bluuebunny/crossref_metadata_2025 huggingface dataset:
Capillary Electrophoresis and Computational analysis of human serum transferrin and its receptor complex for the surface changes in physicochemical properties
Authors: Sakajiri Tetsuya , Yamamura Takaki , Kikuchi Takeshi , Yajima Hirofumi
Published: June 2021

Transferrins (Tfs) are a family of homologous iron‐binding proteins, e.g., serum transferrin (STf), lactoferrin (LTf), ovotransferrin (OTf), that are widely distributed in the animal kingdom. However, the 3D structures of the STf C‐domain and its receptor complex have not yet been determined by X‐ray analysis. In order to definitely understand the physiological functions of STf involving the processes of metal toxicities, it is necessary to clarify the 3D‐structural characteristics of metal‐loaded STfs and the STf‐receptor recognition process. We have predicted the 3D structures of the apo‐ and iron‐loaded STf to study the changes in the structural characteristics. We then investigated the correlation of the theoretical values for some physicochemical properties calculated from the modeled STf and the experimental data, where for reference, those for OTf were included. Furthermore, from the viewpoint of the molecular modeling results, significant factors involved in the STf‐receptor recognition process were scrutinized.
Sumoylation is important for the nuclear transport and antioxidant function of the translationally controlled tumor protein
Authors: Munirathinam Gnanasekar , Kalyanasundaram Ramaswamy
Published: June 2021

Translationally controlled tumor protein (TCTP) is one of the most abundantly expressed proteins in the cell. Several functions have been suggested for TCTP family of proteins ranging from calcium binding to histamine release. Nevertheless, its physiological function within the cell is still a mystery. TCTP is present abundantly in the nucleus despite the absence of a nuclear localization signal. Sequence analysis showed that all TCTPs described to date has small ubiquitin‐like modifier (SUMO) motifs. Since SUMO modification plays an important role in the nuclear transport of proteins, we evaluated whether sumoylation is important for the nuclear transport of TCTP. Initial studies confirmed that sumoylated form of TCTP is present in the cytoplasm and nucleus of mammalian cells. Point mutation studies confirmed that sumoylation is important for the transport of TCTP into the nucleus. Previous studies also showed that the expression of TCTP is increased several fold during oxidative stress. To test whether nuclear TCTP has any role in protecting DNA from oxidative stress, we first reduced TCTP with thioredoxin and used this reduced TCTP in our assays. These studies showed that TCTP can protect DNA from oxidative damage. Targeted point mutation studies showed that certain residues in TCTP are critical for this antioxidant function. Knocking down of the TCTP with SiRNA or mutating the TCTP at the SUMO motif failed to confer the anti‐oxidant protection. These studies thus demonstrate that TCTP has significant antioxidant property within the nucleus. This study is supported by NIH grant AI064745 to Dr. K. Ramaswamy
The MLL SET domain is a histone H3 lysine 4 mono‐methyltransferase that interacts with WDR5 in the pre‐SET ATA2 domain.
Authors: Patel Anamika B. , Dharmarajan Venkat , Cosgrove Michael S
Published: June 2021

Di‐ and Trimethylation of histone H3 Lysine 4 (H3K4) are associated with transcriptional activation, and are deposited by a family of proteins sharing a conserved SET domain. One of the chief regulators of H3K4 methylation is the Mixed Lineage Leukemia protein (MLL), which is required for the regulation of Hox genes in hematopoiesis and development. MLL interacts with WDR5, RbBP5, and Ash2L to form a core complex of proteins that are required for the regulation of MLLâ€™s H3K4 methylation activity. It has been suggested that WDR5 plays a central role in maintaining the structural integrity of the MLL core complex. However, it is not currently understood how WDR5 interacts with MLL and how it regulates MLLâ€™S catalytic properties. Here we report on the hydrodynamic properties of WDR5 and an MLL SET domain construct consisting of residues 3745‐3969 (MLL3745). We show that MLL3745 forms a 1:1 hetero‐dimer with WDR5 with a dissociation constant (Kd) of 0.26 Â± 0.02 ÂμM. Furthermore, this interaction requires conserved residues in the pre‐SET ATA2 region of MLL3745. We also show that the recombinant MLL SET domain is a mono‐methyltransferase, and that interaction with WDR5 does not change MLLâ€™S product specificity, but does enhance MLLâ€™S thermostability. These results reveal a new function for the pre‐Set ATA2 domain of MLL in that it is important for the recruitment of proteins that regulate the catalytic activity of the SET domain.
Analysis of Trypanosoma cruzi Phosphoproteome by Mass Spectrometry
Authors: Gaynor Matthew R , Nakayasu Ernesto S , Almeida Igor C
Published: June 2021

Trypanosoma cruzi is the etiologic agent of Chagas’ disease, which affects over 11 million people in Latin America. Lately, much research has been focused on drug and vaccine development against this parasite. In eukaryotic cells, protein phosphorylation regulates many vital pathways and has been extensively studied as potential molecular target. Here, we analyzed T. cruzi phosphoproteins and mapped the phosphorylation sites through mass spectrometry (MS). Total proteins from T. cruzi epimastigote forms were digested with trypsin/urea and fractionated in a strong cation‐exchange ziptip. Ensuing phosphopeptides were purified by immobilized metal‐affinity chromatography (IMAC) and analyzed by liquid chromatography‐tandem MS (LC‐MS/MS, ‐MS3, ‐pseudoMS3). The resulting spectra were searched against T. cruzi sequences in the GenBank using Sequest algorithm. We were able to identify 143 phosphorylation sites from 98 distinct proteins. The Gene Ontology analysis revealed several proteins with protein‐ and nucleotide‐binding activity, cytoskeleton proteins, and proteins involved signal transduction. To our best knowledge, this is the first phosphoproteome study performed in a pathogenic protozoan. Our data may provide new insights into discovery of new interventions to prevent or treat Chagas’ disease.

Supported by NSF/REU Program (grant#DBI‐0353887), and NIH/SCORE (grant#2SO6GM0812‐37).
VACM‐1/cul5 Function Is Regulated by Its Posttranslational Modification Status
Authors: Bradley Shirley E , Burnatowska‐Hledin Maria A
Published: June 2021

VACM‐1 is a cul‐5 gene product, and when expressed in endothelial and in cancer cells in vitro, it inhibits cellular proliferation and MAPK activated signaling. Structure‐function analysis of VACM‐1 protein sequence identified consensus sites specific for phosphorylation by protein kinases PKA and PKC. Mutations at the PKA specific site resulted in increased cellular growth and an appearance of a larger Mr protein on Western blot, suggesting posttranslational modification of VACM‐1/cul5. The aim of our study was to examine if modification of VACM‐1/cul5 by PKC dependent phosphorylation is under the control of PKA dependent phosphorylation and if these changes can affect the biological activity of VACM‐1 protein. Our results indicate that when PKA phosphorylation site is mutated, VACM‐1/cul5 is phosphorylated by PKC. PMA, a drug which increases PKC activity, induced growth in the endothelial cells transfected with mutated VACM‐1/cul5 when compared to the wt VACM‐1 transfected cells. Similarly, Gö6983, an inhibitor of PKC, significantly decreased cell growth in cells transfected with mutated VACM‐1 when compared to the wt VACM‐1 cDNA transfected cells. These results suggest that biological activity of VACM‐1 is dependent on its phosphorylation by PKA and PKC. This work was supported by a grant from NCI (R15CA104014) and by Dept of Chemistry.
Palmitoylation of CKAP4 by DHHC2 regulates APF‐mediated signaling
Authors: Planey Sonia Lobo , Zhang Jun , Zhang Chen‐Ou , Keay Susan , Zacharias David A
Published: June 2021

We identified CKAP4 as a major substrate of the palmitoyl acyl transferase DHHC2 using a novel proteomic method called Palmitoyl‐cysteine Identification, Capture and Analysis (PICA). CKAP4 is a reversibly palmitoylated and phosphorylated protein that links the ER to the cytoskeleton. It is also a high‐affinity receptor for APF, a small glycosylated peptide secreted from bladder epithelial cells of patients with interstitial cystitis. The ability of DHHC2‐mediated palmitoylation of CKAP4 to regulate the antiproliferative effects of APF in HeLa cells was investigated. Our data show that siRNA‐mediated knockdown of DHHC2 expression and consequent suppression of CKAP4 palmitoylation blocks the ability of APF to regulate proliferation. Immunocytochemistry revealed inhibition of CKAP4 trafficking to the plasma membrane following DHHC2 knockdown. Stable expression of a palmitoylation‐incompetent form of CKAP4 showed reduced binding to microtubules and significantly increased the rate of cell migration. These data suggest an important role for DHHC2‐mediated palmitoylation of CKAP4 in cancer‐related cellular behaviors and support the idea that DHHC2 is a tumor suppressor.
Roles of Arp2/3 complex and mDia2 in actin‐based protrusions
Authors: Svitkina Tatyana , Yang Changsong , Korobova Farida , Gerboth Silke , Scita Giorgio
Published: June 2021

Actin polymerization is a key mechanism of force generation in cells. Actin filament nucleators, formins and the Arp2/3 complex, are believed to have non‐overlapping functions in inducing actin filament bundles and dendritic networks, respectively. We investigated roles of the Arp2/3 complex and an mDia2 formin in lamellipodia and filopodia, cellular protrusions containing dendritic and bundled actin filaments, respectively. Contrary to the above paradigm, we have found that mDia2 is an important player in lamellipodia, while Arp2/3 complex significantly contributes to generation of filopodia. The contribution of mDia2 to lamellipodial protrusion was related to generation of long filaments in the lamellipodial network. These long filaments displayed a high tendency to converge into bundles during elongation, thereby producing filopodia. Depletion of the Arp2/3 complex by siRNA in neuronal cells, as expected, inhibited lamellipodia, but remarkably, also filopodia. Correlative structural and kinetic analyses suggested that Arp2/3‐nucleated filaments become incorporated into filopodial bundles in the course of elongation, and that this process contributes significantly to filopodia initiation. Together, our data show that each of two distinct actin nucleators, Arp2/3 complex and mDia2, jointly participate in generation of both lamellipodia and filopodia. Supported by NIH grant GM 070898 to TMS.
Modeling Tumor Development: the Role of L1
Authors: Brady Owen Adam , Yang Muhua , Galileo Deni S.
Published: June 2021

L1 is a membrane‐bound neural cell adhesion protein involved in the development of the nervous system. However, L1 has been implicated in mediating some of the invasive properties of several cancers including gliomas and breast cancers when proteolyzed to create a soluble ectodomain, which then binds to cell surface integrin receptors in an autocrine/paracrine manner. L1 has also been shown to rescue cancerous cells from apoptosis. It was hypothesized that polyclonal antibodies raised against a 15 amino acid peptide corresponding to a critical integrin binding region of human L1 would slow the migration of rat 9L/LacZ and human T98 G glioma cells in vitro, whereas the peptide alone would stimulate migration. Incubation of cells in the peptide decreased cellular migration, possibly due to competitive inhibition. The effects of the antibody on cell migration were inconclusive. Quail QT6 cells have been stably transfected with an L1 ectodomain‐Fc fusion protein (L1‐Fc) designed to be secreted into the media. Once purified, it will be applied to 9L/LacZ and T98 G cells and analyzed using automated time‐lapse microscopy. Additionally, glioma cell migration will be analyzed while on top of QT6 cells secreting L1‐Fc. Both conditions are hypothesized to increase the migration rates of these cell lines by mimicking the activity of endogenous L1 ectodomains.

Supported by HHMI and INBRE.
Ski is required for normal development of embryonic progenitors of hypaxial muscle
Authors: Zhang Hong , Chen Yishi , Colmenares Clemencia , Stavnezer Edward
Published: June 2021

The Ski encodes a transcriptional co‐regulator that stimulates muscle differentiation. We have found that Ski−/− embryos exhibit selective defect in hypaxial muscles. Hypaxial muscles are derived from progenitor cells that originate in the dermomyotome, migrate to distal sites and commit to a myogenic fate. However, the Ski−/− embryos showed that the dermomyotome developed normally. We found that myogenic determination and terminal differentiation were initiated appropriately in the absence of Ski. However, the number of cells expressing myogenic regulatory factors (MRF) genes was reduced and they were restricted to proximal limbs. Our study shows that this deficiency in number and distribution of MRF+ cells is not caused by abnormal proliferation, apoptosis or differentiation. Further examination of Pax3 and Met, which are important for progenitor cell migration, indicated that this defect resulted from impaired migration of myogenic progenitors.

This work was supported by NIH grants (DE15198 to C.C and E.S, CA43600 to E.S, and HD30728 to C.C).
IQGAP1 regulates actin pedestal formation by enteropathogenic E. coli
Authors: Brown Matthew , Bry Lynn , Li Zhigang , Sacks David
Published: June 2021

Microbial pathogens continue to be a major cause of morbidity and mortality worldwide. Many microbes utilize the host cell cytoskeleton for attachment, entry into cells, movement inside and between cells and vacuole formation. Enteropathogenic E. coli (EPEC) use a type three secretion system to inject into the host cell bacterial toxins, which act through N‐WASP and Arp2/3 to promote actin polymerization (Nat Rev Micro Hayward, 2006; 4:358). This localized polymerization of actin induces the formation of stable actin pedestals that support the EPEC. IQGAP1 is a widely expressed 190kDa protein that binds activated Cdc42 and Rac1 (but not RhoA or Ras), actin, calmodulin, E‐cadherin, β‐catenin, extracellular signal‐regulated kinase 1 (ERK1) and ERK2 (Trends Cell Biol. Brown, 2006; 16:242). A primary function of IQGAP1 is to modulate cytoskeletal architecture through a direct interaction with actin, and also indirectly through the RhoGTPases, Cdc42 and Rac1 and via N‐WASP. We show through immunocytochemistry and confocal microscopy IQGAP1 localises to EPEC‐induced actin pedestals. Analysis with selected IQGAP1 mutant constructs indicates IQGAP1 localises to these structures independently of its interactions with actin, Rac1/Cdc42 and calmodulin. Through immunoprecipitation of endogenous IQGAP1 and detecting co‐immunprecipitated proteins, we observe that infection of HeLa cells with EPEC promotes the interaction of IQGAP1 with calmodulin, concomitantly reducing the interaction of IQGAP1 with Rac1 and Cdc42. Together, these data identify IQGAP1 and calmodulin in a novel signaling pathway inducing actin pedestal formation by EPEC, and playing a role in EPEC pathogenicity.
Oxidative and nitrosative stress in mitochondrial of rat kidney during experimental diabetes mellitus
Authors: Pérez‐Gallardo Rocío V. , Albarrán‐Bravo Sacnité , Clemente‐Guerrero Mónica , Manzo‐Ávalos Salvador , Saavedra‐Molina Alfredo
Published: June 2021

Diabetes mellitus (DM) is a metabolic disorder characterized by hyperglycemia. Within the affected population most of the people are in the adult age. The diabetic nephropathy is characterized by patho‐physiological events, which stimulate and repress the production of nitric oxide (NO). The NO is able to react with metalloproteins that contains hemo groups. Veelken (2000) tested that renal vasodilatation mediated by NO is due to endothelial constitutive nitric oxide synthase activity and contributed to hyperfiltration and renal injury in diabetic rats. The respiratory chain is the main generating source of ROS in mitochondria of mammals at level of complexes I and III. Many evidences exist in where oxidative stress is implied in the etiology of several diabetic complications. Our interest is in the pathophysiologic role of the mitochondrial NO, and its narrow relation with oxidative and nitrosative stress during hyperglycemia. Our results showed a significant increase of NO in rat kidney mitochondrial with 1 and 6 weeks after having injected with streptozotocin, respect to the control rats. While in the week 9 no significant differences were observed. Our results also note that it has been an increase in the degree of lipoperoxidation in rats with 1, 6 and 9 weeks of hyperglycemia, respect to control. Oxidative and nitrosative stress in experimental DM affected rat kidney mitochondria. Supported by Fondos Mixtos CONACYT‐ Gob. Edo. de Michoacán (64277, 64308), COECYT and CIC‐UMSNH (2.16) grants.
Decreases in ATP levels of human natural killer cells induced by exposure to Pentachlorophenol.
Authors: Nnodu Ugo c , Whalen Margaret
Published: June 2021

Pentachlorophenol (PCP) is an organochlorine compound that has been used as wood preservative for power‐line poles and fence poles. Pentachlorophenol (PCP) has been detected in human tissues. Human natural killer (NK) cells play a central role in immune defense against viral infection and tumor development. In previous studies we have shown that exposure to10–0.5μM PCP decreases the tumor‐killing (lytic) function of NK cells. The purpose of the study was to examine whether there was an association between loss of lytic function and decreased ATP levels. The ability of antioxidants to prevent any PCP‐induced decreases in either ATP levels or lytic function was also examined. NK cells were exposed to PCP at concentrations ranging from 10 to 0.5 μM for 24 and 48 h. ATP levels were decreased by 15% in NK cells exposed to 10 μM PCP for 24 h. There was no decrease in ATP levels at 5 μM PCP. Exposure of NK cells to 5 and 2.5 μM PCP for 48 h decreased ATP by 32% and 12%, respectively. Exposure to 10 μM PCP for 24 h decreased lytic function by 69%, while exposure to 5μM PCP for 24 h decreased lytic function by about 45% and by 90% after 48 h. These data indicate that PCP exposure is able to decrease ATP levels in NK cells but that significant decreases in cytotoxic function occur even when there is no decrease in ATP levels. The addition of 1 mM antioxidant was unable to prevent the PCP‐induced decreases in ATP levels or lytic function.
Lipid biosynthesis and the unfolded protein response
Authors: Bommiasamy Hemamalini , Sriburi Rungtawan , Fagone Paolo , Jackowski Suzanne , Brewer Joseph W.
Published: June 2021

Professional secretory cells possess an expansive endoplasmic reticulum (ER) to accommodate high‐rate protein production. ER biogenesis requires phospholipids and phospholipid synthesis requires fatty acids. Yet, the mechanisms that regulate phospholipid and fatty acid supply when demand for ER increases are incompletely understood. A link exists between ER expansion and the unfolded protein response (UPR), a stress‐induced signaling pathway that emanates from the ER. The UPR transcriptional activator XBP‐1(S) is required for proper ER development in specialized secretory cells and is sufficient upon overexpression to induce phospholipid synthesis and ER expansion. We find that enforced expression of ATF6, another UPR transcription factor, upregulates phospholipid synthesis and ER biogenesis in fibroblasts. Furthermore, enforced expression of either XBP‐1(S) or ATF6 results in increased fatty acid biosynthesis. Interestingly, fibroblasts overexpressing either XBP‐1(S) or ATF6 differ in their expression of lipid metabolic genes, suggesting that these two UPR factors have the capacity to regulate lipid and fatty acid biosynthesis by distinct mechanisms. These studies reveal further complexity in the relationship between the UPR, lipid production and ER biogenesis. [supported by NIH Grants GM 61970 (J.W.B.), GM 45737 (S.J.), T32 AI007508 (H. B.) and by ALSAC]
Differential effects of apoE3‐ and apoE4‐associated VLDL lipolysis products on monocyte activation
Authors: Higgins Laura J , Tetali Sarada D , Rutledge John C
Published: June 2021

Atherosclerosis is an inflammatory condition perpetuated by the activation and recruitment of circulating monocytes. Postprandial lipemia, characterized by elevated triglyceride‐rich lipoproteins such as very low‐density lipoproteins (VLDL), has been implicated as a mechanism of chronic vascular injury. We hypothesized that postprandial VLDL lipolysis products (PVLP) would induce a greater inflammatory response from monocytes than fasting VLDL lipolysis products (FVLP), mediated by apoE isoform changes in the postprandial state. VLDL was isolated from human donors, subjected to lipolysis, and treated in combination with LPS on THP‐1 monocytes. We found that FVLP attenuated LPS‐induced TNFα secretion to a greater extent than PVLP. This was partially reversed by blocking apoE on FVLP, but not on PVLP. In addition, apoE3‐containing vesicles attenuated LPS‐induced TNFα and IL‐1β expression, while complexes with apoE4 increased cytokine expression. An apoE ELISA revealed that apoE3 binds to LPS with higher affinity than apoE4, suggesting that apoE3 can sequester LPS away from monocytes more readily, thus attenuating their activation. These results imply that postprandial changes in apoE structure and isoform on VLDL could have dramatic effects on vascular inflammation, with implications for atherogenesis. This work was supported by the Training Program in Biomolecular Technology (T32‐GM08799).
Variants of 5‐lipoxygenase from human neutrophils impact on leukotriene biosynthesis in HEK293 cells
Authors: Boudreau Luc H , Bertin Jonathan O , Surette Marc E
Published: June 2021

5‐lipoxygenase (5‐LO) catalyzes the oxygenation of arachidonic acid and is an essential enzyme forleukotriene (LT) biosynthesis. Human neutrophils (PMN) are important producers of LTB4, a potent chemoattractant involved in the inflammatory response.

In this study, we evaluated whether alternative forms of 5‐LO mRNA exist in human PMN and if they are implicated in the biosynthesis of LTB4. RT−PCR analysis of total mRNA from human PMN revealed several potential variants of 5‐LO mRNA. Molecular cloning and sequencing experiments showed that at least three isoforms are present in human PMN. One containing all 14 exons with the expected splicing sites (full 5‐LO) producing a known protein of 78 kDa. Another isoform retained intron 10 (alpha‐10) resulting in a shift of the open reading frame and produces a protein of expected molecular weight of 56 kDa. A third isoform lacked the first 96 nucleotides of exon 10 (delta‐p10) and produces a protein of expected molecular weight of 74 kDa. HEK293 cells transfected with the full 5‐LO produced LTB4 and 5‐hydroxyeicosatetraenoic acid (5‐HETE) following ionophore stimulation; however, cells transfected with either alternative isoform produced no 5‐LO products. HEK293 cells transfected with the full 5‐LO and co‐transfected with the alpha‐10 or the delta‐p10 isoform produced 25±2% (mean±sem) and 49±7% (mean±sem) less 5‐LO products, respectively, compared to cells expressing only the full 5‐LO. These alternative isoforms of 5‐LO may represent a new mechanism for the regulation of LT biosynthesis in inflammatory cells. (Supported by the Canadian Institutes of Health Research and the Canada Research Chairs Program).
Modulation of Lipid Body Biogenesis in Epithelial Cells
Authors: Diaz Bruno L , Moreira Luciana S , Piva Bruno O , Bozza Patricia T , Bandeira‐Melo Christianne
Published: June 2021

Lipid Bodies are cytoplasmic inclusions mainly formed by triglycerides and cholesterol esters, being also intracellular deposits of arachidonic acid (AA), which can be metabolized for eicosanoids generation. PGE2 is a major AA metabolite produced by epithelial cells and can modulate restoration of epithelium homeostasis after injury. We studied lipid body biogenesis and their role in AA metabolic pathway in epithelial cells. A rat cell line derived from normal intestinal epithelium, IEC‐6, was used. IEC‐6 in sub‐confluent cultures presented 15–20 lipid bodies/cell after staining with by OsO4. Lipid bodies were virtually absent in IEC‐6 upon reaching confluency or after FBS withdraw. Stimulation of confluent IEC‐6 with IL‐1β, PAF, or PMA was unable to induce lipid bodies. In contrast, AA dose‐dependently induced lipid body formation and proliferation foci in confluent cultures independently of its metabolism to PGE2. AA induction of lipid bodies was dependent on signaling through p38, PKC, and PI3K, but not ERK 1/2 or JNK. Lipid body biogenesis facilitates AA release after ionophore activation without modifying cPLA2α expression. Our results suggest a strong relationship between lipid body biogenesis and epithelial cell proliferation, and indicate that these organelles are induced in a stimulus‐specific manner and facilitate AA mobilization in epithelial cells.

Financial support: CNPq, FAPERJ, CAPES
Fatty‐Acid‐Profiles: a comparison of FAME‐GC and Saponification‐ESI‐MS
Authors: Earl Cameron Lee , Roberts Brad A. , Yang Li , Wiest Landon , Moulton Josiah , Nance Brett , Wood Stephen G. , Lindford Matthew R. , Thulin Craig D.
Published: June 2021

Not enough is known about how the fatty‐acid composition of cellular membranes is affected by different medical conditions. Being able to accurately obtain and analyze the fatty‐acid profile of cells provides important information about the state of different cells under given conditions. Such information potentially could be useful in understanding, diagnosing and treating many medical conditions. This study is a comparison of two methods of obtaining such a profile. The first method is fatty‐acid methyl esterification followed by gas chromatography (FAME‐GC) with flame ionization detection. Fatty acids are identified by their retention times. This method is a common approach. The second method—saponification followed by negative‐ion‐mode electrospray mass spectrometry (sapESI‐MS)—has the potential to identify any unusual fatty acids, but is less widely employed. Preliminary indications are that both methods are effective and useful. A quantitative comparison will be presented.
Manipulation of the mevalonate pathway in glial‐derived cells
Authors: Johnson Matthew T , Hohl Raymond J
Published: June 2021

Intermediates in the mevalonate pathway are critical for cell growth and survival. Statin‐induced inhibition of hydroxymethylglutaryl coenzyme A reductase prevents the formation of mevalonate and its derivatives, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). These isoprenoids post‐translationally modify signal transduction proteins. Glial (C6) and glioblastoma (A172) cells were incubated with 0.1 – 10 μM lovastatin without and with 10 μM mevalonate, FPP, or GGPP for 24 hours. Lovastatin alters RAS expression in A172, but not C6 cells. In both lines unmodified RAP1A does accumulate in a dose‐dependent manner from 1 – 10 μM lovastatin. The addition of GGPP, but not FPP, prevents this effect. Rheb protein levels increase with 10 μM lovastatin. This increase is enhanced with 10 μM GGPP. Selective inhibition of geranylgeranyl (10 μM GGTI‐286), but not farnesyl protein transferase (10 μM FTI‐277) results in an upregulation of Rheb in A172 but not C6 cells. The basis for these differences may lie in distinct regulation for the mevalonate pathway between these cell lines. Ongoing investigations will determine this regulatory difference.
Time Course Analysis of Eight Autophosphorylation Sites on p21‐Activated Protein Kinase Pak2
Authors: Jung Jin‐Hun , White Jon , Traugh Jolinda
Published: June 2021

The p21‐activated protein kinase Pak2 is activated under cytostatic or apoptotic conditions. Binding of Cdc42(GTP) stimulates autophosphorylation of Pak2 up to 8 mol/mol, which is required for full activation. There are seven serine sites (S19, S20, S55, S141, S165, S192 and S197) in the regulatory domain, and one threonine (T402) in the activation loop. The time course of autophosphorylation of Cdc42‐activated Pak2 showed a gradual increase from 1 to 30 min. When autophosphorylated Pak2 was subjected to two‐dimensional tryptic phosphopeptide mapping and quantified, S197 was rapidly phosphorylated during first 10 min and constituted about 40% of the autophosphorylation, while the other sites were significantly less. To delineate the effect of each site in the molecular activation of Pak2, the sites were changed to alanine by site‐directed mutagenesis and the level of autophosphorylation was analyzed. In the presence of Cdc42, the four alanine mutants, S141A, S192A, S197A and T402A, showed a 50–60% reduction in autophosphorylation, while there was no effect on S19A/S20A, S55A and S165A, as compared to the level with WT Pak2. Thus, autophosphorylation of S141, S192, S197 and T402 are required to achieve maximum autophosphorylation, and phosphorylation of S197 contributes to promoting further autophosphorylation on other sites.

Supported by NIH grant GM 26738 to J. A. T.
Quantification of myo‐inositol cotransporter expression in control and streptozotocin‐treated rats.
Authors: Aouameur Rym , Cal Sandra Da , Lahjouji Karim , Bissonnette Pierre , Coady Michael J. , Lapointe Jean‐Yves
Published: June 2021

Myo‐inositol (MI) uptakes in cells is essentially performed by two Na+‐coupled transport systems; SMIT1, a basolateral system inducible through hypertonicity and SMIT2, an apical system responsible for MI uptake in the kidney and intestine, amongst other tissues. In diabetes, urinary MI excretion is increased by 1 order of magnitude through a non‐established mechanism. The purpose of this study is thus to investigate the influence of an acute diabetic condition (5‐day streptozotocintreatment) on the expression of both SMIT mRNA's in rat brain, liver, muscle, kidney and intestine through quantitative‐RT‐PCR (qRT‐PCR). Also, SMIT2 activity was determined by measuring MI transport in purified brush border membranes (BBMv) from kidney and intestine. SMIT1 transcripts are mainly found in the kidney where it doubles in diabetic rats. Other tissues show more modest mRNA levels and were not altered in diabetes. SMIT2 transcripts were found in all tested tissues in varying levels but were not modified in diabetes. MI uptakes performed on kidney and intestine BBMv confirmed the qRT‐PCR results showing no variations of transport in STZ rats due to SMIT2. We thus conclude that the increase in MI urinary excretion is not due to a reduction in the apical expression of MI cotransporter SMIT2.
Small Molecules, Chaperones and Neurodegenerative Disease
Authors: Gestwicki Jason E
Published: June 2021

Neurodegenerative disorders, such as Alzheimer's and Huntington's diseases, are associated with the accumulation of misfolded polypeptides. These misfolded proteins have a tendency to self‐associate and these oligomers are believed to cause the memory/behavioral defects seen in patients. The focus of our group's work is to identify new strategies for blocking the toxicity of these oligomers by chemical manipulation of the cell's protein folding machinery. Specifically, we are pursuing small molecules that promote the function of molecular chaperones, such as heat shock protein 70 (Hsp70). Using high throughput screening of focused chemical libraries, in concert with computational and structural approaches, we have identified a number of potential candidate molecules. Interestingly, these candidates control protein aggregation in cell‐based models of neurodegenerative disease. These chemical tools may help elucidate the molecular mechanisms underlying neurodegenerative disorders.
Nonenzymatic glycation of guanosine 5′‐ triphosphate (GTP) by dihydroxyacetone phosphate (DHAP): an in vitro study of AGE formation
Authors: Li Yuyuan , Cohenford Menashi A , Dutta Udayan , Dain Joel A
Published: June 2021

Sugars and their metabolites can nonenzymatically react with purines and pyrimidines altering the nucleic acid structures in DNA and RNA. Previous studies in our laboratory demonstrated that guanosine 5′‐triphosphate (GTP) was highly reactive with D/L‐glyceraldehyde, forming a heterogenous group of glycated end products that were readily resolvable by HPLC. The objective of this investigation was two fold. First, it was to evaluate the susceptibility of nucleotides to nonenzymatic attack by dihydroxy acetone phosphate (DHAP) and secondly, to determine the factors influencing the rate and extent of nucleotide glycation by DHAP. Of the four nucleotide triphosphates (ATP, CTP, GTP and TTP) that were evaluated, only GTP was reactive, forming a wide range of UV and fluorescent products with DHAP. Increases in temperature, pH and nucleotide concentration augmented the heterogeneity of glycated products and enhanced the yield of UV and fluorescent compounds. Capillary electrophoresis, HPLC and mass spectrometry allowed for a thorough analysis of these glycated products and demonstrated that the reaction of DHAP with GTP occurred via the classical amadori pathway. Supported jointly by NCRR/NIH Grant # P20 RR016457.
Coumarin‐Fluorescein – Based FRET Probe for Real Time Quantitation of Thiols Redox State
Authors: jonhera timothy , Pullela Phani kumar , Sem daniel
Published: June 2021

The total concentration of cellular thiols is about 30 mM, and use of current fluorescent chemical probes to quantitate these thiols inside cells is impractical. We recently reported fluorescent dithio probes (donor‐S‐S‐acceptor, DSSA) with unusually low reduction potential (− 0.6 V) that can detect changes in intracellular thiols and cross membranes of bacterial and mammalian cells, but they could not quantify thiol redox state. Here we report a new variant of the DSSA series, which has hydroxy coumarin (Ex: 320 nm, Em: 460 nm) as donor and fluorescein (Ex: 485 nm, Em: 520 nm) as acceptor. The probe was characterized in vitro by titration with glutathione in a physiologically relevant range and monitoring changes in fluorescence emission from both Coumarin and Fluorescein, upon reduction of the disulfide bond that joins this donor‐acceptor FRET pair. The ratio of fluorescence emission band intensity, changes upon reduction with glutathione, and because of the probe's low reduction potential, permits quantitative measurement of glutathione levels in the 1 – 10 mM range. This is the first demonstration of ratiometric (FRET‐based) thiol level measurement and will be enabling for quantitative assessment of thiol redox state (GSG/GSSG) inside cells. Preliminary studies in E. Coli are also presented. This research was supported by a Biomedical Technology Alliance grant (01‐KMS LEG FY06‐12368) to D.S.S
Tyrosine phosphorylation of an adapter protein induced by NK cell inhibitory receptor contributes to inhibition of cytotoxicity
Authors: Peterson Mary E. , Long Eric O.
Published: June 2021

Many cellular responses, including immune autoreactivity, are under negative control by receptors carrying immunoreceptor tyrosine‐based inhibition motifs (ITIM) in their cytoplasmic tail. Natural killer (NK) cell cytotoxicity is blocked by ITIM‐containing receptors through recruitment of tyrosine phosphatases, which dephosphorylate key components of activation pathways, such as Vav1. Here we show that binding of an NK cell inhibitory receptor to its HLA class I ligand on target cells induces selective phosphorylation of an adapter protein and its dissociation from a signaling complex. Inhibition of NK cell cytotoxicity was impaired by expression of a non‐phosphorylated mutant of the adapter, implying a role in the inhibitory signal. These results show that inhibitory receptor engagement leads to specific phosphorylation as well as dephosphorylation events, and suggest the possibility of other signaling functions for ITIM‐containing receptors. This research was supported by the Intramural Research Program of the National Institutes of Health, National Institute of Allergy and Infectious Diseases.
Phosphoinositide 3‐kinase gamma; participates in T cell receptor‐induced T cell activation.
Authors: Alcazar Isabela , Marqués Miriam , Kumar Amit , Hirsch Emilio , Wymann Matthias , Carrera Ana Clara , Barber Domingo Francisco
Published: June 2021

Class I phosphoinositide 3‐kinase (PI3K) is a family of enzymes that generate 3‐phosphorylated poly‐phosphoinositides at the cell membrane following stimulation of protein tyrosine (Tyr) kinase‐associated or G protein‐coupled receptors (GPCR). The class I PI3K are divided into two types, class IA p85/p110 heterodimers, which are activated by Tyr kinases, and the class IB p110γ isoform, which is activated by GPCR. Although the T cell receptor (TCR) is a protein Tyr kinase‐associated receptor, previous studies showed that p110γ deletion affects TCR‐induced T cell stimulation. We examined whether the TCR activates p110γ and the consequences of interfering with p110γ expression or function for T cell activation. We found that after TCR ligation, p110γ interacts with Gαq/11, lymphocyte‐specific tyrosine kinase, and zeta‐associated protein. TCR stimulation activates p110γ, which affects 3‐phosphorylated poly‐phosphoinositides levels at the immunological synapse. We show that TCR‐stimulated p110γ controls Rac1 activity, F‐actin polarization and the interaction between T cells and antigen‐presenting cells, illustrating a crucial role for p110γ in TCR‐induced T cell activation.
Cdc42/Rac function in NK cells and CTLs is variable and governed by spatiotemporal patterning of Cdc42/Rac
Authors: Sinai Parisa , Nguyen Chau , Schatzle John D. , Wuelfing Christoph
Published: June 2021

Natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) are central immune effectors. They activate in a cellular interaction with target cells, leading to target cell lysis and IFN? secretion. These effector functions require effective cellular polarization. We have therefore studied roles of Cdc42 and Rac, two general regulators of cellular polarization, in primary NK cells and CTLs. NK cells were primed with IL‐2, IL‐12/IL‐18, or IL‐15, reflecting different physiological and therapeutically used activation conditions. We expected that Cdc42/Rac by promoting cellular polarization would support effector functions. Surprisingly however, functions of Cdc42/Rac varied dramatically between the different cell types and activation conditions. When we determined the spatiotemporal patterns of Cdc42/Rac activity in NK cell/CTL – target cell interactions, we intriguingly found that Cdc42/Rac could only support effector functions when enriched at the center of the effector/target cell interface. Our data thus establish that Cdc42/Rac function in NK cells and CTLs is not only governed by the activation of these proteins but, as importantly, by the location of this activation.

Supported by the NIH
Important roles for Fyn in CD4 T cell activation and helper functions in vivo
Authors: Yusuf Isharat , McCausland Megan , Tran Hung , Crotty Shane
Published: June 2021

Fyn is a src‐family kinase involved in signaling downstream of the T cell receptor and B cell receptor. Initial reports on the Fyn‐deficient mice revealed no obvious defects in mature CD4 T cells suggesting that its function maybe redundant with Lck. More recently Fyn has been shown to play a critical role downstream of the SLAM‐family receptors through the adaptor protein, S AP/SH2DIA. Here we report that although early CD4 T cell dependent antibody production appears to be normal, Fyn‐deficient mice display a defect in germinal center formation and long‐term antibody production resulting from a lack of long‐lived plasma cells – a defect similar to SAP‐deficient mice. These defects are distinct from Fyn's role downstream of SLAM‐SAP signaling pathway as demonstrated by the use of a SAP mutant incapable of binding Fyn. This non‐Fyn binding mutant SAP is fully capable of rescuing the humoral defect seen in the SAP‐deficient mice. The data here suggest that Fyn may indeed play a critical role in CD4 T cell help to B cells, but Fyn's role in the humoral response is independent of the SLAM‐SAP pathway.

This work was supported by NIH T32 AI060536 grant (UCSD) to IY
Memory phenotype CD4+ T‐cells with innate function selectively develop in the absence of active Itk
Authors: Hu Jianfang , August Avery
Published: June 2021

The Tec family kinase Itk regulates the development of conventional and innate CD8+ T cells, little is known about the role of Itk in the development of CD4+ T cell lineage, although the role of Itk in the T cell activation and function is well defined. We show here that the Itk null mice have increased percentage of memory phenotype CD4+ T cells compared to WT mice. These cells directly arise from thymus. They express high levels of transcripts for the T‐bet and IFN¦Ã and are able to produce IFN¦Ã directly ex vivo in response to stimulation. Itk deficiency greatly decreases the number of CD4+ T cells with naïve phenotype, but shows no effect on the number of memory phenotype CD4+ T cells, which indicates that the development of memory phenotype CD4+ T cells are Itk independent. The development of the naïve phenotype CD4+ T cells is active Itk kinase signaling dependent and can be rescued by over‐expression the hItk in T cells, but not the memory phenotype CD4+ T cells. Our data also show that the Itk is required for the function through TCR signaling of these cells, but not for the innate function in response to stimulation. These results indicate that the development of memory phenotype CD4+ T cells with innate function is Itk independent.
Regulation of IL‐1 Secretion in Response to Cellular Stress: A Mechanism for Sterile Inflammation
Authors: Kwong Christina , Ramu Sivakumar , Ntrivalas Evangelos , Gilman‐Sachs Alice , Beaman Kenneth
Published: June 2021

The inflammatory response is normally thought to be switched on by an antigenic stimulus. However, in certain instances, such as cell injury, pregnancy and tumor growth, sterile inflammation occurs. We have found a protein that responds to cellular stress, the a2 isoform of vacuolar ATPase, and initiates cytokine secretion without antigen involvement. In response to stress, a2V‐ATPase migrates to the plasma membrane and a 20‐kDa [N‐terminus domain of the a2V‐ATPase (a2NTD)] molecule is cleaved and secreted into the extracellular space. Flow cytometric data show intracellular expression of a2V‐ATPase to be 40% in basal PBMC but levels of a2NTD to be much less. The release of a2NTD regulates cytokine production in PBMC. Supernatants from PBMC cultured with a2NTD show increased levels of IL‐1β (451.08 ± 142 pg/ml) compared to controls without a2NTD (5.16 ± 6.89 pg/ml) via ELISA. Flow cytometric analysis of these cells detect an increase in percentage of CD14+/IL‐1β+ monocytes in the presence of a2NTD (88.0% ± 3.12%) compared to CD14+/IL‐1β+ monocytes without a2NTD (0.43% ± 0.29%). There is no difference in intracellular TNF‐α expression in monocytes cultured with or without a2NTD. Fluorescent microscopy of PBMC shows that a majority of monocytes bind a2NTD on their cell surface. These results show a direct role for a2NTD in IL‐1β production and secretion and may play a role in sterile inflammation. It demonstrates that immune regulation via cytokines may begin prior to antigen recognition and stimulation.
Reciprocal Regulation of Cyclooxygenase (COX)‐1 and COX‐2 Expression in Murine Resident and Elicited Peritoneal Macrophages by Lipopolysaccharide
Authors: Tan Xiaoyu , Rakhra Varundeep K , Menon Harry S , Stechschulte Daniel J , Dileepan Kottarappat N
Published: June 2021

Macrophages express COX‐1 and COX‐2 that are involved in the biosynthesis of prostanoids including prostaglandin E2 (PGE2). In this study, we examined the relative expression of COX‐1 and COX‐2 in murine resident (RPM) and thioglycollate‐elicited peritoneal macrophages (EPM) and determined the influence of lipopolysaccharide (LPS) on their gene expression and functions. Freshly harvested RPM and EPM from C57Bl/6J mice were allowed to adhere on cell culture plates and incubated with LPS (0.1 to 100 ng/ml) for 4 h for measuring COX‐1 and COX‐2 mRNA expression by quantitative RT‐PCR. To monitor COX‐1 and COX‐2 protein expression, Western blot analyses were carried out after incubating macrophages with LPS for 2, 4, 8, 16 and 24 h. The production of PGE2 and TNF‐α by RPM and EPM were monitored as functional indices. The study revealed that, in comparison to EPM, RPM constitutively express markedly higher levels of COX‐1 (12‐fold) and COX‐2 (3.5‐fold) mRNA and produce substantially higher quantities of PGE2 when stimulated with LPS. In contrast, EPM generate considerably lower levels of PGE2 and higher levels of TNF‐α than RPM when activated with LPS. Incubation of RPM or EPM with LPS resulted in marked increases in COX‐2 mRNA (up to 1000 fold) and protein expression. On the other hand, LPS challenge significantly inhibited COX‐1 mRNA (60 to 90%) and protein expression in both RPM and EPM. These results demonstrate that LPS reciprocally regulate the expression of COX‐1 and COX‐2 in murine peritoneal macrophages. Furthermore, the newly recruited peritoneal macrophages seem to undergo plausible phenotype switching from high‐TNFα/low‐PGE2 producing EPM to low‐TNFα/high‐PGE2 producing RPM. (Supported by NIH R01‐HL070101 and Carey Arthritis Fund).
Induction of autophagy is associated with acute hepatitis caused by Concanavalin A in mice
Authors: Chang Chih‐Peng , Lei Huan‐Yao
Published: June 2021

Concanavalin A (Con A) is known to induce acute hepatitis that is mediated by activation of NKT and T‐cell and cytokines production in immunocompetent mice. The observation of Con A‐induced autophagic cell death in hepatocytes via a BNIP3‐mediated autophagic pathway made us re‐evaluate the effect of Con A on mice. In this study, we report that Con A can induce acute hepatitis in SCID or SCID/NOD mice, with kinetics similar to that of BALB/c, but requiring a higher dose of Con A. No lymphocyte infiltrations were found in liver of SCID or SCID/NOD mice, and the cytokine productions were different. An autophagy with LC3‐II conversion was demonstrated in the liver post Con A injection with a quicker kinetics in the BALB/c than SCID mice. Con A induces IFN‐£^ production at early time on BALB/c mice, and enhances LC3‐II conversion in hepatocytes with addition of IFN‐£^ in vitro. Due to the mannose/glucose‐specific binding on cell membrane, Con A can exhibit both autophagic cytotoxicity and immunomodulation on T cells. This dual‐property can cause acute hepatitis that is enhanced by the immune status of the host. In conclusion, Con A can induce autophagy formation in both T cell‐dependent or ‐independent acute hepatitis.
Signaling from the B cell antigen receptor (BCR) induces VH replacement in human immature B cells
Authors: Liu Jing , Fan Run , Hong Sang Yong , Yu Zhihong , Su Kaihong , Zhang Zhixin
Published: June 2021

VH replacement occurs through RAG‐mediated recombination between a cryptic RSS (cRSS) from a rearranged VH gene and a 23 bp RSS from an upstream VH gene. We have shown that VH replacement occurs in human bone marrow immature B cells and contributes to about 5% of the primary B cell repertoire. Elevated frequencies of VH replacement products in IgH genes derived from autoimmune diseases and anti‐viral response further suggested an important function of VH replacement. How VH replacement is regulated remains unknown. The human EU12 μHC+ cells undergo spontaneous VH replacement. Crosslinking B cell antigen receptor (BCR) on the EU12 μHC+ cells quickly induces BCR internalization and prevents cell proliferation. As we predicted, crosslinking BCR strongly induces VH replacement as indicated by elevated levels of double stranded DNA breaks at the VH3 cRSS borders and VH3 to VH1 VH replacement excision circles. Blocking BCR‐mediated signaling with tyrosine kinase inhibitor Genistein inhibits BCR‐mediated induction of VH replacement. In addition, we found that VH replacement occurs in the newly emigrated immature B cells in the peripheral blood of healthy donors and tonsillar B cells. Crosslinking BCR further enhances VH replacement in primary immature B cells. These results provide the first evidence that VH replacement in human immature B cells is regulated by BCR mediated signaling.