substrate concentration. Our results suggest that GlfT, a template‐independent polymerase, uses a novel mechanism to attain processive polymerization and to control polymeric length.

This research was supported by the National Institutes of Health (AI063596); JFM was supported by an National Science Foundation Graduate Research Fellowship.
Cordyceps pruinosa induces apoptosis in HeLa cells
Authors: Kim Tae‐woong , Yoon Deok‐Hyo , Lim Mi‐Hee , Kim Ho‐Gyoung , Park Hae‐Il , Sung Jae‐Mo , Hwang Ki‐Chul
Published: June 2021

Cordyceps is a parasitic fungus and has long been used as a Chinese medicine to treat illnesses, promote longevity, increase athletic power, and relieve exhaustion and anticancer. In this study, especially, we focused on the mechanisms of cell death exhibited by Cordyceps pruinosa butanol fraction (CPBF) against human cervical adenocarcinoma cell line, HeLa. CPBF inhibited growth and induced apoptosis in HeLa cells in a dose‐dependent manner as determined by MTT assay, DNA fragmentation and phosphatidyl serine distribution. In western blot analysis, apoptosis in Hela cells was associated with the activation of caspase‐3, ‐9 and proteolytic cleavage of PARP, decrease of Bcl‐2/Bax protein ratio, and release of cytochrome C from mitochondria into cytosol. Apoptosis induced by CPBF could not be prevented by various antioxidants. These results show that apoptosis of CPBF toward HeLa cells is mediated by mitochondria‐dependent death‐signaling pathway regardless of reactive oxygen species. Thus, it indicated that CPBF may be a potential candidate in future research and development of bioactive ingredients as anticancer agents. Supported by grant from the BioGreeen 21 program, Korea
Withaferin A induces apoptosis and inhibits adipogenesis in 3T3‐L1 adipocytes
Authors: Park Hea Jin , Della‐Fera Mary Anne , Ambati Suresh , Baile Clifton A
Published: June 2021

Withaferin A (WA), a highly oxygenated steroidal lactone that is found in the medicinal plant Withania somnifera (ashwagandha) has been reported to have anti‐tumor, anti‐angiogenesis, and pro‐apoptotic activity. We investigated the effects of WA on viability, apoptosis and adipogenesis using 3T3‐L1 adipocytes. Mature adipocytes were treated with WA (1μM to 25μM) for 6, 12 and 24h. Viability and apoptosis were measured by CellTiter‐Blue® Cell Viability Assay and single strand DNA ELISA assay, respectively. WA decreased viability in a dose‐ and time‐dependent manner. WA 10μM and WA 25μM increased apoptosis by 73.2 ± 8.1 % and 104.1 ± 19.7% (p<0.001) after 24 hours. WA's effect on adipogenesis was examined by AdipoRed™ assay after treating with WA (0.1μM – 5μM) during differentiation. WA decreased lipid accumulation in a dose‐dependent manner without decreasing viability up to 1μM. WA 0.1μM, 0.5μM and 1μM decreased lipid accumulation by 6.5 ± 5.1%, 47.0 ± 2.7% and 86.1 ± 0.6% (p<0.001). Treatment with WA during the first 2 days seems critical in lipid accumulation. The effect on apoptosis and lipid accumulation were also confirmed with Hoechst staining and Oil Red O staining, respectively. These results show that WA has a potential to act directly on adipocytes to reduce cell viability and adipogenesis and also induce apoptosis. The outcome of this study may have applications in the treatment of obesity.
Spatiotemporal patterning of T cell signaling as an important regulator of T cell activation at the systems level
Authors: Wuelfing Christoph , Singleton Kentner L. , Roybal Kole T. , Sun Yi
Published: June 2021

Spatiotemporal patterning of various individual proteins in T cell activation, i.e. the formation of an immunological synapse, is well described. However, a comprehensive analysis of multiple proteins in the same system is still lacking, yet critical: Spatiotemporal patterning governs interaction probabilities. Two proteins enriched at the same time and location are more likely to interact than ones with distinct patterns. To thus understand how spatiotemporal patterning guides signaling interactions, we determined the patterning of 25 elements of T cell activation, mostly signaling intermediates, in the activation of the same primary T cells by professional APCs. Spatiotemporal pattering was highly diverse: No two elements of T cell activation showed the same patterns. This diversity is critical in allowing control of spatiotemporal patterning to be a powerful regulator of T cell signaling. We also determined the patterning of key receptors/signaling intermediates using T cells with different TCRs. Here, TCR patterning controlled patterning and efficiency of T cell signaling. Spatiotemporal pattering thus is an important regulator of T cell signaling at the systems level.

Supported by the NIH.
Characterization of a novel basic‐rich signaling motif found in the CD3 ε subunit of the T cell receptor
Authors: DeFord‐Watts Laura M. , Albanesi Joseph P. , Murphy Tara , van Oers Nicolai S. C.
Published: June 2021

The T cell receptor (TCR) complex consists of the ligand‐binding α/β heterodimer as well as four associated signaling chains (CD3 γ, δ, ε, and ζ). Each of the CD3 subunits contains one or more copies of a signaling motif termed an immunoreceptor tyrosine‐based activation motif (ITAM). Phosphorylation of tyrosine residues within the ITAMs is critical for TCR‐mediated signaling events. We recently identified an additional signaling motif in the cytoplasmic tail of CD3 ε that we termed the basic‐rich stretch (BRS). Biochemical studies revealed that the BRS could complex the serine/threonine kinase G protein‐coupled receptor kinase 2 (GRK2). Interactions between the BRS and GRK2 likely contribute to TCR cross‐talk with G protein‐coupled receptors, such as CXCR4. To address the role of the BRS in T cell functions, we generated several murine CD3 ε transgenic lines bearing distinct mutations in the BRS. Preliminary results indicate that select modifications of the BRS can dramatically attenuate T cell development and/or alter phosphoprotein induction. Our findings suggest that in addition to its ITAM, CD3 ε contains a second signaling motif (the BRS) that is critical for T cell functions.

[Work was supported by grants from NIAID 5T32AI005284 and AI042953]
Dendritic cell subset‐specific expression of glucocorticoid receptor translational isoforms
Authors: Cao Yun , Lu Nick Z.
Published: June 2021

Dendritic cells (DC) play important roles in both innate and adaptive immunity. Mature DC, but not immature DC, are killed by the potent anti‐inflammatory agents glucocorticoids, the mechanisms of which are unclear. We reported that GR translational isoforms mediate distinct glucocorticoid sensitivity in bone cells. Here we determined the role of these novel GR isoforms in glucocorticoid sensitivity in DC. Bone marrow derived immature DC are resistant to glucocorticoid killing while mature DC induced by lipopolysaccharide and other toll‐like receptor ligands are killed by glucocorticoids. Immature DC contain the GR‐D isoform, the same isoform that mediates glucocorticoid resistance in bone cells. In contrast, mature DC express the GR‐A, ‐B, and ‐C isoforms, enabling glucocorticoid killing. Selective GR translational isoforms also suppressed maturation stage‐specific DC functions, e.g., antigen uptake in immature DC and cytokine secretion in mature DC. The GR mRNA level is similar between immature and mature DC, indicating that translational mechanisms may be involved in the differential expression of GR isoforms in subsets of DC. These observations may provide a basis for designing GR isoform‐selective glucocorticoids with improved anti‐inflammatory efficacy and reduced side effects.

Supported by divisional fund of Allergy‐Immunology, Northwestern University.
AIMP1 induces activation and maturation of bone marrow‐derived dendritic cells via activation of NFkappaB, leading to a type 1 T cell response
Authors: Kim Eugene , Kim Tae Sung
Published: June 2021

AIMP1, previously known as p43, was initially found as a factor associated with a macromolecular tRNA synthetase complex. Recently, we demonstrated that AIMP1 is also secreted and acts as a novel pleiotropic cytokine. In this study, we investigated whether AIMP1 induces activation and maturation of murine bone marrow‐derived dendritic cells (DCs). AIMP1‐treated DCs showed up‐regulated expression of cell surface molecules, including CD40, CD86 and MHC class II. Additionally, AIMP1 induced IL‐12 production. Importantly, AIMP dose‐dependently increased NF‐κB DNA‐binding activity in BMDC. Pretreatment of BMDC with NF‐κB inhibitors inhibited the AIMP‐induced up‐regulation of cell surface molecules and IL‐12 production. Microarray analysis and RT‐PCR determinations indicated that the expression of known DC maturation genes also increased significantly following treatment with AIMP1. Moreover, treatment of DCs with AIMP1 resulted in a significant increase of antigen‐presenting capability, and also stimulated the proliferation of allogeneic T cells. AIMP1‐treated DCs induced activation of antigen‐specific T helper type 1 (Th1) cells in vitro and in vivo. AIMP1‐stimulated DCs significantly enhanced the IFN‐γ production of co‐cultured CD4+ T cells. These results indicate that AIMP1 may play an essential role in generating DC1s, which skew the immune response toward a Th1 response.
MS4A8B oligomerizes with CD20 (MS4A1) and modulates B cell receptor‐stimulated calcium entry
Authors: Deans Julie , Deng Lili , Zuccolo Jonathan
Published: June 2021

The MS4A gene family comprises over 20 distinct murine and human genes expressed in hematopoeitic and other tissues. The founding member of this family, CD20 (MS4A1), is highly expressed in B lymphocytes and functions in store‐operated calcium influx activated by B cell receptor crosslinking (Li, H. et al J. Biol. Chem. (2003) 278:42427–34). Immune responses in CD20‐deficient mice appear to be normal, highlighting the possibility of compensating function from other members of the same family. In this study we examined the expression and function of a new member of the human MS4A family, MS4A8B, which was reported to be transcribed in B cell lines but not in cell lines from other hematopoeitic lineages. Using a GFP‐tagged construct expressed in the BJAB human B cell line, we found that MS4A8B co‐localised with CD20 at the plasma membrane and co‐immunoprecipitated with CD20 from detergent lysates. Like CD20, MS4A8BGFP co‐capped with the BCR in anti‐IgM stimulated cells and remained at the plasma membrane following internalization of the BCR. Over‐expression of MS4A8B in a human B cell line enhanced calcium influx following BCR stimulation. The expression of endogenous MS4A8B and its association with CD20 in primary B cells were confirmed using a new polyclonal anti‐MS4A8B antibody. Interestingly, expression of MS4A8B (but not CD20) was rapidly down‐regulated after BCR stimulation, suggesting a differential role for MS4A8B in modulating BCR‐activated calcium signalling. Funded by the Canadian Institutes of Health Research (CIHR).
Editing IgH Genes Encoding Anti‐SM Antibodies by VH Replacement
Authors: Fan Run , Liu Jing , Yu Zhihong , Zhang Zhixin
Published: June 2021

VH replacement occurs through RAG‐mediated recombination between a cryptic RSS within a rearranged IgH gene and an upstream VH gene. Our previous studies showed that VH replacement occurs spontaneously in human EU12 cells and generates new IgH genes even after formation of functional IgH genes. Recently, we found that one of the dominantly expressed IgH genes in the EU12 muHC+ cells, A19H, directly encodes anti‐SM antigen antibodies when paired with two endogenous IgLambda chains. SM antigen can be detected from EU12 culture supernatant, presumably released from dead cells in the culture, suggesting that SM antigens might be the initial stimuli of ongoing VH replacement in the EU12 cell system. Indeed, treatment of EU12 muHC+ cells with purified bovine SM antigen strongly induces VH replacement. Moreover, when the A19H gene was artificially paired with a panel of 14 randomly selected light chains to express recombinant antibodies, 10 out of the 14 antibodies still react with SM antigens; 5 of them even have higher affinities against SM antigens. Our results suggest that the SM reactivity contributed by the A19H IgH gene can not be efficiently neutralized by light chain editing and VH replacement might offer a unique mechanism to delete IgH genes encoding anti‐SM antibodies.
Scavenger receptor type AI mediates antigen transfer from human B cells to other APCs
Authors: Harvey Bohdan P , Quan Timothy , Roman Robert , Rudenga Benjamin , Craft Joseph , Mamula Mark J
Published: June 2021

With a capacity to sequester specific antigens, B cells are critical antigen presenting cells in the induction of autoimmunity. Our recent findings with Ig transgenic mice indicate that B cells can initiate an immune response in vivo to autoantigen independently of other antigen‐presenting cells (APCs). However, the participation of other professional APCs, both macrophages and dendritic cells, are important in maintaining chronic autoimmunity. We have now examined with human cells the uptake of antigen by specific B cell receptors followed by the active transport of antigen to other APCs using an in vitro system to monitor the transfer of fluorochrome labeled antigen between APCs. We have identified the ability of both macrophages and DCs to acquire antigen from B cells. The transfer of antigen was dependent on cell‐to‐cell contact; however, B cell death did not play a significant role. Antigen transfer was completely blocked by the presence of blocking antibodies to scavenger receptor‐A (SR‐AI). Moreover, antigen trafficking was only observed in the human monocytic cell line THP‐1 following TPA‐induced SR‐AI expression. Overall, these results provide strong evidence for a novel mechanism by which B cells can transfer autoantigen to other professional APCs and more importantly, provide a novel therapeutic target for treatment of lupus. Funding was provided by the Arthritis Foundation and NIH grant AI‐48120.
Anti‐GITR treatment in collagen‐induced arthritis results in enhanced epitope spreading and increased antibodies to citrullinated antigens
Authors: Cozine Cassy , BenBarak Maya , Robinson William H. , Holers V. Michael
Published: June 2021

Patients with rheumatoid arthritis display highly specific autoantibodies to citrullinated protein Ags (ACPA). We previously demonstrated that pathogenic ACPA develop in collagen‐induced arthritis, but the mechanisms of ACPA generation and regulation remain unknown. We hypothesized that effector and/or regulatory T cells (Tregs) play a role in the generation of ACPA. DBA1/J mice were immunized with bovine type II collagen (bCII) in CFA followed by a booster of bCII in IFA. Treg and T effector cells were modified by treating mice with either anti‐CD25 or anti‐GITR mAb. Anti‐GITR treated mice exhibited a significant increase in clinical disease activity. While none of the treatment groups demonstrated a change in autoantibody levels to bovine or murine type II collagen, ACPA titers were significantly elevated in anti‐GITR treated mice. Synovial antigen arrays further demonstrated that anti‐GITR treatment led to an increase in epitope spreading to citrullinated and non‐citrullinated Ags. Furthermore, splenocytes from anti‐GITR treated mice showed an increased proliferative response to bCII and citrullinated peptide. These results suggest that co‐stimulatory effects of anti‐GITR on effector T cells drive autoreactive T cells to expand subsets of pathogenic autoantibodies, particularly ACPA. This work was supported by: NIH R01 AR51749.
IL‐10 is a death signal for newly committed monocyte‐dendritic cell precursors
Authors: Santiago‐Schwarz Frances , Lubin Jean Bernard , Espaillat Mel Pilar , Martin Carla A. , Kurkowski Danielle L.
Published: June 2021

Molecular processes governing the suppression of immunogenic dendritic cell (DC) activity are under intense study. Based on the role of apoptosis in regulating DC development and the ability of IL‐10 to promote apoptosis, we postulated that IL‐10 inhibits DC function by killing monocyte (mono)‐DC precursors upon commitment to the DC lineage. In support of this idea, inclusion of IL‐10 in mono‐derived DC cultures from the outset instituted dose dependent apoptosis during the earliest phases of DC growth (days 2–4). After day 4, IL‐10 mediated apoptosis was no longer confirmable. Flow cytometric analysis verified that IL‐10 killed mono‐DC precursors once they acquired DC surface markers (day 2), while sparing a population of mono‐like cells not yet fully committed to DC growth. These surviving cells possessed remarkable phagocytic ability (including ingestion of apoptotic DC) but lacked the capacity to stimulate naïve T cells, which their counterpart DC cultured without IL‐10 demonstrated. We suggest that IL‐10 imposes innate versus acquired immunity by enhancing mono‐macrophage survival and function while killing mono‐DC precursors newly committed to DC growth. Our results are in agreement with the premise that IL‐10 monitoring of DC activity is not strictly due to maturation arrest. The relevance of our findings to autoimmune disease and immune escape mechanisms employed by pathogens and tumors is discussed.
Subunit Dynamics in the IL‐17 Receptor Complex: Identification of a Pre‐ligand Assembly Domain (PLAD) and Ligand Binding site in IL‐17RA
Authors: Gaffen Sarah , Kramer Jill M , Hanel Walter , Shen Fang , Malone James P , Maitra Amarnath , Isik Nilgun , Swart David , Tocker Joel , Jin Tian
Published: June 2021

IL‐17 is the defining cytokine of the “Th17” subset, and is implicated in the pathogenesis of numerous autoimmune diseases. The first IL‐17 binding protein, IL‐17RA, is the founder of a unique receptor superfamily, and there is considerable interest in targeting IL‐17 or its receptor for anti‐cytokine therapeutics. However, the composition, subunit dynamics and ligand contact sites of the IL‐17R complex remain poorly defined. We show by fluorescence resonance energy transfer (FRET) microscopy that IL‐17RA oligomerizes in the absence of ligand, suggesting that an unknown region mediates pre‐assembly. Using computational modeling, we identified 2 fibronectin‐like III (FN) domains connected by a non‐structured linker. Because FN domains are known to aggregate, we hypothesized that these were responsible for IL‐17RA self‐interactions. The membrane‐proximal FN domain (FN2) but not the membrane‐distal (FN1) domain was found to form homomeric interactions. Thus, FN2 constitutes a “pre‐ligand assembly domain” (PLAD). Interestingly, FN2 fused to the linker but not FN2 alone retained IL‐17‐dependent signaling activity, indicating that the FN2/linker domain contains the IL‐17 binding site. Therefore, while the PLAD is located entirely within FN2, effective ligand binding also involves contributions from the linker region and the FN1 domain.